Microencapsulation of flavour-enhancing enzymes

for acceleration of Cheddar cheese ripening

By

Kavya Anjani

A thesis submitted in fulfilment of the requirements for the

degree of Doctor of Philosophy

Centre for Plant and Food Science

University of Western Sydney
Australia

December 2007
 Statement of Authentication

The work presented in this thesis is, to the best of my knowledge and belief, original

except as acknowledged in the text. I hereby declare that I have not submitted this

material, either in whole or in part, for a degree at any other institution.

Kavya Anjani

i
 Acknowledgements

I would like to sincerely thank A/Prof. Kasipathy Kailasapathy for giving me an

opportunity to work on this project and for all the encouragement, support and

guidance that has helped me complete this study.

I wish to covey my heartfelt gratitude to Dr. Michael Phillips for all his support,

understanding and for guiding me through the course of this study. Thank you,

Michael for sharing your knowledge.

My sincere thanks go to Mohan Bhargava and Nigel Barrette at Dairy Farmers for

giving me an opportunity to conduct cheese trials at Dairy Farmers’ facility and

assisting me in cheese grading and also for sharing their expertise.

I am grateful to the Australian Research Council, Dairy Farmers and University of

Western Sydney for funding this study and my scholarship.

The successful completion of this project is also owed to the valuable support,

understanding and cooperation of Dr. Jim Hourigan, Dr. Saman Seneweera, Dr.

Minh Nguyen and Dr. Linh Vu, thank you all.

It would not have been possible to complete this study on schedule, without the

support and co-operation of all the technical staff from horticulture, microbiology,

science, food and agriculture. I take this opportunity to convey my heartfelt gratitude

to all of them. I am also thankful to all the administrative staff at Centre for

Advanced Food Research and Centre for Plant and Food Science for assisting me

during this study. I wish to thank all my colleagues for their friendship, help, support,

understanding and co-operation.

Lastly I would like to thank my family; it would not have been possible to complete

this study without their love, encouragement and support.

ii
 List of Publications

Journal articles

Anjani, K., Kailasapathy, K. and Phillips, M., (2007). Microencapsulation of

enzymes for potential application in acceleration of cheese ripening. International

Dairy journal, 17; 79-86.

Anjani, K., Kailasapathy, K. and Phillips, M. Effect of alginate-chitosan

encapsulated Flavourzyme on peptide and amino acid profile in Cheddar-type cheese

(manuscript under preparation).

Conference presentations

Anjani, K., Kailasapathy, K., Phillips, M. and Nguyen, M. Microencapsulation of

enzymes for accelerated cheese ripening, 13th International Workshop on

Bioencapsulation, June 24th – 26th, 2005, Kingston, Ontario, Canada (Oral

presentation).

Anjani, K. Microencapsulation of enzymes for accelerated cheese ripening, in the

proceedings of the Malcolm Bird award, 38th Annual Australian Institute of Food

Science & Technology Convention, 10-13th July, 2005 Sydney Convention &

Exhibition Centre, Sydney, Australia (oral presentation).

iii
Anjani, K., Kailasapathy, K., Phillips, M. and Nguyen, M. Encapsulated enzymes:

effect on peptide profile of Cheddar cheese, 39th Annual Australian Institute of Food

Science & Technology Convention, 9-12th July, 2006, Adelaide Convention Centre,

Adelaide, Australia (poster presentation).

Anjani, K., Phillips, M. and Kailasapathy, K. Acceleration of Cheddar cheese

ripening with encapsulated enzymes, Cheese Science 2006, 24th – 26th July, 2006,

Sydney, Australia (poster presentation).

Awards

Runner up, Septimus Birrell Award, 22nd May 2005, Dairy Industry Association of

Australia.

Runner up, CSTE Innovation conference 8th June, 2005, University of western

Sydney.

Travel award by International Society of Bioencapsulation for oral presentation at the

13th International Workshop on Bioencapsulation, June, 2005.

Runner up, Best poster at the 38th Annual Australian Institute of Food Science &

Technology Convention, 10-13th July, 2005, Sydney, Australia.

iv
Table of Contents

Statement of Authentication...................................................................................... i
Acknowledgements.................................................................................................. ii
List of Publications ................................................................................................. iii
Table of Contents......................................................................................................v
List of Figures....................................................................................................... viii
List of Tables........................................................................................................ xiii
List of Abbreviations .............................................................................................xiv

Abstract...................................................................................................................1

1 Introduction ......................................................................................................4
1.1 Aim .........................................................................................................8
1.2 Objectives ...............................................................................................8

2 REVIEW OF LITERATURE...........................................................................9
2.1 History and background of cheese .........................................................10
2.2 Cheese production .................................................................................11
2.2.1 Cheese manufacture...............................................................................11
2.2.2 Cheese ripening .....................................................................................13
2.2.2.1 Microbiology of cheese ripening...................................................14
2.2.2.2 The biochemistry of cheese ripening .............................................15
2.2.2.2.1 Proteolysis .............................................................................17
2.2.2.2.1.1 Catabolism of amino acids during cheese ripening...........21
2.2.2.2.2 Lipolysis ................................................................................26
2.2.2.2.3 Glycolysis ..............................................................................29
2.2.2.3 Texture .........................................................................................32
2.2.2.4 Flavour .........................................................................................33
2.3 Acceleration of cheese ripening .............................................................34
2.3.1 Elevation of ripening temperature..........................................................35
2.3.2 High pressure processing .......................................................................36
2.3.3 Attenuated starters .................................................................................37
2.3.4 Starter selection .....................................................................................38
2.3.5 Genetically modified starters .................................................................39
2.3.6 Adjunct cultures ....................................................................................40
2.3.7 Exogenous enzymes ..............................................................................41
2.3.7.1 Proteolytic enzymes ......................................................................42
2.3.7.2 Lipolytic enzymes.........................................................................43
2.3.7.3 Microencapsulation of enzymes for acceleration of cheese ripening.
....................................................................................................45
2.4 Microencapsulation ...............................................................................47
2.4.1 Techniques of Microencapsulation ........................................................48
2.4.2 Hydrogels..............................................................................................49
2.4.3 Alginate.................................................................................................50
2.4.4 Carrageenan ..........................................................................................52
2.4.5 Chitosan ................................................................................................52
2.4.6 Starch ..................................................................................................53
2.4.7 Pectin ..................................................................................................53

v
 2.4.8 Poly-L-lysine.........................................................................................53
2.5 Analysis and evaluation of cheese ripening............................................54
2.6 Factors affecting cheese quality .............................................................56
2.7 Summary...............................................................................................57

3 SCREENING AND SELECTION OF POLYMERS FOR ENZYME

ENCAPSULATION..............................................................................................59
3.1 Abstract.................................................................................................60
3.2 Introduction...........................................................................................62
3.3 Materials and methods...........................................................................64
3.3.1 Flavourzyme® assay ..............................................................................64
3.3.2 Palatase® assay ......................................................................................65
3.3.3 Selection of polymers for enzyme encapsulation....................................65
3.3.4 Polymer combinations for improved Flavourzyme retention in capsules 66
3.3.5 Optimisation of polymer concentration..................................................67
3.3.6 Treatment of cationic solution with chitosan and optimisation ...............69
3.3.7 Coating of capsules for improved enzyme retention and stability...........69
3.3.8 Optimisation of gelling duration ............................................................70
3.3.9 Optimisation of releasing solution concentration ...................................70
3.3.10 Shelf life of encapsulated Flavourzyme...............................................70
3.3.11 Morphology of enzyme microcapsules................................................71
3.3.12 Statistical analysis ..............................................................................71
3.4 Results...................................................................................................72
3.4.1 Selection of polymers for enzyme encapsulation....................................72
3.4.2 Effect of chitosan concentration in gelling solution on encapsulation
efficiency of Flavourzyme .....................................................................77
3.4.3 Weight of Flavourzyme microcapsules ..................................................77
3.4.4 Optimisation of polymer concentration..................................................80
3.4.5 Effect of coating on Flavourzyme retention in capsules .........................80
3.4.6 Optimisation of gelling duration ............................................................80
3.4.7 Optimisation of tri-sodium citrate concentration for in vitro release of
encapsulated enzyme .............................................................................85
3.4.8 Shelf life of freeze-dried, air-dried and frozen Flavourzyme
microcapsules........................................................................................86
3.4.9 Morphology of uncoated and poly-L-lysine coated Flavourzyme
microcapsules........................................................................................87
3.5 Discussion .............................................................................................92
3.6 Conclusion ..........................................................................................100

4 APPLICATION OF ENCAPSULATED ENZYMES IN CHEDDAR

CHEESE MANUFACTURE ..............................................................................101
4.1 Abstract...............................................................................................102
4.2 Introduction.........................................................................................103
4.3 Materials and methods.........................................................................106
4.3.1 Leakage of encapsulated Flavourzyme in milk.....................................106
4.3.2 Stability of encapsulated Flavourzyme under simulated cheese press
conditions............................................................................................107
4.3.3 Cheddar cheese manufacture with microencapsulated Flavourzyme and
Palatase ...............................................................................................109

vi
 4.3.4 Effect of stage of capsule addition during cheese manufacture on the
distribution of enzyme capsules in cheese matrix.................................112
4.3.5 Analysis of Cheddar cheese .................................................................112
4.3.5.1 Moisture .....................................................................................113
4.3.5.2 Fat ..............................................................................................113
4.3.5.3 pH...............................................................................................113
4.3.5.4 Microbiological status of cheese samples ....................................114
4.3.5.5 Statistical analysis.......................................................................114
4.4 Results.................................................................................................115
4.4.1 Stability of encapsulated Flavourzyme.................................................115
4.4.2 Determination of stage of capsule addition during cheese manufacture 116
4.4.3 Composition of cheese.........................................................................117
4.4.4 Microbiological status of cheese ..........................................................117
4.5 Discussion ...........................................................................................124
4.6 Conclusion ..........................................................................................129

5 MONITORING AND EVALUATION OF CHEESE RIPENING..............130

5.1 Abstract...............................................................................................131
5.2 Introduction.........................................................................................133
5.3 Materials and methods.........................................................................135
5.3.1 Monitoring of proteolysis in cheese .....................................................135
5.3.1.1 Extraction of water-soluble peptides ...........................................135
5.3.1.2 Analysis of water-soluble peptides..............................................135
5.3.1.3 Extraction of water-insoluble peptides ........................................136
5.3.1.4 Analysis of water-insoluble peptides ...........................................136
5.3.1.5 Extraction of amino acids............................................................137
5.3.1.6 Derivatization of free amino acids and analysis by RP-UPLC .....137
5.3.1.7 Determination of total amino acids by spectrophotometer ...........138
5.3.1.8 Preparation of cadmium-ninhydrin reagent for the determination of
total amino acid concentration by spectrophotometer.................................138
5.3.2 Monitoring of lipolysis in cheese .........................................................139
5.3.2.1 Extraction and esterification of fatty acids from cheese for Gas
Chromatography analysis ..........................................................................139
5.3.2.2 Analysis of fatty acid methyl esters by GC..................................139
5.3.3 Cheese grading ....................................................................................140
5.4 Results.................................................................................................142
5.4.1 Assessment of proteolysis....................................................................142
5.4.1.1 Peptide profile: water-soluble and water-insoluble ......................142
5.4.1.2 Free and total amino acid composition ........................................150
5.4.2 Assessment of lipolysis........................................................................153
5.4.2.1 Fatty acid methyl ester profile of Cheddar-type cheese................153
5.4.3 Grading of control and accelerated ripened Cheddar-type cheeses .......159
5.5 Discussion ...........................................................................................166
5.6 Conclusion ..........................................................................................180

6 CONCLUDING SUMMARY .......................................................................181

7 FURTURE DIRECTIONS ...........................................................................188

8 REFERENCES..............................................................................................191

vii
List of Figures

Figure 2.1 Overview of biochemistry of cheese ripening (Law, 2001). .....................16

Figure 2.2 Proteolytic agents in cheese during ripening (Sousa et al., 2001). ............18

Figure 2.3 Schematic representation of the action of peptidases found in lactic

acid bacteria (Upadhyay et al., 2004a). ...................................................20

Figure 2.4 Schematic diagram of amino acid catabolism pathways found in

different microorganisms and some chemical reactions (dotted line)
occurring in cheese during ripening. HA-DH: hydroxyacid
dehydrogenase, -KADH: -ketoacid dehydrogenase, -KADC: -
ketoacid decarboxylase, aldDH: aldehyde dehydrogenase, alcoDH:
alcohol dehydrogenase, MGL: methionine -lyase, CGL:
cystathionine -lyase, CBL: cystathionine -lyase, TPL: tyrosine-
phenol lyase, TIL: tryptophan-indole lyase (Yvon and Rijnen, 2001;
modified by Curtin and McSweeney, 2004). ...........................................22

Figure 2.5 Catabolism of leucine initiated by transaminase, deaminase or

decarboxylase action and volatile flavour compounds that may be
formed from this amino acid. Similar catabolic pathways operate for
the other branched-chain aliphatic amino acids (isoleucine and
valine) (McSweeney, 2004). ...................................................................23

Figure 2.6 Methionine degradation pathways by starter cultures. PLP: pyridoxal

phosphate; TPP thiamine pyrophosphate (Smit et al., 2000)....................24

Figure 2.7 Pathways for the production of flavour compounds from fatty acids
during cheese ripening (McSweeney, 2004)............................................28

Figure 2.8 Pathways by which lactate is metabolised in cheese during ripening.

(1) racemization, (2) metabolism by Propionibacterium freudereichii
in Swiss cheese, (3) oxidative metabolism of lactate, (4) conversion
to formate, (5) anaerobic metabolism of lactate to butyrate and H2,
which leads to late gas blowing (McSweeney and Fox, 2004). ................30

Figure 2.9 Pathways for citrate-positive strains of Lactococcus and Leuconostoc

sp (McSweeney and Fox, 2004)..............................................................31

Figure 2.10 Structure of alginate. Alginate molecules are linear block co-
polymers of β-D-mannuronic acid (M) and -L-guluronic acids (G)
with a variation in composition and sequential arrangements (de Vos
et al., 2006).............................................................................................51

Figure 3.1 Schematic representation of enzyme encapsulation in calcium-

alginate by extrusion (Anjani et al., 2007)...............................................68

Figure 3.2 Encapsulation efficiency of Flavourzyme immobilised in alginate by

gel bead formation in 0.1M CaCl2 and the effect of alginate

viii
 concentration on encapsulation efficiency. Error bars indicate
standard error of mean. ...........................................................................74

Figure 3.3 Encapsulation efficiency of Flavourzyme immobilised in alginate-

starch mixture by gel bead formation in 0.1M CaCl2 and the effect of
varying alginate-starch concentration and proportion (in 100mL
water) on encapsulation efficiency. Error bars indicate standard error
of mean. .................................................................................................75

Figure 3.4 Encapsulation efficiency of Flavourzyme immobilised in alginate-

pectin mixture by gel bead formation in 0.1M CaCl2 and effect of
varying concentration and proportion of alginate and pectin (in
100mL water) on encapsulation efficiency. Error bars indicate
standard error of mean. ...........................................................................76

Figure 3.5 Encapsulation efficiency of Flavourzyme immobilised in 2% (w/v)

alginate with gel bead formation in cationic solution of 0.1M CaCl2
containing chitosan and the effect of chitosan concentration on
encapsulation efficiency (results are a mean of 5 replicates, * data
obtained from Figure 3.2, mean of 6 replicates). Error bars indicate
standard error of mean. ...........................................................................78

Figure 3.6 Effect of alginate concentration on encapsulation efficiency of

Flavourzyme gel beads formed in 0.1M CaCl2 containing 0.1% (w/v)
chitosan. Error bar indicates standard error of mean................................82

Figure 3.7 Flavourzyme activity in capsules produced from 2% (w/v) alginate

allowed to gel in cationic solution of 0.1M CaCl2 containing 0.1%
(w/v) chitosan and coated with 0.15% alginate and 0.05% poly-L-
lysine for 10 min. Error bars indicate standard error of mean. .................83

Figure 3.8 Effect of gelling duration in 0.1M CaCl2 containing 0.1% (w/v)
chitosan on Flavourzyme retention in (a) uncoated and (b) poly-L-
lysine coated capsules made from 1.6, 1.8 and 2.0% (w/v) alginate.
Error bar indicates standard error of mean. .............................................84

Figure 3.9 Effect of tri-sodium citrate concentration on in vitro release of

encapsulated Flavourzyme from (a) uncoated and (b) poly-L-lysine
coated capsules produced from 1.6, 1.8 and 2.0 % (w/v) alginate and
allowed to gel in 0.1M CaCl2 containing 0.1% (w/v) chitosan. Error
bar indicates standard error of mean. ......................................................88

Figure 3.10 Shelf life of Flavourzyme in uncoated capsules immobilised in 1.6

(i), 1.8 (ii) and 2.0 % (w/v) (iii) alginate by gel bead formation in
0.1M CaCl2 containing 0.1% (w/v) chitosan, stored by freeze-drying
(a), air-drying (b) and freezing (c) over a period of 10 weeks. Error
bar indicates standard deviation ..............................................................89

Figure 3.10 Shelf life of Flavourzyme in poly-L-lysine coated capsules

immobilised in 1.6 (iv), 1.8 (v) and 2.0 % (w/v) (vi) alginate by gel

ix
 bead formation in 0.1M CaCl2 containing 0.1% (w/v) chitosan,
stored by freeze-drying (a), air-drying (b) and freezing (c) over a
period of 10 weeks. Error bar indicates standard deviation......................90

Figure 4.1 Application of simulated cheese press pressure on Flavourzyme

capsules................................................................................................108

Figure 4.2 Flow process chart of Cheddar cheese manufacture (adapted from
Madziva, 2006).....................................................................................111

Figure 4.3 Retention of Flavourzyme activity in capsules produced from 1.6, 1.8
and 2.0% (W/V) alginate and gelling in 0.1M CaCl2 containing 0.1%
(w/v) chitosan suspended in milk at pH 5.0 and 6.0 at 32°C (a) and
38°C (b) for 10 min. The Flavourzyme activity in the capsules prior
to suspension in acidified milk is labelled initial and the difference in
the activity initially and at different pH and temperature treatments is
equivalent to the loss of activity as a result of leakage of
encapsulated Flavourzyme in milk. Error bars indicate standard error
of mean. ...............................................................................................118

Figure 4.4 Retention of Flavourzyme activity in the capsules produced from 1.6,
1.8 and 2.0% (w/v) alginate and gelling in 0.1M CaCl2 containing
0.1% (w/v) chitosan subjected to simulated cheese press pressure in
a texture analyser for 4, 8 and 16h. The Flavourzyme activity was
measured before and after subjecting to the simulated cheese press
pressure and the difference in the initial and final Flavourzyme
activity is presented as the retained activity after simulated cheese
press. Error bars indicate standard error of mean...................................119

Figure 4.5 Distribution of stained Flavourzyme capsules in Cheddar cheese, (a)

capsule addition to milk prior to rennetting and (b) capsule addition
to curd prior to salting ..........................................................................120

Figure 5.1 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 30 days of ripening .........................145

Figure 5.2 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 90 days of ripening .........................146

Figure 5.3 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 180 days of ripening .......................147

Figure 5.4 Chromatograms of the water-soluble peptides from cheeses

manufactured with encapsulated Flavourzyme at concentrations of
0.5 (F50), 0.75 (F75) and 1.0 LAPU/g milk protein (F100) after 60
days of ripening....................................................................................148

x
Figure 5.5 Chromatograms of the water-insoluble peptides from cheese F50
with encapsulated Flavourzyme at a concentration of 0.5 LAPU/g
milk protein, cheese C with no added enzymes extracted after 180
days of ripening, -casein and -casein standards at concentration of
15mg/mL..............................................................................................149

Figure 5.6 Composition of free amino acids from control cheese C with no
added enzymes and cheese F75 with encapsulated Flavourzyme.
Free amino acid composition in cheeses C and F75 after (a) 30 days
ripening (b) 90 days ripening ................................................................151

Figure 5.7. Total amino acid concentration measured by spectrophotometer in

the following cheeses; C, D, F50, F75, F100, PF50, PF75, PF100 &
P...........................................................................................................152

Figure 5.8 Profile of fatty acid methyl esters of control cheese C (KAA90B
with no added enzymes) and cheese P (KAI90B, with
encapsulated Palatase) extracted after 90 days of ripening ....................155

Figure 5.9 Profile of fatty acid methyl esters of cheeses with encapsulated
Flavourzyme (F75; KAD90B ) and with encapsulated
Flavourzyme and Palatase (PF75; KAG90B ) extracted after 90
days of ripening....................................................................................156

Figure 5.10 Profile of fatty acid methyl esters of cheeses; PF75 with
encapsulated Flavourzyme and Palatase (KAG90B ) P and with
encapsulated Palatase (KAI90B ) extracted after 90 days of
ripening ................................................................................................157

Figure 5.11 Profile of Standard fatty acid methyl esters (KAFAME7 ) and
FAME from cheese P with encapsulated Palatase (KAI90B )
extracted after 90 days of ripening ........................................................158

Figure 5.12 Comparison of grading score of cheeses (a) C, D and F75, (b) F75
and PF75, (c) PF75 and PF100 based on colour and condition on a
scale of 1-20. C – no added enzymes, D - free Flavourzyme at a
concentration of 0.75LAPU/g milk protein, F75 – encapsulated
Flavourzyme at a concentration of 0.75LAPU/g milk protein, PF75 –
encapsulated Flavourzyme at 0.75LAPU/g milk protein and
encapsulated Palatase at 0.5LU/g milk fat and PF100 - encapsulated
Flavourzyme at 1.0LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat. ..............................................................................162

Figure 5.13 Comparison of grading score of cheeses (a) C, D and F75, (b) F75
and PF75, (c) PF75 and PF100 based on body and texture on a scale
of 1-30. C – no added enzymes, D - free Flavourzyme at a
concentration of 0.75LAPU/g milk protein, F75 – encapsulated
Flavourzyme at a concentration of 0.75LAPU/g milk protein, PF75 –
encapsulated Flavourzyme at 0.75LAPU/g milk protein and

xi
 encapsulated Palatase at 0.5LU/g milk fat and PF100 - encapsulated
Flavourzyme at 1.0LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat. ..............................................................................163

Figure 5.14 Comparison of grading score of cheeses (a) C, D and F75, (b) F75
and PF75, (c) PF75 and PF100 based on flavour on a scale of 1-50.
C – no added enzymes, D - free Flavourzyme at a concentration of
0.75LAPU/g milk protein, F75 – encapsulated Flavourzyme at a
concentration of 0.75LAPU/g milk protein, PF75 – encapsulated
Flavourzyme at 0.75LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at
1.0LAPU/g milk protein and encapsulated Palatase at 0.5LU/g milk
fat.........................................................................................................164

Figure 5.15 Comparison of the final grading score of cheeses (a) C, D and F75,
(b) F75 and PF75, (c) PF75 and PF100 on a scale of 1-100. C – no
added enzymes, D - free Flavourzyme at a concentration of
0.75LAPU/g milk protein, F75 – encapsulated Flavourzyme at a
concentration of 0.75LAPU/g milk protein, PF75 – encapsulated
Flavourzyme at 0.75LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at
1.0LAPU/g milk protein and encapsulated Palatase at 0.5LU/g milk
fat.........................................................................................................165

xii
List of Tables

Table 2.1 Flavour compounds derived from amino acids via -keto acids by the
transaminase pathway. Compounds in italics are formed by non-
enzymatic reactions (Adapted from Smit, 2005). ....................................25

Table 3.1 Weight of capsules obtained from 100mL polymer-Flavourzyme

(90mL:2mL, 8mL Flavourzyme diluent) mixture allowed to gel in
cationic solution of 0.1M CaCl2 with and without chitosan for 10
min. * treatment with varying concentrations..........................................79

Table 3.2 Size and shape of alginate Flavourzyme capsules allowed to gel in
0.1M CaCl2 containing 0.1% (w/v) chitosan (mean ± standard
deviation, average of 20 replicates).........................................................91

Table 4.1 Capsule distribution represented by ratio of number of pixels to

number of particles, * mean of 30 fields ± standard deviation ...............121

Table 4.2 Fat, moisture and final pH of cheeses with and without flavour-
enhancing enzymes * mean of 3 samples ± standard error of mean .......122

Table 4.3 Pathogens tested in control and experimental cheeses produced for
grading (acceptable microbiological level in a cheese sample (AS),
other dairy products (M) as per the Australian Standard 1.6.1)..............123

Table 5.1 Defects in Cheddar cheese considered while grading ..............................141

xiii
List of Abbreviations

ALP Alkaline phosphatase

ANOVA Analysis of variance
BSNP Bacillus subtilis neutral proteinase
EE Encapsulation efficiency
ELISA Enzyme linked immuno sorbent assay
CFE Cell free extract
FAME Fatty acid methyl esters
FAA Free amino acids
FFA Free fatty acids
FITC Fluorescein-iso-thio-cyanate
FTIR Fourier transform infrared (spectroscopy)
GC Gas chromatography
HTST High-temperature short-time (pasteurisation)
LAB Lactic acid bacteria
LAPU Leucine amino peptidase units
LC-ESI-MS/MS Liquid chromatography electrospray ionisation tandem mass
spectrometry
LU Lipase units
LPL Lipoprotein lipase
MALDI-TOF Matrix-assisted laser desorption/ionisation time-of-flight
(mass spectrometry)
MFGM Milk fat globule membrane
MLV Multilamellar vesicles
NSLAB Non starter lactic acid bacteria
PCA Principal component analysis
PLL Poly-L-lysine
RP-HPLC Reverse phase high performance liquid chromatography
RP-UPLC Reverse phase ultra performance liquid chromatography
SLAB Starter lactic acid bacteria
SPSS Statistical Package for Social Sciences
SSA Sulphosalicylic acid
SUV Small unilamellar vesicles
TFA Tri fluoro acetic acid
TMAH Tetra methyl ammonium hydroxide

xiv
 Abstract

Abstract

Commercial flavour-enhancing enzymes were delivered in an encapsulated form to

accelerate Cheddar cheese ripening. Polymers such as alginate, chitosan and κ-

Carrageenan were screened to be used as encapsulant material for

microencapsulation of the commercial protease enzyme, Flavourzyme®. Alginate

was found to be a suitable polymer for Flavourzyme encapsulation using the

Inotech® encapsulator while -Carrageenan and chitosan were too viscous for

extrusion through the encapsulator nozzle. Gelling of alginate-Flavourzyme

microcapsules in 0.1M CaCl2 resulted in poor encapsulation efficiency (ranging 17-

18% depending on the alginate concentration). Incorporation of Hi-Maize™ starch or

pectin as filler materials into the alginate-Flavourzyme encapsulation matrix to

increase encapsulation efficiency by minimising porosity also resulted in poor

encapsulation efficiency.

An alternative approach to the modification of the cationic gelling solution, by

adding chitosan, significantly increased the encapsulation efficiency to 70-88% and

produced mostly spherical capsules with an average diameter of 500 m.

Encapsulation efficiency increased with an increase in chitosan concentration from

0.1 to 0.3% (w/v) in the cationic gelling solution of 0.1M CaCl2. Though gelling of

alginate-Flavourzyme microcapsules in gelling solution of 0.1M CaCl2 containing

0.3% (w/v) chitosan resulted in higher encapsulation efficiency, a chitosan

concentration of 0.1% (w/v) was chosen for further work as higher concentrations of

chitosan in the gelling solution resulted in aggregation of capsules during formation.

Gelling time of 10 min and alginate concentrations in the range 1.6 to 2.0% (w/v)

were found to be optimal encapsulation parameters for Flavourzyme encapsulation

1
 Abstract

while 2.0% (w/v) solution of trisodium citrate was found to be optimal for in vitro

release of encapsulated enzymes for measurement of enzyme activity.

Flavourzyme capsules stored frozen or freeze-dried were shelf stable for at least 10

weeks retaining about 80% of the initial enzyme activity as opposed to retention of

25-34% activity in air-dried capsules.

Leakage of encapsulated Flavourzyme prepared from 1.6% (w/v) alginate was

slightly higher than those prepared from 1.8 and 2.0% (w/v) alginate in cheese milk.

Flavourzyme-alginate capsules prepared from 1.6, 1.8 and 2.0% (w/v) alginate

retained over 70% of the initial enzyme activity under simulated cheese-press

pressure. Concentration of alginate had no significant effect (p > 0.05) on the

retention of encapsulated Flavourzyme when the capsules were pressed for 4h;

however when the simulated cheese press duration increased to 8 and 16h the

retention of encapsulated Flavourzyme was significantly higher (p < 0.01) in

capsules produced from 2.0% (w/v) alginate.

Incorporation of encapsulated enzymes into the milk prior to rennetting resulted in an

even distribution of capsules in the cheese matrix compared to aggregation of

capsules, when added to milled curd prior to salting. All cheeses; control with no

added enzymes and experimental cheeses with free and encapsulated Flavourzyme

and/or Palatase showed higher levels of moisture and lower levels of fat compared to

standard Cheddar cheese due to the variation in the manufacturing protocol. There

was no significant difference (p > 0.05) in fat and final pH between control and

experimental cheeses and there was no difference in the numbers of coliforms,

E.coli, Salmonella, Listeria, coagulase positive staphylococci, Bacillus cereus, yeast

and moulds in control or experimental cheeses.

2
 Abstract

Increased and prolonged proteolysis was observed in cheeses with encapsulated

Flavourzyme showing increased release of several peptides, also with the formation

of new peptides absent in the control cheese with no added enzymes. Accumulation

of high molecular weight/hydrophobic peptides was higher in cheeses with free

Flavourzyme followed by cheeses with encapsulated Flavourzyme. Concentration of

water-soluble peptides increased with the increase in the concentration of

encapsulated Flavourzyme in the cheese. Concentration of water-insoluble peptides

was higher in control cheese compared to cheeses with encapsulated Flavourzyme

even after 180 days ripening. After 30 days of ripening, concentration of most free

amino acids was about 3 times greater in cheeses with encapsulated Flavourzyme

than in control and about 7 times higher after 90 days ripening. Concentration of total

amino acids was consistently higher in cheeses with encapsulated Flavourzyme

compared to control.

Cheese grading scores for body, texture and appearance of all cheeses with

encapsulated enzymes were lower than control and free enzyme treated cheeses

during the entire grading period of about 100 days due to crumbly and pasty texture.

Control and cheeses with added Flavourzyme received high overall score for flavour.

Flavour score of cheese with encapsulated Flavourzyme at a concentration of 0.75

LAPU/g milk protein was higher than all cheeses around 50 days with better overall

flavour score until about 94 days ripening with improved flavour and elimination of

bitterness. However the flavour of enzyme treated cheeses deteriorated with time and

the control cheese scored the highest for flavour.

Though increased concentration of free fatty acids was detected in cheeses treated

with encapsulated lipase; Palatase, these cheeses developed rancid, unpleasant,

strong lipolytic flavours as early as 55 days ripening.

3
 Introduction

1 Introduction

Cheese is a popular dairy product in most regions of the world with a growing

consumer demand. Consistent growth in cheese consumption has been the result of

good dairying and marketing ability of the cheese industry and also due to increased

awareness of the nutritional benefits of cheese. Cheese is made from coagulation of

milk followed by fermentation, dehydration and salting, though bacterial activity

continues throughout the ripening of cheese.

Several groups of cheese have evolved over time with diverse varieties dominating

different markets. Cheddar has been the most popular cheese variety in Australia as it

accounted for more than 50% of the total cheese manufactured in 2005/2006 and

total Cheddar (natural (76.6%) and processed (23.4%)) constituting 71.7% of the

total cheese supermarket sales in 2005/2006 (Anon, 2007b, 2007d).

Cheddar cheese is usually ripened around 8°C (Fox, 1988). Flavour development in

cheese is the result of biochemical processes occurring during ripening which are

proteolysis: breakdown of milk proteins to peptides and amino acids, lipolysis:

breakdown of milk fat to fatty acids and glycolysis: hydrolysis of milk sugars mainly

lactose and citrate to smaller compounds and catabolism of these products to smaller

compounds by cheese microflora. All these processes lead to development of

characteristic flavour and texture during ripening (Adda et al., 1982; Forde and

Fitzgerald, 2000; McSweeny, 2004). Flavour formation in low-moisture varieties like

Cheddar range from 2 months to over 2 years depending on the intensity of flavour

sought (McSweeny, 2004).

Long ripening duration in varieties like Cheddar implies additional expenses in

maintenance, refrigeration and risk of low yield due to contamination and syneresis

4
 Introduction

thus increasing the overall production costs (Cliffe and Law, 1990; Picon et al.,

1997). Demand for consistent low-cost high-intensity flavour in cheese has lead to an

intensive research on potential means for acceleration of ripening. Several attempts

at minimising the ripening duration have been reported and those techniques include

elevation of ripening temperature, high pressure processing, use of attenuated

starters, addition of adjunct cultures, use of genetically modified starters and addition

of exogenous enzymes.

Elevation of ripening temperature was thought to be a straightforward approach

towards accelerating cheese ripening but it was limited as ripening at high

temperatures led to increased risk of contamination and syneresis leading to textural

defects and low yield (Wilkinson, 1993, Fox et al., 1996; Hannon et al., 2005). High

pressure treatment of cheeses has been reported for acceleration of ripening however,

there are no known reports of successful commercial application. Use of genetically

modified starters to produce excess proteolytic enzymes was also limited as it was

technically complex and has problems with acceptance by the consumer. Addition of

adjunct cultures known to produce proteolytic enzymes similar to the starter and non

starter lactic acid bacteria is limited by the availability of strains also with the risk of

development of off-flavours. Addition of cultures attenuated by various ways such as

heat, lysozyme, and freeze-thaw treatments resulting in earlier lysis and release of

proteolytic enzymes during ripening leading to an accelerated flavour development

was reported however, this approach was not very successful due to low specificity.

Addition of exogenous enzymes to catalyse the biochemical reactions occurring

during cheese ripening was studied by many researchers with the aim of accelerating

ripening (Fox and Stepaniak, 1993). Addition of free enzymes (mainly proteolytic

enzymes) led to premature and extensive proteolysis with the development of off-

5
 Introduction

flavours and bitterness in addition to contamination of whey (Law and Kirby, 1987;

Wilkinson and Kilcawley, 2005).

Microencapsulation is a well known technique used for protection, isolation and

controlled release. However, its application to deliver enzymes into cheese system

for acceleration of ripening is a recent technique and is innovative. Encapsulation of

enzymes was thought to prevent immediate proteolysis and contamination of whey

also aiding in an even distribution of the enzymes in cheese matrix resulting in

prolonged and relatively controlled release of the enzymes during ripening.

Several materials such as milk fat, food gums and liposomes were tested for

delivering enzymes into the cheese system to accelerate ripening with reports of poor

encapsulation efficiency, poor stability of capsules in cheese and very little

understanding of the release mechanism of the encapsulated enzymes during ripening

(Perols et al., 1997; Picon et al., 1997; Kailasapathy et al., 1998; Laloy et al., 1998;

Kheadr et al., 2000, 2003).

Potential of alternative materials and techniques for enzyme microencapsulation for

application in cheese has not been well reported. Though a large number of

techniques and ingredients are used for microencapsulation, suitability of a technique

largely depends on the substance subjected to microencapsulation and its application.

In the current study microencapsulated enzymes were delivered into cheese for

acceleration of ripening, as per this requirement it was necessary to deliver the

enzymes in inert, non-fat capsules that do not alter the composition of cheese, in line

with the focus of this study to microencapsulate flavour-enhancing enzymes for

controlled release in Cheddar cheese during ripening.

Entrapment of biological material in hydrogels or polymers for the purpose of

protection, isolation or controlled release is very popular. Use of the encapsulator for

6
 Introduction

enzyme encapsulation eliminated the need for solvents and emulsifying agents

otherwise used in common encapsulation procedures. Microencapsulation of

enzymes in food-grade polymers using the encapsulator was found to be ideal for this

study. Suitability of food gums for microencapsulation of flavour-enhancing

enzymes was investigated in this study for application into cheese for acceleration of

ripening.

7
 Introduction

1.1 Aim

The aim of this study was to evaluate an accelerated Cheddar cheese ripening process

by microencapsulating flavour-enhancing enzymes in food gums and delivering into

cheese for controlled release during ripening.

1.2 Objectives

i. Screening of food gums suitable for enzyme encapsulation using the Inotech®

encapsulator followed by optimisation of enzyme encapsulation, shelf life

study of encapsulated enzymes and morphological characterisation of enzyme

capsules

ii. Studies on the stability of the enzyme capsules under simulated conditions of

cheese manufacture

iii. Evaluation of enzyme delivery into cheese for maximum retention and an

even distribution

iv. Monitoring of cheese ripening by analysis of water-soluble and water-

insoluble peptides, free amino acids and free fatty acids

v. Organoleptic evaluation of accelerated ripened Cheddar cheese

8
2 REVIEW OF LITERATURE

9
 Review of Literature

2.1 History and background of cheese

Cheese is one of the oldest, diverse and most widely consumed groups of dairy

products with high nutritional value. Cheese is made from coagulation of milk

followed by separation of whey and pressing the curd. The pressed curd can be

consumed fresh or after a period of ripening depending on the variety of cheese and

intensity of the flavour sought.

Cheese is said to have evolved about 8000 years ago in the region around southern

Turkey and spread to rest of the world with the civilization (Fox and McSweeney,

2004). Cheese was the only means of milk preservation for centuries and some long-

ripened varieties can be considered as long-shelf life products (Adda et al., 1982).

Originally cheese was manufactured using milk from all forms of domesticated

animals however, in recent times cheese is mostly made using bovine milk. Curdling

of milk stored in the bags made of young animal’s stomach lead to the discovery of

milk coagulating properties of digestive juices from the stomach tissue. It is now

known that rennet is the milk coagulating extract obtained from the fourth stomach

of young animals. Shortage of rennet lead to the development of rennet substitutes

for cheese manufacture; yet rennet is the principal milk coagulating agent used to

date.

Cheese is a popular dairy product globally and several varieties have evolved over

time depending on the source of milk, origin, variation in the manufacturing process

and maturation. Cheddar is the most popular cheese variety in Australia as it

accounted for over 51% of the total cheese manufactured while 71% of the cheese

supermarket sales accounted for total Cheddar (natural and processed) in 2005/2006

(Anon, 2007b, 2007d). Natural Cheddar alone accounted for about 54% of the total

cheese supermarket sales demonstrating the popularity of this variety in Australia.

10
 Review of Literature

2.2 Cheese production

Cheese is inherently, biologically and biochemically unstable in contrast to many

other dairy products. Cheeses can be produced by acid-coagulation or rennet-

coagulation. Most acid-coagulated cheeses (eg. Cottage) are consumed fresh, soon

after manufacture; while most rennet-coagulated cheeses (eg. Cheddar, Parmesan)

are consumed after ripening, the production of these involve two stages; manufacture

and ripening (Fox and McSweeney, 2004).

2.2.1 Cheese manufacture

Cheese manufacture is mainly a dehydration process involving concentration of milk

fat and casein between 6 and 12 folds depending on the variety and may last for 5-

24h. The basic steps involved in cheese manufacture are acidification, coagulation,

dehydration, salting and pressing or moulding. Stage of milk supply such as early or

late lactation milk also affects the quality of cheese, increased proteolysis was

observed in cheeses made from late lactation milk. Increased level of free fatty acids

(FFA) was also noted in late lactation milk and hence higher in cheeses

manufactured from it (Hickey et al., 2006). In modern production, milk of high

microbiological quality, free from antibiotics is standardised for required amount of

casein and fat ratio followed by pasteurisation. Microorganisms in milk that survive

pasteurisation contribute to the population of non-starter lactic acid bacteria

(NSLAB) in cheese. In current dairy processing, pasteurised milk is stored in cool

rooms until manufacture. Pasteurised cheese milk is heated to about 30°C depending

on the variety being manufactured (Fox and McSweeny, 2004).

Acidification during cheese manufacture is a crucial step for certain varieties and is

achieved by encouraging production of lactic acid from the starter culture. For

11
 Review of Literature

varieties such as Cheddar that are cooked not more than 40°C Lactococcus lactis

subsp cremoris and/or Lc. lactis subsp lactis are the most commonly used starter

cultures whereas Streptococcus thermophilus is used as a starter in high temperature

cooked cheeses and other Lactobacillus spp are also used as starter lactic acid

bacteria (SLAB) in most varieties of cheese. Time required to attain the end pH

during cheese manufacture can vary depending on the variety. Calcium chloride is

added early during cheese manufacture, it influences acid development, coagulation

properties and gel syneresis (Fox and McSweeney, 2004).

Coagulation is one of the most important stages of cheese manufacture. Most fresh

cheeses are acid-coagulated while long-ripened cheeses are rennet-coagulated.

Rennet is extracted from the stomach of young animals; the principal enzyme in

rennet is chymosin, accounting for about 95% of the milk-clotting activity along with

small amount of pepsin which hydrolyse milk proteins (mainly caseins). Casein

micelles constituting about 80% of bovine milk proteins is composed of several

thousand individual molecules of all four major types of caseins (αs1, αs2, β and κ)

with κ-casein located on the outer surface of the micelle exerting a stabilising effect

on the micelles, preventing them from coagulating. The κ-casein constitutes

hydrophobic and hydrophilic moieties, and is linked to the micelle by the

hydrophobic moiety while the hydrophilic moiety interacts with the solvent

stabilising the casein micelle (Dalgleish, 1993). Milk-clotting enzymes such as

chymosin (and pepsin) hydrolyse κ-casein at the junction of hydrophobic para-κ-

casein and hydrophilic macropeptide moieties, the macropeptide diffuses into the

solution following hydrolysis and the stabilising influence of κ-casein is lost leading

to coagulation of casein micelles. This marks the initial coagulation of milk after

rennetting while β-casein is untouched by rennet. This leads to a curd formation.

12
 Review of Literature

The curd is cut into distinct size depending on the variety. Cutting, stirring, size of

the curd and temperature at which it is cut greatly influences the rate of expulsion of

whey; determining the loss of milk components in whey and also affecting moisture

content and water activity of cheese (Lawrence et al., 2004). The curd is cooked to

specific temperature and subjected to processes such as cheddaring, kneading or

stretching depending on the variety and the whey is expelled. The curd is either dry

salted or brined. Salt concentration in cheese can vary between 0.7 and 6% and can

greatly influence moisture, water activity, shelf life, growth and activity of SLAB

and NSLAB thus indirectly affecting the protein and fat stability, enzyme activity

during ripening and hence the quality of cheese (Laan et al., 1998). The salted,

dehydrated pressed curd is (treated with secondary starters for some varieties)

moulded, packed and allowed to ripen.

2.2.2 Cheese ripening

Some varieties of cheese are consumed fresh while certain varieties are consumed

after ripening, which can range from two weeks (e.g. Mozzarella) to two or more

years (e.g. Parmigiano-Reggiano or vintage Cheddar) (McSweeny, 2004). The

diversity in the properties of different varieties of cheese is mainly due to the

variation in the source of milk, starter cultures, manufacturing processes, ripening

time and temperature and secondary cultures. The flavour of freshly produced cheese

curd is bland, characteristic cheese flavour develops during ripening due to the

production of a wide range of sapid compounds by biochemical pathways (Adda et

al., 1982; McSweeny, 2004). A range of biochemical and microbiological events

contribute to the flavour and texture development of the cheese during ripening.

13
 Review of Literature

2.2.2.1 Microbiology of cheese ripening

Starter lactic acid bacteria are primarily used for acid production and their growth

ceases soon after manufacture due to low pH, but continue to contribute towards

ripening as they produce proteolytic and lipolytic enzymes responsible for

conversion of peptides, amino acids and fatty acids to flavour compounds (Fox and

Wallace, 1997) however, most starter enzymes are intracellular and are released only

upon cell lysis during ripening.

Non starter lactic acid bacteria (NSLAB) also form a significant portion of the

microbial load in most ripened cheese varieties (Beresford and Williams, 2004). The

population of NSLAB is greater and diverse in cheeses made from raw milk and their

source can range from milk to manufacturing equipment. The number of NSLAB is

highest around 3 months of ripening and is stable for rest of the maturation period.

Different strains may dominate during different stages of ripening while some persist

during the entire ripening period. Lactobacilli are the predominant species of

NSLAB in Cheddar cheese during ripening. Non starter lactic acid bacteria like

SLAB contribute to ripening through release of intracellular and extracellular

enzymes responsible for conversion of peptides, amino acids and fatty acids to

flavour compounds (McSweeney et al., 1993).

Secondary starters like Penicillium roqueforti in blue cheeses such as Roquefort,

Gorgonzola, Stilton and Danish blue; Penicillium camemberti in surface-mould

ripened cheeses such as Brie and Camembert; Propionic acid bacteria in Swiss type

cheeses are also used for development of characteristic flavour and texture. However

there is no deliberate addition of secondary starters in Cheddar cheese and hence will

not be discussed further.

14
 Review of Literature

2.2.2.2 The biochemistry of cheese ripening

In mature cheeses such as Cheddar, development of flavour and texture is largely

controlled by complex biochemical reactions occurring during ripening (Forde and

Fitzgerald, 2000). These biochemical reactions can be grouped into three major

categories;

i. Proteolysis: breakdown of casein to peptides and amino acids and their

catabolism

ii. Lipolysis: breakdown of milk fat to free fatty acids (short, medium and long

chain length) and their catabolism and

iii. Glycolysis: metabolism of lactose, lactate and citrate by SLAB and NSLAB

with the development of flavour compounds (McSweeny, 2004) (Figure 2.1).

Enzymes that catalyse these biochemical reactions during ripening originate from

milk, coagulant, SLAB, NSLAB, secondary starters (in certain varieties) and

exogenous enzymes (McSweeney, 2004; Wilkinson and Kilcawley, 2005).

15
 Review of Literature

Figure 2.1 Overview of biochemistry of cheese ripening (Law, 2001).

16
 Review of Literature

2.2.2.2.1 Proteolysis

Proteolysis is the most complex and most important biochemical event occurring

during ripening in cheese varieties such as Cheddar (Forde and Fitzgerald, 2000;

Upadhyay et al., 2004a). This topic has been extensively reviewed (Fox, 1989; Fox

and Law, 1991; Fox and McSweeny, 1996; Sousa et al., 2001; Upadhyay et al.,

2004a). Proteolysis is responsible for the change in pH, water activity, and protein

structure of cheese thus affecting the texture and flavour (Upadhyay et al., 2004a).

During ripening, proteins in cheese curd are hydrolysed to smaller peptides and

amino acids. The mixture of peptides and amino acids not only contribute directly to

the flavour and mouth feel of the cheese, but also act as substrates for further

metabolism by SLAB and NSLAB generating a range of flavour compounds (Law,

2001; Upadhyay et al., 2004a).

Proteolysis in cheese during ripening is catalysed by proteinases and peptidases from

milk, coagulant, SLAB, NSLAB, secondary starters and in some cases exogenous

enzymes (Fox and McSweeney, 1996; Law, 2001; Sousa et al., 2001 and

McSweeney, 2004) (Figure 2.2).

17
 Review of Literature

Figure 2.2 Proteolytic agents in cheese during ripening (Sousa et al., 2001).

18
 Review of Literature

Rennet is the principal coagulant used in cheese manufacture and consists of

chymosin, pepsin and other proteinases. Chymosin is the principal proteinase in most

commercial rennets as it accounts for about 95% of the milk-clotting activity of

rennet. It hydrolyses at the Phe105-Met106 bond of -casein leading to destabilisation

of casein micelle and coagulation of the milk. Though most of the rennet is lost

during whey drainage, a small amount is trapped in the cheese curd. The residual

chymosin from the trapped rennet in cheese curd cleaves s1-casein at various sites

(McSweeney et al., 1993) influenced by pH and ionic strength (Mulvihill and Fox,

1977). Pepsins are also present in considerable amount in rennet and their activity is

similar to chymosin.

The main proteolytic agents from SLAB are proteinases and a range of intracellular

peptidases. Lactocepin, a cell envelop associated proteinase, degrades casein to short

peptides. The degradation of these peptides to amino acids is catalysed by

intracellular peptidases (Christensen et al., 1999 as cited by Upadhyay et al., 2004a).

While low pH of curd inhibits further growth of SLAB in cheese, its intracellular

enzymes are released into cheese upon lysis. A range of endopeptidases, di- and

tripeptidases and aminopeptidases have been studied from LAB; both SLAB and

NSLAB and carboxypeptidases in a few strains of lactobacilli have also been studied

(Figure 2.3) (El Soda et al., 1987; Upadhyay et al., 2004a).

There is no contribution of secondary starters to the ripening of traditional Cheddar

cheese as there is no deliberate addition of secondary cultures during manufacture.

19
 Review of Literature

Figure 2.3 Schematic representation of the action of peptidases found in lactic

acid bacteria (Upadhyay et al., 2004a).

20
 Review of Literature

2.2.2.2.1.1 Catabolism of amino acids during cheese ripening

Catabolism of amino acids is a major flavour-forming process during ripening (Yvon

and Rijnen, 2001; Ardo, 2006), which can be initiated by decarboxylases, lyases,

transaminases, deaminases and dehydratases (Ardo, 2006). The major pathway for

degradation of amino acids by LAB is an -keto acid pathway initiated by

transamination catalysed by aminotransferases (Yvon and Rijnen, 2001; Williams et

al., 2001). Aminotransfereases catalyse amino acid transamination to -keto acid

which is further degraded to a range of compounds (Yvon and Rijnen, 2001; Curtin

and McSweeney, 2004) (Figure 2.4, Figure 2.5, Table 2.1). Alternative amino acid

catabolism pathways include cleavage of amino acid side chains by amino acid

lyases; formation of amines by decarboxylases and ammonia by deaminases and

hydrolysis of –OH and –SH groups in amino acids by dehydratases (Figure 2.6)

(Smit et al., 2000, Smit, 2005; Ardo, 2006). Aminotransferase activity was detected

in all LAB (SLAB and NSLAB) isolates from Cheddar cheese (Williams et al., 2001)

indicating the role of LAB and their enzymes in flavour formation during ripening;

by catabolism of amino acids.

21
 Review of Literature

Figure 2.4 Schematic diagram of amino acid catabolism pathways found in

different microorganisms and some chemical reactions (dotted line) occurring in
cheese during ripening. HA-DH: hydroxyacid dehydrogenase, -KADH: -
ketoacid dehydrogenase, -KADC: -ketoacid decarboxylase, aldDH: aldehyde
dehydrogenase, alcoDH: alcohol dehydrogenase, MGL: methionine -lyase,
CGL: cystathionine -lyase, CBL: cystathionine -lyase, TPL: tyrosine-phenol
lyase, TIL: tryptophan-indole lyase (Yvon and Rijnen, 2001; modified by Curtin
and McSweeney, 2004).

22
 Review of Literature

Figure 2.5 Catabolism of leucine initiated by transaminase, deaminase or decarboxylase action and volatile flavour compounds that may
be formed from this amino acid. Similar catabolic pathways operate for the other branched-chain aliphatic amino acids (isoleucine and
valine) (McSweeney, 2004).

23
 Review of Literature

Figure 2.6 Methionine degradation pathways by starter cultures. PLP: pyridoxal phosphate; TPP thiamine pyrophosphate (Smit et al.,
2000).

24
 Review of Literature

Table 2.1 Flavour compounds derived from amino acids via -keto acids by the transaminase pathway. Compounds in italics are formed
by non-enzymatic reactions (Adapted from Smit, 2005).

Amino Keto acid Aldehyde Organic acid Alcohol (thiol) Esters (examples)

acid

Ile -keto-3-methyl- 2-methylbutanal 2-methylbutyric acid 2-methyl butanol

pentanoic acid

Leu -ketoisocaproic acid 3-methylbutanal 3-methylbutyriccacid 2-methyl propanol Ethyl-3-methylbutanoate

2-methylpropanal 2-methyl propanoic acid 2-methyl propanol

Val -ketoisovaleric acid 2-methylpropanal 2-methyl propanoic acid 2-methyl propanol Ethyl isobutanoate

Phe Phenyl pyruvate Benzaldehyde Benzoic acid Phenyl methanol Ethyl benzoate

Phenyl acetaldehyde Phenylacetic acid Phenyl ethanol Phenylethyl acetate

Trp Indole-3-pyruvate Indole-3-acetaldehyde Indole-3-acetic acid

Met -keto methylthio Methional Methylthiobutyricacid Methionol Ethyl-3-methylthiopropionate

butyrate

Methylthio-acetaldehyde Methanethiol Methylthioacetate

25
 Review of Literature

2.2.2.2.2 Lipolysis

Lipolysis during cheese ripening mainly refers to the enzymatic hydrolysis of

triglycerides to fatty acids and glycerol, mono-, di- or triglycerides and is essential

for flavour formation in varieties such as Blue but in cheeses such as Cheddar due to

low level of lipolysis, its influence on flavour has received little attention

(McSweeney and Sousa, 2000). Extensive lipolysis is characteristic of mould-ripened

(Gripon, 1993; Spinnler and Gripon, 2004) and Italian cheeses (Bosset and Gauch,

1993), and only moderate lipolysis occurs in Cheddar cheese while extensive

lipolysis is considered undesirable in Cheddar (McSweeney and Fox, 1993;

McSweeney and Sousa, 2000). Lipolysis in cheese influences flavour through

formation of free fatty acids (FFA), which act as precursors for formation of several

flavour compounds such as lactones in Cheddar cheese. Lipids also act as solvents

for many sapid flavour compounds in cheese and also influence the texture of cheese

(Figure 2.7) (Adda et al., 1982; McSweeney, 2004).

Lipolytic enzymes; lipases and esterases can originate from milk, rennet, SLAB,

NSLAB, secondary starters and exogenous preparations (Fox and Wallace, 1997).

Milk contains indigenous lipoprotein lipase (LPL) which under optimum conditions

can hydrolyse milk fat and release FFA causing rancidity, however, this does not

occur as triglycerides in milk are surrounded by milk fat globule membrane (MFGM)

and LPL is associated with casein micelles (Collins et al., 2004). High-temperature

short-time (HTST) pasteurisation (72°C for 15s) extensively inactivates this enzyme

however, it may still contribute to lipolysis during ripening as heating at 78°C for

10s is required for its complete in activation (Driessen, 1989 as cited by Collins et

al., 2004).

26
 Review of Literature

Rennet pastes contain lipolytic enzymes however, commercial rennet preparations

used in Cheddar cheese manufacture are free from lipolytic enzymes. Hence the

principal lipolytic agents in Cheddar cheese originate from LAB (Fox et al., 2000).

Lipolytic agents from LAB can hydrolyse a range of FFA, mono-, di- and tri-

glyceride substrates (Fox and Wallace, 1997). Though LAB are weakly lipolytic their

existence in large numbers over a long period of time can cause substantial amount

of lipolysis with liberation of FFA in cheeses without secondary lipolytic starter

cultures (Collins et al., 2004). Numerous studies indicate that the lipolytic enzymes

of LAB are intracellular; hence lysis of LAB in cheese during ripening causes

considerable lipolysis. Cheddar cheese made with highly autolytic starter Lc. lactis

subsp cremoris AM2 developed significantly higher levels of caprylate (C8:0),

myristate (C14:0), palmitate (C16:0) and stearate (C18:0) during ripening compared

to cheese made with less autolytic strain Lc. lactis subsp cremoris HP demonstrating

that these lipolytic enzymes of LAB are intracellular as cell free extracts of both

strains had similar activities (Collins et al., 2003). Catabolism of FFA is important

for formation of flavour compounds; esters impart buttery or fruity aroma, however,

thioesters formed by the reaction of short-chain fatty acids with methional impart

cheesy aroma to Cheddar cheese (Arora et al., 1995). Important lipolytic flavour

compounds such as methyl ketones (Dimos, 1992; Engels et al., 1997), secondary

alcohols (Engels et al., 1997) and lactones (Wong et al., 1975) have also been

reported in Cheddar cheese.

27
 Review of Literature

Figure 2.7 Pathways for the production of flavour compounds from fatty acids during cheese ripening (McSweeney, 2004).

28
 Review of Literature

2.2.2.2.3 Glycolysis

During cheese ripening, metabolism of lactose, lactate and citrate are collectively

referred to as glycolysis. Lactose is the primary milk sugar, during manufacture of

cheeses such as Cheddar; lactose is converted to L-lactic acid by SLAB. The rate and

extent of acidification influences the pH, water activity and enzymatic reactions

during ripening. Although most of the lactose is lost during whey drainage, small

amount still remain in curd (Huffman and Kristoffersen, 1984). This lactose is

immediately metabolised to L-lactate by SLAB at low concentration of S/M (salt-in-

moisture) and low populations of NSLAB. However, in cheeses with high NSLAB

populations such as raw milk cheeses at high S/M, lactose is converted to DL-lactate

by NSLAB. High numbers of NSLAB are found to be responsible for racemization

of L-lactate in Cheddar and similar cheeses (Crow et al., 2001). Racemization is

thought to favour development of Ca-lactate crystals as Ca-DL-lactate is less soluble

than Ca-L-lactate (Thomas and Crow, 1983; Dybing et al., 1988). Though

racemization does not affect flavour significantly it may result in increased formation

of Ca-lactate crystals in cheese resulting in undesirable appearance. Lactate can be

oxidised to acetate, formate and ethanol (Figure 2.8) (Fox et al., 2000; McSweeney

and Fox, 2004). However, formation of propionate and CO2 is of importance in

Swiss-type cheese for the characteristic eye formation (Piveteau, 1999).

Milk contains 1750mg of citrate per litre, though most of it is lost in whey (Fox et

al., 1993), Cheddar cheese curd contains 0.2%-0.5% citrate and is metabolised by a

few citrate positive lactococci to diacetyl, acetate, acetoin and CO2 contributing to

the cheese flavour (Figure 2.9) (McSweeney and Fox, 2004).

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 Review of Literature

Figure 2.8 Pathways by which lactate is metabolised in cheese during ripening.

(1) racemization, (2) metabolism by Propionibacterium freudereichii in Swiss
cheese, (3) oxidative metabolism of lactate, (4) conversion to formate, (5)
anaerobic metabolism of lactate to butyrate and H2, which leads to late gas
blowing (McSweeney and Fox, 2004).

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 Review of Literature

Figure 2.9 Pathways for citrate-positive strains of Lactococcus and Leuconostoc

sp (McSweeney and Fox, 2004).

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 Review of Literature

2.2.2.3 Texture

Flavour and texture are the main characteristics responsible for the identification of

cheese by consumers. Cheese texture is defined as “the attribute of a cheese resulting

from a combination of physical properties including size, shape, number, nature and

conformation of the constituent structural elements that are perceived by a

combination of the senses of touch (tactile texture), vision (visual texture) and

hearing (auditory texture)” (Delahunty and Drake, 2004). Requirements of Cheddar

cheese texture vary with the utility, commercial cheeses sold sliced need to be firm

where as those used as ingredients can be used even when extremely crumbly

(Lawrence et al., 2004). Cheese texture is influenced by various factors such as pH,

moisture, salt, calcium, fat and protein (Lawrence et al., 2004).

The lactic acid produced by SLAB early in cheese production drops the pH of the

curd and at low pH the proteins assume increasingly compact structure resulting in

firmer texture of the young cheese. Cheddar cheese in most countries is

manufactured with a final pH around 5.1 to 5.3 as compared to 4.9 of the traditional

English Cheddar, giving these cheeses a softer texture. The effect of pH on texture

can be modified by factors such as moisture, salt and calcium. In the pH range of 5.1

and 5.5, colloidal calcium phosphate and casein are dissociated from sub-micelles

and these changes in sub-micelle size and characteristics increases its water binding

ability and solubilisation of the micelle by NaCl decreases as the calcium

concentration increases (Creamer, 1985). Excessive salting in Cheddar cheese can

result in firm-textured drier cheese (Van Slyke and Price, 1952; as cited by Lawrence

et al., 2004). Salt concentration is also known to influence enzyme activity and

conformation of s1- and -casein (Fox and Walley, 1971). In addition enzyme

32
 Review of Literature

activity, hydration of protein network and interaction of calcium with casein are also

influenced by salt concentration (Delahunty and Drake, 2004).

Protein matrix is responsible for the rigid form of the cheese, thus the increased

amount of protein in reduced fat cheese leads to firmer and more elastic texture

compared to full fat Cheddar cheese (Lawrence et al., 2004). Breakdown of s1-

casein during ripening considerably softens the texture of the curd. Breakdown of

peptide bonds as a result of proteolysis results in formation of water molecules with

ionic groups. As ripening progresses and with increase in proteolysis, water available

for protein solvation is increasingly bound by new ionic groups leading to firm and

easily deformed cheese. Change in texture of the cheese depends on extent of

proteolysis which in turn depends on duration of ripening (Lawrence et al., 2004).

Increase in fat (Fenelon and Guinee, 2000) and moisture content in cheese results in

slightly softer cheese.

2.2.2.4 Flavour

Flavour constitutes the olfactory and taste perception of cheese with aroma being the

initial flavour perception. Texture and flavour are considered to be the most

important attributes of Cheddar cheese (Jack et al., 1993) and vary considerably

between cheese varieties. Raw milk cheeses are intensely flavoured and have

atypical flavours (Lawrence et al., 2004). Cheese flavour and texture also vary to a

lesser extent within the same batch and even between two regions for the same

cheese. Cheddar cheeses are low in flavour at 2 months ripening and have intense

flavour at 2 years of ripening. Extent of proteolysis with the release of peptides,

amino acids and these acting as substrates for production of a range of compounds

contribute to the flavour (McSweeney and Sousa, 2000). Key aroma compounds in

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 Review of Literature

Cheddar cheese are known to originate from amino acids (Urbach, 1995). Bitterness

is a very common flavour defect in Cheddar cheese caused by accumulation of low-

molecular weight hydrophobic peptides and has been reviewed (Fox, 1989; Habibi-

Najafi and Lee, 1996); now several exopeptidases are used to counter bitterness in

cheese (Raksakulthai and Haard, 2003).

Low-fat Cheddar cheese lack typical Cheddar flavour (Foda et al., 1974; Olson and

Johnson, 1990) indicating the contribution of fat to the Cheddar cheese flavour.

Substituting milk fat with vegetable or mineral oil also resulted in atypical flavours

with a degree of Cheddar flavour (Foda et al., 1974), suggesting that water-fat

interface in cheese is important for flavour development and the flavour components

are dissolved and retained in fat (Lawrence et al., 2004). Fatty acids, methyl ketones,

keto acids, esters and lactones are the lipolytic flavour compounds in Cheddar cheese

derived from milk fat; some may contribute directly to the flavour while some may

contribute only to the background flavour (McSweeney and Sousa, 2000).

Starter lactic acid bacteria, NSLAB and secondary starters also contribute to the

flavour of cheese by metabolising peptides, amino acids and fatty acids to smaller

flavour compounds. Enzymes from cheese microflora are largely responsible for

biochemical reactions leading to formation of flavour compounds.

2.3 Acceleration of cheese ripening

Ripening of certain cheese varieties involve complex and often slow biochemical

processes (Fox et al., 1993; Fox and McSweeney, 1996; Fox and Wallace, 1997) and

represents a significant proportion of the total cost of cheese production (Cliffe and

Law, 1990; Picon et al., 1997). Long ripening implies expenses in refrigeration and

maintenance with the risk of contamination and low yield, thus increasing the overall

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 Review of Literature

production cost. Any attempt at accelerating cheese ripening is appreciated and is of

economic interest (Cliffe and Law, 1990) in addition to the technological advantage

it offers the cheese makers. Various methods employed to reduce the ripening time

of cheese have been extensively reviewed (Wilkinson, 1993; Law, 2001; Azarnia et

al., 2006). With an understanding of the biochemical processes occurring during

ripening, many researchers have attempted to accelerate cheese ripening by various

ways as summarised below.

2.3.1 Elevation of ripening temperature

Most varieties of cheese are ripened at low temperatures, for instance around 6-8oC

for Cheddar (Fox, 1988). At this low temperature the biochemical processes occur at

a very slow rate thus prolonging flavour generation. Increasing the ripening

temperature can increase the rate of biochemical reactions occurring during ripening.

Ripening of cheese at 13-15°C instead of 5-8°C accelerated the ripening time without

any development of off-flavours (El soda, 1993). Folkertsma et al. (1996) reported

accelerated proteolysis and lipolysis with improved flavour scores for Cheddar

cheese ripened at 16°C but suggested ripening at 12°C, as the cheeses ripened at

16°C deteriorated after 6 months. Law (2001) proposed that cheese manufactured

from good quality milk can be ripened at 15°C; while ripening of Cheddar cheese at

12°C can reduce the ripening time by 60-75% without any defects in body. A well

balanced and accelerated flavour development in Cheddar cheese was achieved by

ripening at 20°C or 12°C for 1 or 6 weeks respectively (Hannon et al., 2005). Though

elevation of ripening temperature appear to be a simple technique for acceleration of

cheese ripening, this method has several drawbacks such as risk of microbial

contamination leading to development of off-flavours and unhygienic cheese in

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 Review of Literature

addition to risk of syneresis and fat exudation (Wilkinson, 1993, Fox et al., 1996).

Use of this technique is recommended only for cheeses manufactured with high

microbiological standards (El soda and Pandian, 1991; El soda, 1993; Wilkinson,

1993).

2.3.2 High pressure processing

Subjecting cheeses to high pressure of 50Mpa have shown to accelerate ripening as

reviewed by Law (2001). It appears that increase in pressure may stimulate cell lysis;

releasing intracellular enzymes leading to acceleration of ripening. La Serena cheeses

treated with high pressure (300-400Mpa 10 min at 10°C) on day 2 showed higher

levels of hydrophilic peptides and lower levels of hydrophobic peptides, increased

levels of free amino acids than control and also showed delayed breakdown of

caseins. Cheeses treated at 400Mpa on day 2 had the lowest flavour score while the

other high pressure (300Mpa) treated cheeses did not differ from controls in flavour

scores (Garde et al., 2007). In a similar study confocal laser scanning microscopy

showed differences in the structure of high pressure treated and control cheeses,

temperature of pressurisation was suggested as a significant determinant of the

overall effect of high pressure treatment (O’Reilly et al., 2003). Increased hydrolysis

of -casein was reported for cheese treated to high pressure on day 15 of ripening,

increased levels of free fatty acids were noted compared to control (Juan et al., 2008)

suggesting the importance of time of high pressure treatment in acceleration of

cheese ripening. However, there are very few reports of application of high pressure

to accelerate cheese ripening and its utility is uncertain at this stage.

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 Review of Literature

2.3.3 Attenuated starters

Starters are known to strongly influence flavour and texture development in cheese

(Shakeel-Ur-Rehman et al., 1999) and thus are an easy and obvious choice for

manipulation for controlled acceleration of ripening (Law, 2001). The main function

of starters during cheese manufacture is production of acid at a standard rate, while

the enzymes from starters also contribute to ripening. The growth of starters cease

immediately after salting and pressing, and the cells lyse and release intracellular and

extracellular enzymes contributing to ripening. Increased number of starters can

potentially accelerate ripening but also lead to increased acid production and low pH

which can be undesirable. Addition of starter bacteria unable to produce significant

levels of lactic acid but able to deliver enzymes during cheese ripening can

supplement ripening thus resulting in acceleration of ripening. These starters are

attenuated by treatments like heating, freezing, spray-drying, freeze-drying or

weakened by lysozyme or solvents. Use of attenuated starters to accelerate cheese

ripening has been well reviewed by Klein and Lortal (1999) and also by Madkor et

al. (2000), Law (2001) and Azarnia et al. (2006).

Heating is the most widely used form of cell attenuation (Klein and Lortal, 1999).

Though heat attenuated starters were widely used for acceleration of cheese ripening

(Kim et al., 1994; Skeie et al., 1995, 1997; Asensio et al., 1996), only a few studies

reported reduction in ripening time (Abdel Baky et al., 1986; Aly, 1990; Kebary et

al., 1997). A 90% reduction in acid production also with 60% reduction in cell wall

proteinase activity and 10% reduction in amino peptidase activity was noted for

Lactobacillus helveticus attenuated at 64°C, increasing the temperature to 66°C

caused considerable reduction in proteinase and peptidase activities (Castaneda et al.,

1990) and development of off-flavours was also reported (Castaneda et al., 1990;

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 Review of Literature

Johnson et al., 1995). Alternative starter attenuation processes used for acceleration

of cheese ripening include spray/freeze-drying (Johnson and Etzel, 1995), freezing

(Johnson and Etzel, 1995; Verdini et al., 2005), freeze-shock (freeze and rapid

thawing) (Spangler et al., 1989; Aly, 1990, El soda et al., 1991, 1992; Johnson et al.,

1995; Kebary et al., 1997; Madkor et al., 2000), exposure to lysozyme (Law et al.,

1976) and exposure to bacteriocin (Morgan et al., 1995, 1997). Though increased

proteolysis, improved flavour score and reduction in bitterness was reported,

reduction in actual ripening time was reported only in a few studies also with

development of uncharacteristic and off-flavours (Johnson and Etzel, 1995; Johnson

et al., 1995). Cheddar cheese manufactured with high pressure attenuated starters Lc.

lactis ssp cremoris HP and Lc. lactis ssp cremoris 303 had higher levels of amino

acids compared to controls (Upadhyay et al., 2007) accelerating secondary

proteolysis. Success of this approach towards acceleration of cheese ripening has

prompted development of Flavour control FCTM (Chr. Hansen) and EnzobatTM

(Medipharm), commercial attenuated starters for enhancement of flavour.

2.3.4 Starter selection

The primary function of starters is acid production during cheese manufacture, strain

selection offers opportunity to supplement the enzyme system thus potentially

accelerating ripening. Muir et al. (1996), Shakeel-Ur-Rehman et al. (1999) and Pripp

et al. (1999) suggested use of single strain of Lactococcus as the starter for improved

cheese quality. Starter lysis ensures the involvement of intracellular enzymes in the

gradual process of cheese flavour formation (Meijer et al., 2004); endogenous cell

wall associated enzymes, phage encoded enzymes, autolysins, and endolysins can

induce this phenomenon (Lortal and Chapot-Chartier, 2005). Premature lysis of

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 Review of Literature

starters can cause early release of intracellular enzymes; hence these starters have the

potential to accelerate ripening. Phage-induced lysis of Lactococcus in cheese leads

to accelerated release of amino acids and reduced bitterness (Crow et al., 1995a,

1995b). Use of fast lysing and heat sensitive stains of Lactococcus (Feritag and

McKay, 1987), Lc. lactis subsp cremoris AM2 (Chapot-Chartier et al., 1994),

thermophilic Lactobacillus spp. and Streptococcus thermophilus (Lortal et al., 1997),

Lc. lactis subsp cremoris (Collins et al., 2003) have been shown to increase flavour

development in cheese during ripening.

2.3.5 Genetically modified starters

Genetically modified starters over expressing genes for proteolytic or lipolytic

systems were developed to augment starter enzyme system during ripening. Robust

proteinase genes from non-cheese microorganisms were cloned into starters for

increased production of proteolytic enzymes for accelerated ripening.

Bacillus subtilis neutral proteinase (BSNP) gene cloned in Lc. Lactis UC 317 caused

extensive proteolysis in Cheddar cheese within 2 weeks at 8°C; blend of 80:20

unmodified:modified cells were recommended for a more controlled proteolysis

(McGarry et al., 1994; as cited by Fox and Tobin, 1999). Proteolysis or texture of

Cheddar cheese made from recombinant chymosin from E. coli (Green et al., 1985)

or Kluyveromyces lactis (Bines et al., 1989) did not differ significantly from control

cheeses with chymosin from calf rennet. Common peptidases from NSLAB have

been expressed in starters for increased proteolytic activity to supplement the

contribution of NSLAB to proteolysis during ripening. Lc. lactis subsp. cremoris

MG1363 was genetically modified to over-express the genes pepC, pepN, pepO,

pepV or the lysin gene of the bacteriophage vML3 (Guldfeldt et al., 2001). Cheeses

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 Review of Literature

produced with strains over expressing pepN or pepC scored well for flavour with

reduced bitterness while cheeses produced with strains over expressing pepO or

pepV were indistinguishable from controls and also in the cheeses made from strains

with lysin gene were found to be different and it was concluded that lytic property of

the recipient strain was important for the flavour development (Guldfeldt et al.,

2001). Cheeses made from Lc. lactis starters enriched with lactobacilli peptidases led

to 3-fold increase in amino acid levels (Courtin et al., 2002). Starter modification for

increased sensitivity to lysis has been exploited for early lysis and release of

intracellular enzymes during ripening. A three-strain starter system was proposed by

Morgan et al. (2002), comprising of a bacteriocin producing strain, a bacteriocin

sensitive strain for lysis upon exposure to bacteriocin to release the intracellular

enzymes thus accelerating ripening and a bacteriocin insensitive strain for

development of acid during manufacture. A Lactococcus starter strain PRA270

genetically modified to produce streptokinase, an extracellular protein produced by

Streptococcus uberis is known to activate plasminogen to plasmin, the active

indigenous milk proteinase. Cheddar-type cheeses made with PRA270 showed

increased plasmin activity, accelerated hydrolysis of -casein resulting in increased

concentrations of -casein during ripening (Upadhyay et al., 2004b). Though genetic

modification appears to be a specific approach it is technically complex and is

weakly accepted by many consumers.

2.3.6 Adjunct cultures

Contribution of NSLAB to cheese ripening is well known and this knowledge has

been applied for development of adjunct cultures for acceleration of cheese ripening.

Selected strains of NSLAB are deliberately added during cheese manufacture, as

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 Review of Literature

adjunct cultures in the aim of accelerating ripening (McSweeney et al., 1993).

Addition of cell free extracts of brevibacteria caused accelerated flavour

development in Cheddar cheese (Law, 1987).

Cheese manufactured with adjunct cultures such as are Brevibacterium linens

(Weimer et al., 1997), NSLAB (Swearingen et al., 2001), Lactobacillus lactis

(Andersen and Madsen, 2004), probiotic cultures of Lb. acidophilus, Lb casei, Lb

paracasei and Bifidobacterium spp. (Ong et al., 2006), Lb. acidophilus and Lb.

paracasei subsp. paracasei (Bergamini et al., 2006), autolytic or fast lysing strains of

Lb. helveticus (Hannon et al., 2003; Kenny et al., 2006) NSLAB; Lb. plantarum

(Tavaria et al., 2006) are reported to have improved flavour development. In addition

to use of SLAB and NSLAB as adjunct cultures, yeasts have also been exploited for

contribution to cheese ripening; Geotrichum candidum (Berger et al., 1999; Martin et

al., 1999), Debryomyces hansenii and Yarrowia lipolytica (Ferreira and Viljoen,

2003; De Wit et al., 2005).

Although increased levels of flavour compounds were detected in cheeses with

adjunct cultures, there are no reports of reduction in ripening time or successful

commercial application. Few studies have also reported development of off-flavours

in cheeses with adjunct cultures.

2.3.7 Exogenous enzymes

Cheese ripening is mainly due to the action of enzymes from coagulant, milk, SLAB

and NSLAB. Addition of proteolytic and lipolytic enzymes was considered as a more

defined and simpler approach to accelerate cheese ripening and has been reviewed by

Fox and Stepaniak (1993).

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 Review of Literature

2.3.7.1 Proteolytic enzymes

Early attempts of exogenous enzyme addition to milk to accelerate cheese ripening

were not completely successful as bitterness, soft body, brittleness and other defects

were associated with increased proteolysis (Sood and Kosikowski, 1979; Law and

Wigmore, 1982, 1983; Fernandez-Garcia et al., 1988; Guinee et al., 1991). Flavour

intensity, formation of soluble nitrogen, liberation of FAA and FFA were enhanced

in Ras cheese made from -galactosidase treated milk (Farahat et al., 1984).

Addition of cheese slurries to cheese curd have also shown to accelerate ripening in

Cheddar (Dulley, 1976; Cliffe and Law, 1990), Swiss (Singh and Kristoffersen,

1971), Ras (Abdel Baky et al., 1982) and Blue (Rabie, 1989) cheeses. Kilcawley et

al. (2006) reported development of a novel method to generate enzyme modified

cheese (EMC) and Hannon et al. (2006) reported accelerated Cheddar cheese

ripening due to addition of EMC powder.

Extracts from crustacean munida was used as coagulant in miniature Cheddar cheese

manufacture and complex proteolytic products were observed during ripening

compared to cheeses manufactured with chymosin as the coagulant (Rossano et al.,

2005).

Hydrolysis of -casein by plasmin can be augmented by use of plasminogen

activators such as urokinase and streptokinase. Exogenous addition of urokinase, a

serine proteinase that cleaves plasminogen to plasmin to accelerate cheese ripening

resulted in increased proteolysis in Havarti (Bastian et al., 1991), St-Paulin, Swiss

(Bastian et al., 1997) and Cheddar cheeses (Barrett et al., 1999). Cheddar cheese

manufactured from milk supplemented with semi-purified streptokinase, a

plasminogen activator, showed a ~3 fold increase in plasmin activity compared to

control with accelerated hydrolysis of -casein and increased concentrations of -

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 Review of Literature

casein. However the levels of FAA were unaffected and increased concentration of

hydrophobic peptides were reported (Upadhyay et al., 2004b).

Combined action of proteinases and phosphatases are responsible for complete

degradation of casein during cheese ripening, hence phosphatases may play a major

role in cheese maturation and flavour development (Fox and Stepaniak, 1993).

Different peptide pattern was noted while no significant difference in protein or

nitrogen content was observed when miniature Cheddar cheeses were manufactured

with exogenous preparation of alkaline phosphatase (Shakeel-Ur-Rehman et al.,

2006).

Amino acid catabolism during cheese ripening is known to be responsible for

formation of flavour compounds, Yvon et al. (1998) and Banks et al. (2001) reported

enhanced amino acid catabolism in Cheddar cheese upon exogenous addition of

transaminase, -ketoglutarate. Several commercial enzymes such as Flavourzyme,

(Novozymes), Neutrase (Novozymes), Accelase (Danisco) have been recommended

by the companies for debittering and accelerated flavour development in cheese and

these claims need to be validated.

2.3.7.2 Lipolytic enzymes

Contribution of lipolysis to flavour and aroma of Cheddar cheese is not well

understood and proteolysis is thought to be the main contributor of flavour

compounds in Cheddar cheese. Enzymes from Aspergillus oryzae containing both

proteolytic and lipolytic fraction, was found to produce Cheddar aroma in cream, and

lipase was thought to be the main contributor of the aroma (Arbige, 1986).This was

later developed into a commercial enzyme FlavorAge (Chr. Hansen, Inc.) and

recommended for acceleration of cheese ripening. Free fatty acid composition of

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 Review of Literature

cheeses treated with this enzyme was similar to control but in higher concentrations

(Arbige, 1986). Piccantase (Gist Brocades), Palatase (Novozymes), Capalase

(Dairyland) and kid/lamb lipase-esterase (Chr. Hansen) are other commercial lipases

recommended for acceleration of cheese ripening. Addition of commercial lipases to

milk during cheese manufacture have shown to enhance the FFA profile (Law and

Wigmore, 1985) and also has been recommended for acceleration of cheeses such as

Tulum (Yilmaz et al., 2005).

Enzymes ranging from proteinases, exopeptidases, plasminogen activators, lipases,

-galactosidases, phosphatase and enzymes involved in amino acid catabolism have

been exogenously added to cheese in the aim of increased flavour development,

reduction of bitterness and accelerated ripening. Accelerated proteolysis was

achieved, often with textural defects or with no significant improvement in flavour

profile. Though commercial proteinases have been successful in reducing bitterness,

attaining balanced textural and flavour profiles still remain a challenge (Wilkinson

and Kilcawley, 2005). Addition of free enzyme to the milk, with rennet or starter

culture results in loss of nearly 90% of the enzyme at whey drainage (Law and

Kirby, 1987). Addition of enzymes to cheese curd at dry salting stage would raise the

problem of uneven distribution of small amount of enzyme in large masses of curd

(Law, 2001; Wilkinson and Kilcawley, 2005). Uncontrolled premature and extensive

proteolysis with development of bitterness and off-flavours in addition to whey

contamination and loss of yield are some of the problems associated with addition of

free enzymes to accelerate cheese ripening (Law, 2001).

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 Review of Literature

2.3.7.3 Microencapsulation of enzymes for acceleration of cheese ripening

Encapsulation involves confinement of substance/substances of interest within a

membrane or a matrix for isolation (Dziezak, 1988), protection from environment

(Gibbs et al., 1999) or for controlled delivery (Skeie, 1994) of the entrapped material.

The technique of microencapsulation has been studied by a number of groups for

delivering flavour-enhancing enzymes for acceleration of cheese ripening and is an

alternative approach towards accelerating cheese ripening. This method of adding

exogenous enzymes is free from the problems associated with addition of free

enzymes (Wilkinson and Kilcawley, 2005). Several proteolytic/lipolytic enzymes

have been microencapsulated for this purpose and this subject has been reviewed (El

Soda et al., 1989; Skeie, 1994; Wilkinson and Kilcawley, 2005).

Early attempt at acceleration of cheese ripening by microencapsulated cell free

extracts (CFE) of Lc. lactis ssp diacetylactis in milk fat was by Magee et al. (1981),

cooking cheese at 30°C or 33°C disrupted the capsules causing early release of CFE.

More heat stable milk fat capsules with CFE of Lc. lactis var. maltigens and

Gluconobacter oxydans were used and higher levels of flavour compounds were

reported. These capsules were found to be stable at 42°C for only 60 min.

Hydrogel beads designed to melt at <10°C and hence ideal for release of enzymes

during cheese ripening was proposed by Perols et al. (1997) with an encapsulation

efficiency of ~50%; however, bead syneresis interfered with the enzyme release.

Kailasapathy et al. (1998) used food gums such as agar, alginate, gellan, and -

Carrageenan and -Carrageenan-locust bean gum for application of encapsulated

enzymes into cheese for acceleration of ripening. Encapsulation efficiencies ranging

form 29-57% and capsule retention in curd ranging from 74-91% was reported.

Increased proteolysis, lower -casein and higher amino acid levels were observed in

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 Review of Literature

cheeses treated with enzyme capsules. Flavour of experimental cheeses did not differ

from controls. Cheeses made with enzyme capsules scored lower for texture while

bitterness was highest in cheeses with -Carrageenan capsules (Kailasapathy and

Lam, 2005).

Alternative method of enzyme encapsulation in liposomes was developed by Law

and King, (1985). Liposomes are phospholipid bilayers that form spherical structures

upon dispersion in aqueous solutions and these vesicles can be unilamellar or

multilamellar (MLV) depending on the manufacturing method (Walde and Ichikawa,

2001). This method has evolved as the dominant method of enzyme encapsulation to

accelerate cheese ripening and many researchers have reported acceleration of

proteolysis in cheese during ripening by addition of liposome-encapsulated enzymes.

Encapsulation efficiency in small unilamellar vesicles (SUV) was very poor at ~1-

3% (Kirby et al., 1987), hence most of the studies have focused on multilamellar

vesicles (MLV) and they can be produced in various ways. Encapsulation efficiency

of ~30% was achieved with multilamellar vesicles produced by reverse phase

evaporation technique (Alkhalaf et al., 1989). Multilamellar vesicles, produced by

hydration had encapsulation efficiencies 45-70% (Fresta et al., 1995); Multilamellar

vesicles produced by microfluidization had encapsulation efficiencies 11-22%

(Lariviere et al., 1991). Multilamellar vesicles produced using dehydration-

rehydration technique had encapsulation efficiencies ~35% (Skeie et al., 1995).

Enhanced proteolysis, no bitterness and firmer texture were characteristic of cheeses

with Bacillus subtilis neutral proteinase (BSNP) entrapped in liposomes by

dehydration-rehydration with encapsulation efficiency of 54.3% (Picon et al., 1995).

Encapsulation efficiencies of proteinases encapsulated in stimulated release

liposomes were 14.5% for chymosin, 27.5% for BSNP and 11.7% for cyprosins

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 Review of Literature

(Picon et al., 1997). Multilamellar vesicles produced from proliposomes had

encapsulation efficiencies up to 96% and curd entrapment level of 64% (Dufour et

al., 1996). Release of liposome-encapsulated enzymes in cheese is influenced by pH,

fat content and temperature. Release of enzymes from liposomes was significantly

stimulated by increasing the fat content from 0 to 20% and pH from 4.9 to 5.5;

however increasing the ripening temperature had no significant effect on the release

of enzymes from liposomes and the principles involved in the release of liposome

encapsulated enzymes could not be completely explained (Laloy et al., 1994, 1998).

Acceleration of Cheddar cheese ripening with liposome encapsulated bacterial and

fungal proteinases was achieved with encapsulation efficiencies of 32% and 33%

respectively, improved organoleptic properties and no bitterness was detected

(Kheadr et al., 2000). Enzyme mixtures containing bacterial protease and lipase,

fungal protease and lipase and Flavourzyme® and lipase encapsulated in liposomes,

accelerated Cheddar cheese ripening with higher flavour intensity in short period

without texture, rancid or bitter defects (Kheadr et al., 2003).

Majority of the studies involving enzyme encapsulation for acceleration of cheese

ripening have used liposomes as the encapsulating material often with low

encapsulation efficiency and poor understanding of the release mechanism of the

enzymes during ripening.

2.4 Microencapsulation

While liposomes have been the predominant materials used for enzyme

encapsulation for acceleration of cheese ripening they have poor encapsulation

efficiencies and also are expensive ingredients to be applied on a commercial scale.

Though a few researchers have reported use of alternative materials like food gums

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 Review of Literature

and milk fat for enzyme encapsulation for acceleration of cheese ripening, the

potential of other materials for this purpose has not been exploited. Currently almost

any substance can be encapsulated for isolation, purification, protection or controlled

release.

2.4.1 Techniques of Microencapsulation

Microencapsulation techniques currently used in the food industry are spray- drying,

spray-chilling or spray-cooling, fluidized-bed coating, liposome entrapment,

coacervation, inclusion complexation, rotational or centrifugal suspension separation

and extrusion as reviewed by Gibbs et al. (1999).

Spray-drying involves homogenisation of the material of interest with the carrier

material in 1:4 ratios and the mixture is fed into a dryer through a nozzle as opposed

to cooling of the mixture in cool air by spray-chilling/cooling. The latter requires

vegetable oil or hydrogenated oil.

In fluidized-bed coating, solid particles are suspended and the coating material is

atomised, the amount of coating depends on the length of time the particles are in

chamber.

Extrusion mainly involves dispersion of materials in to a liquid; the dispersed

material solidifies on contact with the liquid trapping the particles within a matrix.

Liposomes consist of one or more layers of phospholipids, hydrophilic portion

orients towards aqueous phase while the hydrophobic portion associates with

hydrophobic portions of other lipid molecules. Folding of the lipid sheet into a

spherical shape forms stable capsules and this folding can be induced by various

solvents or aqueous solutions.

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 Review of Literature

Coacervation involves emulsification of the material followed by separation of the

liquid phase which is used to coat the core material. This method though efficient can

be expensive.

Microencapsulation by inclusion complexation involves entrapment of materials in

the hydrophobic core of -Cyclodextrin with hydrophilic surface.

Centrifugal suspension separation is a relatively new technique and involves mixing

of the core and wall materials and then adding to a rotating disk. The core materials

then leave the disk with a coating of residual liquid. The microcapsules are then dried

or chilled after removal from the disk.

2.4.2 Hydrogels

Hydrogels are formed from hydrophilic polymers which may absorb from 10-20% to

thousands of times their dry weight in water. They can be chemically stable or

degradable. When a polyelectrolyte is combined with a multivalent ion of opposite

charge, it may form a hydrogel known as an ionotropic hydrogel, for example

calcium alginate. When polyelectrolytes are mixed they may gel or precipitate. Pores

may be formed in hydrogels by phase separation during synthesis or may exist as

smaller pores in the matrix (Hoffman, 2002). Labelled molecular probes of a range of

molecular weights are used to probe the pore size in hydrogels (Dong et al., 1994).

Polymers such as alginate, -Carrageenan, chitosan form firm hydrogels and could

also be potential entrapment materials for enzymes for application in acceleration of

cheese ripening.

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 Review of Literature

2.4.3 Alginate

Alginate is a polysaccharide that can form a hydrogel. It is available as the sodium

salt of polyuronic acid containing varying proportions of 1 4 linked -D-

mannuronic acid (M) and -L-guluronic acid (G). An ionic network developed in the

presence of Ca2+ or other multivalent cations forms a gel (Betigeri and Neau, 2002).

CaCl2 is the commonly used cation for gelation of alginate. It is widely used in

matrix immobilisation of wide range of biological materials ranging from bacterial

cells (Champagne et al., 1992; Kailasapathy, 2002), mammalian cells (Koch et al.,

2003), enzymes (Blandino et al., 2000), vitamins (Madziva et al., 2006) and also for

controlled drug delivery (Sugawara et al., 1994). The wide application of this

polymer can be attributed to the mild conditions involved in the preparation and low

cost (Fundueanu et al., 1999).

Properties of Ca-alginate hydrogels can be easily modified on treatment with starch

(Sultana et al., 2000), pectin (Madziva et al., 2006), xanthan (Elcin, 1995), poly-L-

lysine (Strand et al., 2002), polyethylene glycol (Chandy et al., 1999) or chitosan

(Sezer and Akbuga, 1999) to suit the application it is being prepared for and is a

potential entrapment material for enzymes for application in acceleration of cheese

ripening.

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 Review of Literature

Figure 2.10 Structure of alginate. Alginate molecules are linear block co-
polymers of β -D-mannuronic acid (M) and -L-guluronic acids (G) with a
variation in composition and sequential arrangements (De Vos et al., 2006).

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 Review of Literature

2.4.4 Carrageenan

Carrageen is a food grade, biocompatible high molecular weight polysaccharide

extracted from seaweed that forms a hydrogel (Velde et al., 2002). Its primary

structure is made up of alternating -(1,3)-D-galactose-4-sulphate and -(1,4)-3,6-

anhydro-D-galactose residues. -Carrageenan forms a thermoreversible gel on

cooling and also in the presence of salts; mainly potassium, which bind specifically

to the helices. Molecules undergo a coil-helix transition followed by aggregation of

helices and salts reduce electrostatic repulsion between chains thus promoting

aggregation leading to gel formation (Williams and Phillips 2000). Sodium salt of -

Carrageenan is soluble in cold water and sets into a gel on cooling between 40-60°C

depending on the cations present. It is used in the encapsulation of cells (Cassidy et

al., 1997), enzymes like -chymotripsin (Belyaeva et al., 2004) and its other

applications for immobilisation has been reviewed by Velde et al. (2002).

2.4.5 Chitosan

Chitosan is a polymer of (1 4)-2-amino-2-deoxy-D-glucose obtained from chitin

by N-deacetylation. Chitosan is insoluble in aqueous solution at pH 6 except for low

molecular weight samples (Terbojevich and Muzzarelli, 2000). Chitosan gels on

addition of cross-linking reagent or by ionotropic gelation with certain anionic

counterions (Cabral and Kennedy, 1991). It is a non-toxic, biocompatible,

antibacterial and biodegradable polyelectrolyte and for this reason has been used for

a variety of applications including controlled drug delivery (Borzacchielo et al.,

2001). Cross linking of chitosan with other polymers has shown to improve its

physical and mechanical properties. Chitosan has several applications including cell

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 Review of Literature

encapsulation (Zielinski and Aebischer, 1994; Chandy et al., 1999), drug delivery

(Sezer and Akbuga, 1999) and plasmid delivery (Bozkir and Saka, 2004). It appears

to be a suitable material for enzyme encapsulation for acceleration of cheese

ripening.

2.4.6 Starch

Starch is the second most naturally abundant polysaccharide of plant origin. It is

made up of glucose polymers. Linear polymer of glucose forms the amylose

molecule while the branched polymer forms the amylopectin molecule. Starch is

made up of insoluble microscopic semi crystalline granules 1 - 100µm in size

(Murphy, 2000). It is used with hydrogels for encapsulation of bacteria (Sultana et

al., 2000).

2.4.7 Pectin

Pectins are hetero-polysaccharides with at least 65% of galacturonic acid units. The

acid group may be free or esterified with methanol. De-esterification yields a range

of ‘low methoxyl’ pectins that have greater reactivity towards calcium ions which

will cause gelation under suitable conditions of soluble solids and pH (May, 2000). It

has been used with alginate for encapsulation of folic acid (Madziva et al., 2006).

2.4.8 Poly-L-lysine

Poly-L-lysine (PLL) is a naturally occurring cationic amino acid polymer (Aynie et

al, 1999) and the most widely studied polycation for alginate bead coating (Orive et

al., 2006). Addition of PLL is known to strengthen the alginate gel structure (Lim

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 Review of Literature

and Moss, 1981; Liu and Krishnan, 1999). Lambert et al. (2001) recommended

addition of PLL for formation of stable alginate nanospheres. It binds to alginate by

forming complexes with M-G sequences on the surface of the alginate beads

(Bystricky et al., 1990). PLL binding to intermediate-G alginate is higher than to

high-G alginate due to the availability of higher number of binding sites in

intermediate-G alginates (De Vos et al., 2002, 2003).

2.5 Analysis and evaluation of cheese ripening

The demand for constant supply of consistent low-cost high intensity flavour in

cheese had led to development of novel ways for monitoring cheese ripening. At first

basic analytical techniques were developed to measure proteolysis and lipolysis; the

major biochemical processes occurring in cheese during ripening. Proteolysis during

cheese ripening was initially monitored by spectroscopy/colorimetry (trinitrobenzene

sulphonic acid soluble fraction, trichloroacetic acid soluble nitrogen and Kjeldahl

method for determination of total nitrogen) for measurement of amino acids and

peptides. Advanced techniques involving electrophoresis (capillary electrophoresis,

polyacrylamide gel electrophoresis) and chromatography (reverse phase - high

performance liquid chromatography, gas chromatography) are now widely used for

characterisation and quantification of peptides, fatty acids and amino acids from

cheese (Cliffe et al., 1989; Cliffe and law, 1990; Verdini et al., 2004; Veloso et al.,

2004). Moisture and textural changes in cheese during ripening were monitored by

measuring the ultrasonic velocity of Mahon cheese (Benedito et al., 2000, 2002).

Dupont et al. (2003) used rabbit polyclonal anti-peptide antibodies against the

cleavage sites to measure the extent of cleavage of peptide bonds by protease,

proposing the use of ELISA with the specific antibodies for assessing proteolysis.

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 Review of Literature

Statistical methods have also been employed to analyse data to predict cheese

ripening. Ripening time of Manchego cheese was predicted using partial least

squares (PLS) regression (Poveda et al., 2004). Coker et al. (2005) statistically

analysed proteolysis data by principal component analysis (PCA) and multiple linear

regression to predict ripening of cheese. Advanced spectroscopy and proteomics

were employed by Gagnaire et al. (2004) to investigate the release of enzymes in

Emmental cheese aqueous phase by using methodology based on size exclusion

chromatography, 2D-electrophoresis, MALDI-TOF MS (matrix assisted laser

desorption/ionisation – time of flight mass spectrometry) and LC-ESI-MS/MS (liquid

chromatography electrospray ionisation tandem mass spectrometry) and identified

five functional groups of proteins involved in 1) proteolysis, 2) lipolysis, 3) stress

response, 4) DNA and RNA repair of LAB and 5) oxido-reduction. Proteomics is fast

evolving as a powerful tool for monitoring cheese ripening, reports of use of

proteomics for analysis in cheese science is constantly increasing. Piraino et al.

(2007) developed a rapid method using (MALDI-TOF MS) for analysis of peptides

from the ripening cheese, the analysis time was reported to be ~ 3 min compared to

100 min for analysis by RP-HPLC. Infrared spectroscopy has also been exploited as

a potential tool for measuring cheese composition (Rodriguez-Soana et al., 2006) and

texture (Blasquez et al., 2006; Karoui et al., 2007). Applying similar technique,

Martin-del-campo et al. (2007) followed proteolysis, lipolysis and glycolysis by

Fourier transform infrared (FTIR) spectroscopy to monitor cheese ripening. Analysis

of variance (ANOVA) showed significant changes on spectra sets and two different

steps of ripening were described by principal component analysis of the spectral data.

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 Review of Literature

Sensory methods are also employed to forecast ripening trend and also for quality

control. These methods involve a scoring system with the grant of maximum score

based on desirable qualities of the cheese and deduction of score for undesirable

characteristics or sensory evaluation by a panel of trained judges (Delahunty and

Drake, 2004).

2.6 Factors affecting cheese quality

Ripening of cheese is due to the action of coagulant, microorganisms and their

enzymes. Every batch of cheese varies even though all the variables such as starters,

coagulant and ripening atmosphere are constant. This variability in the final cheese

quality can be attributed to several factors and their interaction (Fox and Cogan,

2004). Source and quality of milk, coagulant, NSLAB and ripening temperature are

the major factors affecting cheese quality.

Milk quality, stage of lactation and animal nutrition affect cheese quality as pasteur-

fed animal milk contain higher levels of carotene and hence impart deeper colour to

cheese. Increased proteolysis and higher levels of free fatty acids may be present in

late-lactation milk (Hickey et al., 2006) and pathogens from milk of poor

microbiological quality can cause off-flavours in cheese (Fox and Cogan, 2004).

Acidification may be insufficient if the milk is contaminated with excessive

antibiotics or if inactive starters are used thus affecting coagulation, syneresis,

flavour and texture. In modern practice standard casein-fat composition is used for

consistent cheese quality.

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 Review of Literature

2.7 Summary

Cheese is an important part of the daily diet with high per capita consumption in

Australia and most other parts of the world. Most cheeses are consumed after

ripening, an important stage of cheese production. It involves complex biochemical

processes occurring in cheese curd after manufacture. Cheese ripening can range

from a few weeks to over 3 years depending on the variety, thus contributing

considerably to the cost of cheese production. Any attempt at minimising the

ripening duration has attracted attention. Though researchers have been trying over

decades to understand cheese ripening process in order to accelerate ripening, there

are several steps during ripening that are not yet well understood, thus limiting the

efforts at acceleration of ripening. There are several reports of acceleration of cheese

ripening in various varieties using a range of methods but very few reports of

successful commercial application possibly due to complexity of the methods and

poor results at industrial scale. Addition of exogenous enzymes during cheese

manufacture appears to be a specific and simple approach towards accelerating

ripening. Extensive proteolysis and bitterness associated with addition of free

enzymes can be avoided by addition of microencapsulated enzymes. Though several

commercial lipases and proteases are recommended for acceleration of cheese

ripening, very few reports have tested these enzymes. Enzymes for cheese ripening

have been mostly encapsulated in liposomes, which are expensive ingredients and

hence are not feasible for commercial application. Reports of enzyme encapsulation

in other economic ingredients such as milk fats and food gums show poor efficiency

of the enzyme in addition to poor understanding of the release mechanism of

encapsulated enzymes in cheese during ripening. Inert economical food grade

polymers such as alginate, chitosan and -Carrageenan have been extensively used

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 Review of Literature

for encapsulation of biological and biochemical materials like bacterial and

mammalian cells, tissues, enzymes, vitamins and also for controlled drug delivery.

These polymers can be used to microencapsulate enzymes to be delivered into cheese

for acceleration of ripening.

58
3 SCREENING AND SELECTION OF POLYMERS FOR

ENZYME ENCAPSULATION

59
 Enzyme encapsulation

3.1 Abstract

Screening of alginate, chitosan and κ-Carrageenan for microencapsulation of

Flavourzyme showed that alginate was a suitable polymer for enzyme encapsulation

using the Inotech® encapsulator, as chitosan and κ-Carrageenan were too viscous

even at low concentrations to give satisfactory capsules. Encapsulation efficiency of

Flavourzyme-alginate gel beads formed in 0.1M CaCl2 was found to be ~17-18%.

Poor encapsulation efficiency was attributed to the high porosity of alginate gel

beads and possible diffusion of Flavourzyme during encapsulation. Starch (Hi-

Maize™) and pectin were mixed with alginate to minimise the porosity of the gel

beads however there was no improvement in the encapsulation efficiency of

Flavourzyme. Gelling of the alginate-Flavourzyme beads in the chitosan treated

cationic solution of 0.1M CaCl2 significantly increased (p < 0.05) the encapsulation

efficiency to 70-88% depending on the chitosan concentration. Though encapsulation

efficiency increased with the increase in chitosan concentration, 0.1% was used as

higher concentrations caused clumping of capsules during formation. Optimum

gelling time of the capsules in chitosan treated cationic solution was found to be 10

min. Alginate concentration in the range 1.6 to 2.0% (w/v) was found to be suitable

for Flavourzyme encapsulation and 2% (w/v) tri-sodium citrate was found to release

most of the encapsulated enzymes in vitro for measurement of enzyme activity.

Coating of the Flavourzyme capsules in 0.15% (w/v) alginate or 0.05% (w/v) poly-L-

lysine showed no significant difference (p > 0.05) in enzyme retention compared to

those without any coating. Enzyme capsules produced from 1.6, 1.8 and 2.0% (w/v)

alginate allowed to gel in 0.1M CaCl2 containing 0.1% chitosan, stored frozen and

freeze-dried were found to be shelf-stable for at least 10 weeks retaining about 80%

of the initial enzyme activity unlike capsules stored after air-drying which retained

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 Enzyme encapsulation

only 25-50% of the initial enzyme activity. Morphological assessment of enzyme

capsules gelled in cationic solution containing chitosan revealed mainly spherical

capsules with an average diameter of 500 m.

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 Enzyme encapsulation

3.2 Introduction

Cheese is a popular dairy product with some varieties requiring extended ripening

which is an economical burden (Picon et al., 1997). Though several techniques were

developed for acceleration of cheese ripening, not many have been successful at

commercial scale. Exogenous addition of enzymes that catalyse the biochemical

reactions occurring during cheese ripening appears to be a simple approach but has

lead to premature extensive proteolysis in addition to contamination of whey.

Enzyme delivery in the microencapsulated form was found to be a solution for the

problems associated with the use of free enzymes (Wilkinson and Kilcawley, 2005).

Encapsulation in milk fat (Magee et al., 1981; Braun and Olson, 1984), hydrogel

(Perols et al. 1997), food gums (Kailasapathy et al. 1998; Kailasapathy and Lam,

2005) and liposomes (Law and King 1985; Picon et al., 1995, 1997; Dufour et al.,

1996; Laloy et al., 1998; Kheadr et al., 2000, 2003) have been used for enzyme

encapsulation for acceleration of cheese ripening. Liposomes appear to be the

popular material for this purpose as a number of studies have reported use of

liposome-encapsulated enzymes for acceleration of cheese ripening. Liposomes are

expensive to be used on an industrial scale and hence not feasible for commercial

acceleration of cheese ripening.

Though there have been significant developments in the encapsulation technology

for application in biological science, very few techniques have been studied for

delivery of enzymes into cheese for acceleration of ripening. Polysaccharides such as

alginate, chitosan and -Carrageenan are food-grade polymers that form firm gels

with ions of opposite charge. These polymers are inert and inexpensive and have

been widely used for encapsulation of biological and biochemical substances ranging

from cells, enzymes and drugs to plasmid for the purpose of protection, isolation or

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 Enzyme encapsulation

controlled delivery (Champagne et al., 1992; Sezer and Akbuga, 1999; Blandino et

al., 2000; Koch et al., 2003; Bozkir and Saka, 2004). These polymers can be used to

encapsulate flavour-enhancing enzymes for acceleration of Cheddar cheese ripening.

Food-grade polymers were screened in this study for the encapsulation of flavour-

enhancing enzymes. Though several advanced techniques for efficient enzyme

encapsulation have been reported, many techniques involve use of strong chemicals,

solvents or fats which is undesirable for application in cheese due increased reports

of obesity and related illnesses and also the risk of rejection by consumers. Hence the

Inotech® encapsulator was chosen for enzyme encapsulation as it requires mild

conditions such as only enzyme-polymer mixture and gelling solution for enzyme

encapsulation.

Polymers such as alginate, -Carrageenan and chitosan were screened for

encapsulation of Flavourzyme (flavour-enhancing enzyme) using the Inotech®

encapsulator. After selection of a suitable polymer, encapsulation parameters such as

concentration of polymer and gelling solution, gelling duration and in vitro release

were optimised. This study was aimed at assessing the suitability of selected

polymers/hydrogels for microencapsulation of flavour-enhancing enzymes and

optimisation of the encapsulation parameters to enhance the encapsulation efficiency

and enzyme retention after manufacture and sustained release during ripening.

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3.3 Materials and methods

Milli Q water (Millipore, Massachusetts, USA) was used for all preparations unless

otherwise mentioned.

3.3.1 Flavourzyme® assay

Flavourzyme® (Novozymes, Sydney, Australia) a fungal protease/peptidase complex

from Aspergillus oryzae, known to contain both endoprotease and exopeptidase

activities and highly recommended for debittering was employed in this study to

accelerate Cheddar cheese ripening. This enzyme is standardised in leucine amino

peptidase units (LAPU) with declared activity of 500LAPU/g of 500L solution or

1000LAPU/g of 1000L solution. One LAPU is the amount of enzyme which

hydrolyses 1 µmol of L-leucine-p-nitroanilide per min. Flavourzyme activity was

measured based on the rate of reaction of the enzyme with the substrate L-leucine-p-

nitroanilide (Fluka, Sydney, Australia). The assay sensitivity was determined to be in

the range 0.02 to 0.1LAPU/mL hence all the samples and standards were diluted in

this range prior to assay. All standards and samples were diluted with 0.2mM ZnCl2

solution (Sigma, Sydney, Australia) prepared with 0.15% brij, (Sigma, Sydney,

Australia) and 80µL of this diluted enzyme mixture was incubated with 840µL of

assay working solution of 0.1M tris buffer, pH 8.0 (Sigma, Sydney, Australia) for 10

min at 40°C followed by addition of 80µL of reagent; 26mM L-leucine-p-

nitroanilide (Fluka, Sydney, Australia) prepared in absolute ethanol (Sigma, Sydney,

Australia). The absorbance was read at first and tenth min at 405nm, the change in

the absorbance of the samples was compared to that of known standards to estimate

the enzyme activity. This Flavourzyme assay is a modified adaptation of the method

supplied by Novozymes® Australia Pty Ltd.

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 Enzyme encapsulation

3.3.2 Palatase® assay

Palatase® 20000L a lipase from Novozymes (Sydney, Australia) was tested for

acceleration of flavour development during Cheddar cheese ripening. It is

standardised in lipase units (LU) with declared activity of 20000LU/g of preparation.

Standards and samples were diluted with 0.1M sodium phosphate buffer, pH 7.4

which was also used as the assay working solution and 20µL of standard or samples

were added to 200µL of phosphate buffer in a 96 well plate and heated to 30°C,

followed by addition of 10µL of the reagent p-nitrophenyl butyrate; 25µL dissolved

in 10mL 2-propanol (Sigma, Sydney, Australia) and the enzyme kinetics was read at

410nm at 30°C in the Multiskan Spectrum Microplate Spectrophotometer 1500

(Thermo Lab systems, Vantaa, Finland). The Multiskan Spectrum software was used

to interpret the data and calculate the Palatase activity in unknown samples using the

standard curve.

3.3.3 Selection of polymers for enzyme encapsulation

Flavourzyme (500LAPU), 2mL was diluted in 8mL ZnCl2-brij solution and added to

90mL filtered Alginate (molecular weight range of 12000 to 80000, viscosity of

~250cP, 2% (25°C) L and composed of approximately 61% mannuronic acid and

39% guluronic acid with a M/G ratio of 1.56 from Sigma, Sydney, Australia) or

chitosan (minimum 85% deacetylated from Sigma, Sydney, Australia) or -

Carrageenan (Fluka, Sydney, Australia) solutions of 1.0, 0.1 and 0.1% (w/v)

respectively and extruded through the 300 nozzle of the Inotech® encapsulator. The

droplets from alginate, -Carrageenan and chitosan were allowed to gel for 10 min in

0.1M CaCl2, 0.1M KCl and 0.1M sodium phosphate solutions respectively. The

gelling solution was drained and the enzyme microcapsules were washed with water

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 Enzyme encapsulation

and 1g dissolved in 9mL of 2.0% (w/v) tri-sodium citrate and the Flavourzyme

activity was measured as described in section 3.3.1 to determine the encapsulation

efficiency (EE) which was expressed as;

Encapsulation efficiency = Enzyme activity recovered from the capsules X 100

Initial enzyme activity*

* (In the polymer-enzyme solution)

Note: Enzyme activity recovered from the capsules explained in section 3.3.4

All polymers were prepared in higher concentration so as to yield desired

concentration when made up to 100mL from 90mL to compensate for dilution of the

polymer upon mixing with diluted enzyme. Enzyme polymer mixture (100mL) and

the capsules obtained from this mixture were weighed to estimate the loss of water

from the hydrogel beads during formation as a result of diffusion. The difference in

the weight of capsules and enzyme-polymer mixture was used to correct the final

enzyme activity, as undiluted Flavourzyme was used initially and later enzyme

microcapsules were used for the measurement of EE and all further experiments.

3.3.4 Polymer combinations for improved Flavourzyme retention in capsules

Flavourzyme (500LAPU) 2mL, was mixed with polymer combinations of

alginate:starch (Hi-Maize) (Starch Australasia Ltd, Sydney, Australia) in the

concentrations 1.0:1.0, 1.5:1.0, 2.0:1.0 and 2.0:1.5 g in 90mL water and

alginate:pectin (esterified potassium salt, Sigma, Sydney, Australia) in the

concentrations 1.6:0.4 and 1.4:0.6g in 90mL water and the enzyme gel beads were

produced by gelling the droplets produced from the encapsulator in the cationic

solution of 0.1M CaCl2 for 10 min. The enzyme microcapsules were harvested after

draining the cationic solution and 1g was dissolved in 9mL of 2% (w/v) tri-sodium

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 Enzyme encapsulation

citrate and the Flavourzyme activity was measured as described in 3.3.1 after

appropriate dilution.

3.3.5 Optimisation of polymer concentration

Flavourzyme (500LAPU) 2mL was mixed with 1.4, 1.6, 1.8 and 2.0 % (w/v) alginate

and extruded through the 300 m nozzle of the Inotech® encapsulator. The

Flavourzyme-alginate droplets were allowed to gel in the cationic solution of 0.1M

CaCl2 for 10 min. The enzyme micro capsules were harvested after draining the

cationic solution and 1g was dissolved in 9mL of 2.0 % (w/v) tri-sodium citrate and

Flavourzyme activity was measured as described in 3.3.1 after appropriate dilutions.

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 Enzyme encapsulation

Figure 3.1 Schematic representation of enzyme encapsulation in calcium-

alginate by extrusion method (Anjani et al., 2007)

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 Enzyme encapsulation

3.3.6 Treatment of cationic solution with chitosan and optimisation

Alginate (2g) - Flavourzyme 500L (2.0mL ) (in 100mL), droplets from the

encapsulator were allowed to gel in a modified cationic solution of 0.1M CaCl2

containing 0.1% (w/v) chitosan, a modification of the method reported by Sezer and

Akbuga (1999). The pH of the cationic solution containing chitosan was set at 6.2 as

it is the optimal pH of Flavourzyme (Flavourzyme data sheet, Novozymes).

Chitosan, 0.1g was dissolved in 5mL glacial acetic acid (Ajax chemicals, Sydney,

Australia) and the volume was made up to 70mL with Milli Q water and the pH was

adjusted to 6.2 using NaOH (1.0M and 0.1M) (Ajax chemicals, Sydney, Australia)

followed by addition of 1.1g CaCl2 (Sigma, Sydney, Australia) dissolved in 10mL

Milli Q water and the final volume of the solution was made up to 100mL, this

solution was then refrigerated. Gelling solutions of CaCl2 containing 0.1, 0.2 and

0.3% (w/v) chitosan were studied for maximising EE.

Flavourzyme-alginate gel beads produced from 2mL 500LAPU Flavourzyme and

1.4, 1.6, 1.8 and 2.0% (w/v) alginate were allowed to gel in cationic solution of 0.1M

CaCl2 with 0.1% (w/v) chitosan for 10 min and the Flavourzyme activity was

measured in the dissolved beads to optimise the alginate concentration.

3.3.7 Coating of capsules for improved enzyme retention and stability

Flavourzyme-alginate gel beads formed in 0.1M CaCl2 containing 0.1% (w/v)

chitosan was rinsed with Milli Q water and 2g was allowed to stand in 5mL of 0.15%

(w/v) alginate or 0.05% (w/v) poly-L-lysine (PLL) solution for 10 min at room

temperature with swivelling of the containers every 2 min (Champagne et al., 1992;

Quong et al., 1999). After 10 min the capsules were rinsed with Milli Q water and 1g

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 Enzyme encapsulation

capsules were dissolved in 9mL 2% (w/v) tri-sodium citrate and the Flavourzyme

activity was measured as described in 3.3.1 after appropriate dilution.

3.3.8 Optimisation of gelling duration

The Flavourzyme-alginate droplets from the encapsulator resulting from 2mL

500LAPU Flavourzyme and 1.6, 1.8 and 2.0% alginate with and without PLL

coating were allowed to gel in the cationic solution of 0.1M CaCl2 containing 0.1%

(w/v) chitosan for 10, 20 and 40 min to optimise the gelling duration of the capsules

for maximum EE. Flavourzyme gel beads (1g) were dissolved in 9mL 2% tri-sodium

citrate and Flavourzyme activity was measured as described in 3.3.1.

3.3.9 Optimisation of releasing solution concentration

Tri-sodium citrate in the concentration of 0.5, 1.0 and 2.0% (w/v) was tested for

complete in vitro release of encapsulated enzyme. Flavourzyme gel beads (1g) were

dissolved in 0.5, 1.0 and 2.0 % (w/v) tri-sodium citrate and the Flavourzyme activity

was measured as described in 3.3.1 after appropriate dilution. Tri-sodium citrate is

used only for in vitro release of encapsulated enzyme for measurement of enzyme

activity, however, in cheese; the encapsulated enzymes will be released by diffusion

of encapsulated enzymes from the capsules.

3.3.10 Shelf life of encapsulated Flavourzyme

Uncoated and PLL-coated Flavourzyme capsules both in 1g batches were freeze-

dried for 16h in an Alpha-1-4 freeze drier with controller LDC-1M (CHRIST®

Gefriertrocknungsanlagen, Osterode am Harz, Germany), frozen in a –20°C freezer

overnight and air-dried at room temperature (25°C) for 24h. Freeze-dried and air-

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 Enzyme encapsulation

dried capsules were stored at 4°C while frozen capsules were stored at –20°C for 10

weeks and the shelf-stability of these capsules was assessed by monitoring the

Flavourzyme activity every week by dissolving the capsules in 2% tri-sodium citrate

and measuring the activity as described in 3.3.1 after appropriate dilution.

3.3.11 Morphology of enzyme microcapsules

Size of alginate immobilised Flavourzyme gel beads was measured using an optical

microscope (Nikon microscope, Labophot-2, Nikon Corporation, Kanagawa, Japan)

fitted with a stage occulometer of 10X magnification. The average size reported is a

mean of 20 randomly selected gel beads.

3.3.12 Statistical analysis

All results are a mean of 6 replicates unless otherwise mentioned and expressed with

standard error of mean or standard deviation as mentioned. Data were analysed using

one (two) factorial analysis of variance (ANOVA) general linear SPSS® for

WindowsTM Version 14 (SPSS Inc. 2005). The assumption of normal distribution

was checked using P-P plot and homogeneity of variance using Levene’s test of

equality of error variances. Treatments were tested against each other using Ryan-

Einot-Gabriel-Welsch Q test if the assumption of equality of variance was met or

Dunnett’s T-test if the assumption was not met.

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 Enzyme encapsulation

3.4 Results

3.4.1 Selection of polymers for enzyme encapsulation

Alginate, -Carrageenan and chitosan were studied for encapsulation of

Flavourzyme; a flavour-enhancing enzyme, for controlled release of Flavourzyme in

Cheddar cheese for acceleration of ripening. The Inotech® encapsulator was selected

for enzyme encapsulation as it involved mild conditions for capsule production;

hence it was required to pump only relatively low viscosity solutions through the

nozzle of the encapsulator as per instructions from the manufacturer in order to allow

easy flow of the polymer-enzyme mixture. Chitosan was too viscous and could not

be pumped through the nozzle of encapsulator hence was unsuitable for Flavourzyme

encapsulation through using the encapsulator. Similarly -Carrageenan turned too

viscous upon cooling below 65°C and this high temperature was likely to denature

the enzyme and hence reduce Flavourzyme activity therefore -Carrageenan was also

eliminated as a suitable polymer for Flavourzyme encapsulation using this

encapsulator. Thus after preliminary screening it was determined that alginate was a

suitable polymer for enzyme encapsulation through this encapsulator. Alginate

concentrations of 1.4, 1.6, 1.8 and 2.0% (w/v) were considered for Flavourzyme

encapsulation with average EE of 16.3, 19.2, 17.4 and 18.6 % respectively (Figure

3.2). There was no significant difference (p > 0.05) in the EE on increasing the

alginate concentration. Incorporation of starch or pectin into alginate-Flavourzyme

mixture prior to encapsulation resulted in the EE ranging from about ~7 to 12% for

all combinations of alginate/starch (Figure 3.3) to about ~9% for alginate/pectin

(Figure 3.4) tested in this study. Though increase in the alginate and starch

concentrations significantly (p < 0.05) improved the EE of Flavourzyme, it was

72
 Enzyme encapsulation

however lower than that obtained when encapsulated in alginate alone. Hence

alginate was used for enzyme encapsulation.

73
 Enzyme encapsulation

24

20 18.4 18.6
17.8 17.2
EE of flavourzyme (%)
16

12

0
1.4 1.6 1.8 2.0
Alginate conc (% w/v)

Figure 3.2 Encapsulation efficiency of Flavourzyme immobilised in alginate by

gel bead formation in 0.1M CaCl2 and the effect of alginate concentration on
encapsulation efficiency. Error bars indicate standard error of mean.

74
 Enzyme encapsulation

14.0

11.9
12.0

10.0
EE of flavourzyme (%)
9.2
8.1
8.0 7.3

6.0

4.0

2.0

0.0
1.0:1.0 1.5:1.0 2.0:1.0 2.0:1.5
Alginate:starch (% w/v)

Figure 3.3 Encapsulation efficiency of Flavourzyme immobilised in alginate-

starch mixture by gel bead formation in 0.1M CaCl2 and the effect of varying
alginate-starch concentration and proportion (in 100mL water) on
encapsulation efficiency. Error bars indicate standard error of mean.

75
 Enzyme encapsulation

12.0
10.8
10.2

9.0
EE of flavourzyme (%)

6.0

3.0

0.0
70/30 80/20
Alginate:pectin (% w/v)

Figure 3.4 Encapsulation efficiency of Flavourzyme immobilised in alginate-

pectin mixture by gel bead formation in 0.1M CaCl2 and effect of varying
concentration and proportion of alginate and pectin (in 100mL water) on
encapsulation efficiency. Error bars indicate standard error of mean.

76
 Enzyme encapsulation

3.4.2 Effect of chitosan concentration in gelling solution on encapsulation

efficiency of Flavourzyme

Addition of chitosan to the gelling solution of 0.1M CaCl2 significantly increased the

EE of Flavourzyme immobilized in 2 % (w/v) alginate (F2, 6 = 19.013, p < 0.001). EE

of Flavourzyme capsules formed in cationic solution of 0.1M CaCl2 with 0, 0.1, 0.2

and 0.3% (w/v) chitosan was 18.6a, 71.9b, 75.7c and 85.0d % respectively (Figure

3.5). Results are a mean of 5 replicates unless mentioned otherwise and the error bars

indicate standard error of mean. Means followed by different superscript letter are

significantly different.

3.4.3 Weight of Flavourzyme microcapsules

Since Flavourzyme was supplied in liquid form it was measured in millilitres for all

experiments including encapsulation, however; when encapsulated, the weight of

Flavourzyme capsules could only be measured in grams before dissolving to measure

the enzyme activity. All enzyme capsules were of 1g weight for the ease of dilution

and calculations. To adjust for this variation in the measurement, enzyme

microcapsules obtained from 100mL enzyme-polymer mixture was weighed and this

weight was used to adjust the enzyme activity of the capsules accordingly. For

example if 2mL Flavourzyme in 100mL enzyme-polymer mixture yielded 70g

capsules then 1mL yields 0.7g, hence enzyme activity in 0.7g was calculated. Weight

of Flavourzyme-alginate capsules from 100mL mixture ranged from 58.9, 61.1, 61.5

and 62.9g for 1.4, 1.6, 1.8 and 2.0 % (w/v) alginate respectively while that from

100mL solution of alginate-starch ranged 49-80g and alginate-pectin ~64g. Weight

of Flavourzyme capsules formed in the gelling solution containing chitosan was 77.4,

85.5 and 88.0g for 0.1, 0.2 and 0.3% (w/v) chitosan respectively (Table 3.1).

77
 Enzyme encapsulation

90
84.0

80 76.3
72.2
70

60
EE of flavourzyme (%)

50

40

30 *
18.6
20

10

0
0.0 0.1 0.2 0.3
Chitosan concentration in gelling solution (% w/v)

Figure 3.5 Encapsulation efficiency of Flavourzyme immobilised in 2% (w/v)

alginate with gel bead formation in cationic solution of 0.1M CaCl2 containing
chitosan and the effect of chitosan concentration on encapsulation efficiency
(results are a mean of 5 replicates, * data obtained from Figure 3.2, mean of 6
replicates). Error bars indicate standard error of mean.

78
 Enzyme encapsulation

Table 3.1 Weight of capsules obtained from 100mL polymer-Flavourzyme

(90mL:2mL, 8mL Flavourzyme diluent) mixture allowed to gel in cationic
solution of 0.1M CaCl2 with and without chitosan for 10 min. * treatment with
varying concentrations

Polymer/gelling solution Concentration (% w/v) Weight (g)

Alginate*/ 0.1M CaCl2 1.4 58.9 ± 0.2

1.6 61.1 ± 0.25

1.8 61.5 ± 0.15

2.0
62.9 ± 0.55

Alginate-pectin*/ 0.1M CaCl2 1.4:0.6 63.4 ± 0.66

1.6:0.4 63.0 ± 1.0

1.8:0.2 64.0 ± 0.4

Alginate-starch*/ 0.1M CaCl2 1.0:1.0 49.0 ± 0.5a

1.5:1.0 51.9 ± 0.05a

2.0:1.0 60.1 ± 0.46b

2.0:1.5
80.2 ± 1.1c

2.0% Alginate/ 0.1M CaCl2- 0.1 77.4 ± 0.7a

chitosan* 0.2 85.5 ± 0.6b

0.3 88.0 ± 0.8b

abc
Means within the same column followed by different superscript letter are

significantly different (p < 0.05).

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 Enzyme encapsulation

3.4.4 Optimisation of polymer concentration

Varying alginate concentrations were studied for optimisation of Flavourzyme

encapsulation. EE of Flavourzyme-alginate gel beads formed in cationic solution of

0.1M CaCl2 containing 0.1% (w/v) chitosan was 62.4a, 69.7bc, 70.2bc and 72.3c for

1.4, 1.6, 1.8 and 2.0% (w/v) alginate respectively (Figure 3.6). Means followed by

different superscript letter are significantly different (F3, 8 = 193.754, p < 0.01).

3.4.5 Effect of coating on Flavourzyme retention in capsules

Flavourzyme capsules were coated with 0.15% alginate and 0.05% poly-L-lysine for

10 min in an attempt to minimise enzyme loss. Flavourzyme activity in the capsules

produced from 1.6, 1.8 and 2.0% (w/v) alginate was recorded before and after

coating with alginate and PLL. The enzyme activity ranged from 84.4 to 85.9 LAPU

for alginate and 86.1 to 87.0 LAPU for PLL-coated Flavourzyme microcapsules

(Figure 3.7). There was no significant difference (p > 0.05) in the enzyme activity of

capsules produced from 1.6, 1.8 and 2.0% (w/v) alginate or those coated in alginate

or PLL.

3.4.6 Optimisation of gelling duration

Alginate form gel on contact with cations and is important for efficient entrapment.

Properties of alginate microcapsules are influenced by the concentration of cationic

solution in which the gel is formed. Hence varying concentrations of chitosan were

studied for efficient Flavourzyme encapsulation in uncoated and PLL-coated

capsules. Flavourzyme activity decreased with the increase in gelling duration for

both uncoated (Figure 3.8a) and PLL-coated capsules (Figure 3.8b). The enzyme

retention in uncoated capsules produced from 1.6, 1.8 and 2.0 % (w/v) alginate was

not significantly different (p > 0.05) but increase in the gelling duration of 10a, 20b

80
 Enzyme encapsulation

and 40c min caused significant (F2, 44 = 12.321, p < 0.01) reduction in enzyme

retention.

The enzyme activity of PLL-coated capsules produced from 1.6 % alginate was

significantly different (F2, 44 = 33.742, p < 0.01) from those produced from 1.8 or

2.0% alginate. The enzyme retention in capsules allowed to gel for 10a, 20ab and 40b

min was significantly different (F2, 44 = 10.059, p < 0.01), means followed by

different superscript letter are significantly different.

81
 Enzyme encapsulation

80
72.4
69.7 70.3
70
62.5
EE of flavourzyme (%)

60

50

40

30

20
1.4 1.6 1.8 2.0
Alginate concentration (% w/v)

Figure 3.6 Effect of alginate concentration on encapsulation efficiency of

Flavourzyme gel beads formed in 0.1M CaCl2 containing 0.1% (w/v) chitosan.
Error bar indicates standard error of mean.

82
 Enzyme encapsulation

95

90

Flavourzyme activity (LAPU/mL) 85

80

75

70

65

60

55

50
1.4 1.6 1.8 2.0
Alginate Concentration (% w/v)

Alginate PLL

Figure 3.7 Flavourzyme activity in capsules produced from 2% (w/v) alginate

allowed to gel in cationic solution of 0.1M CaCl2 containing 0.1% (w/v) chitosan
and coated with 0.15% alginate and 0.05% poly-L-lysine for 10 min. Error bars
indicate standard error of mean.

83
 Enzyme encapsulation

90 110

105
Flavourzyme activity (LAPU/mL)

Flavourzyme activity (LAPU/mL)

85
100

80
95

90
75

85
70
80

65 75
10 20 40 10 20 40
Gelling duration (min) Gelling duration (min)

1.6 1.8 2.0 1.6 1.8 2.0

(a) (b)

Figure 3.8 Effect of gelling duration in 0.1M CaCl2 containing 0.1% (w/v) chitosan on Flavourzyme retention in (a) uncoated and (b)
poly-L-lysine coated capsules made from 1.6, 1.8 and 2.0% (w/v) alginate. Error bar indicates standard error of mean.

84
 Enzyme encapsulation

3.4.7 Optimisation of tri-sodium citrate concentration for in vitro release of

encapsulated enzyme

Activity of encapsulated enzyme can only be measured upon complete release from

the capsules. Encapsulated Flavourzyme was released in vitro by dissolution of

capsules in tri-sodium citrate solution. Complete dissolution of the capsules and

subsequent release of entrapped enzyme is greatly influenced by the concentration of

tri-sodium citrate solution; hence 0.5, 1.0 and 2.0% (w/v) tri-sodium citrate were

studied for complete in vitro release of encapsulated Flavourzyme. Flavourzyme

release from both uncoated (Figure 3.9a) and PLL-coated capsules (Figure 3.9b)

increased with increase in tri-sodium citrate concentration. There was significant

interaction (F4, 44 = 892.830, p < 0.01) between the concentration of alginate and

concentration of tri-sodium citrate solution hence the differences between buffers

were tested for each concentration of alginate separately.

In uncoated capsules produced from 1.6 and 2.0 % alginate, the enzyme released

from 0.5% tri-sodium citrate was significantly lower (1.6% tri-sodium citrate; F2, 14 =

986.560, p < 0.01) (2.0% tri-sodium citrate; F2, 14 = 10320.078, p < 0.01) than that

released from 1.0 and 2.0% tri-sodium citrate but there was no significant difference

(p > 0.05) in the enzyme released from 1.0 and 2.0% tri-sodium citrate. Where as for

the capsules produced from 1.8% alginate the enzyme released from 0.5a, 1.0b and

2.0c % (w/v) tri-sodium citrate was significantly different (F2, 14 = 4381.913, p <

0.01), means followed by different superscript letter are significantly different.

In PLL-coated capsules produced from 1.6, 1.8 and 2.0% (w/v) alginate, the enzyme

released from 0.5% tri-sodium citrate was significantly different (1.6% alginate; F2, 14

= 30.572, p < 0.01), (1.8% alginate; F2, 14 = 23.146, p < 0.01), (2.0% alginate; F2, 14 =

30.288, p < 0.01) from that released from 1.0 and 2.0% tri-sodium citrate while there

85
 Enzyme encapsulation

was no significant difference (p > 0.05) in the enzyme released from 1.0 and 2.0%

tri-sodium citrate.

3.4.8 Shelf life of freeze-dried, air-dried and frozen Flavourzyme

microcapsules

Flavourzyme encapsulated in 1.6, 1.8 and 2.0% alginate by gelling in 0.1M CaCl2

solution containing 0.1% chitosan, both uncoated and PLL-coated were stored in

three ways; freeze-drying, air-drying and freezing and the effect of these processes

and shelf-stability was evaluated by monitoring the activity of encapsulated enzyme

over a 10 week storage period. Uncoated capsules from 1.6 (Figure 3.10i), 1.8

(Figure 3.10ii) and 2% alginate (Figure 3.10iii), stored at 4°C after freeze-drying and

at -20°C after freezing retained over 80% of the initial enzyme activity while those

stored at 4°C after air-drying retained only about 50% of the initial enzyme activity.

In PLL-coated capsules from 1.6, 1.8 and 2.0% alginate a similar trend was

observed, freeze-dried and frozen capsules retained over 80% of the initial enzyme

activity (Figure 3.10iv, v and vi) while air-dried capsules from 1.6 % alginate

retained about 34% (Figure 3.10iv), from 1.8% alginate retained about 32% (Figure

3.10v) and from 2.0% alginate retained only about 25% (Figure 3.10vi) of the initial

enzyme activity. Loss of enzyme activity appears to occur mainly during the process

of air-drying.

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 Enzyme encapsulation

3.4.9 Morphology of uncoated and poly-L-lysine coated Flavourzyme

microcapsules

Microscopic examination of Flavourzyme capsules showed mainly spherical

capsules for both uncoated and PLL-coated capsules while the number of non-

spherical capsules was slightly higher in PLL-coated capsules (Table 3.2).

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 Enzyme encapsulation

120 120

100 115

Flavourzyme activity (LAPU/mL)

Flavourzyme activity (LAPU/mL)

80 110

60 105

40 100

20 95

0 90
0.5 1.0 2.0 0.5 1.0 2.0
Sodiumcitrate concentration (% w/v) Sodiumcitrate concentration (% w/v)

1.6 1.8 2.0 1.6 1.8 2.0

(a) (b)

Figure 3.9 Effect of tri-sodium citrate concentration on in vitro release of encapsulated Flavourzyme from (a) uncoated and (b) poly-L-
lysine coated capsules produced from 1.6, 1.8 and 2.0 % (w/v) alginate and allowed to gel in 0.1M CaCl2 containing 0.1% (w/v) chitosan.
Error bar indicates standard error of mean.

88
 Enzyme encapsulation

90 90 90

Flavourzyme activity (LAPU/mL)

Flavourzyme activity (LAPU/mL)
80 80
Flavourzyme activity (LAPU/mL)

80
70 70 70

60 60 60

50 50 50

40 40 40

30 30 30

20 20 20

10 10 10

0 0 0

10
10
zero hrs

9
zero hrs
Initial

9
zero hrs

Initial
Initial
10
1

Storage duration (weeks) Storage duration (weeks) Storage duration (weeks)

a 1.6 b 1.6 c 1.6 a 1.8 b 1.8 c 1.8 a 2.0 b 2.0 c 2.0

(i) (ii) (iii)

Figure 3.10 Shelf life of Flavourzyme in uncoated capsules immobilised in 1.6 (i), 1.8 (ii) and 2.0 % (w/v) (iii) alginate by gel bead
formation in 0.1M CaCl2 containing 0.1% (w/v) chitosan, stored by freeze-drying (a), air-drying (b) and freezing (c) over a period of 10
weeks. Error bar indicates standard deviation

89
 Enzyme encapsulation

90 90 90
Flavourzyme activity (LAPU/mL)

Flavourzyme activity (LAPU/mL)

Flavourzyme activity (LAPU/mL)

80 80 80
70 70 70
60 60 60
50 50 50
40 40 40
30 30 30
20 20 20
10 10 10
0 0 0

10
10

9
1

zero hrs
zero hrs

Initial

zero hrs
Initial

Initial

10
1

9
Storage duration (weeks) Storage duration (weeks) Storage duration (weeks)

a 1.6 b 1.6 c 1.6 a 1.8 b 1.8 c 1.8 a 2.0 b 2.0 c 2.0

(iv) (v) (vi)

Figure 3.10 Shelf life of Flavourzyme in poly-L-lysine coated capsules immobilised in 1.6 (iv), 1.8 (v) and 2.0 % (w/v) (vi) alginate by gel
bead formation in 0.1M CaCl2 containing 0.1% (w/v) chitosan, stored by freeze-drying (a), air-drying (b) and freezing (c) over a period
of 10 weeks. Error bar indicates standard deviation

90
 Enzyme encapsulation

Table 3.2 Size and shape of alginate Flavourzyme capsules allowed to gel in
0.1M CaCl2 containing 0.1% (w/v) chitosan (mean ± standard deviation,
average of 20 replicates)

Treatment Size (in µm) Shape

Alginate Range Mean ± SD Spherical Tear Pear Oval

concentration (% drop

w/v)

Uncoated

1.6 410-520 501 ± 32.8 +++ + ++ +

1.8 500-530 511 ± 11.0 +++ + + +

2.0 510-570 541 ± 19.1 +++ + - +

0.05% PLL coated

1.6 440-500 476 ± 21.1 +++ ++ + ++

1.8 460-550 496 ± 25.9 +++ ++ + ++

2.0 460-620 529 ± 42.5 +++ ++ - +

91
 Enzyme encapsulation

3.5 Discussion

Flavourzyme and Palatase were the two enzymes used in this study for acceleration

of Cheddar cheese ripening; all the encapsulation and optimisation studies were

performed with Flavourzyme which has similar molecular weight to Palatase

(communication from Novozymes). Alginate, chitosan and -Carrageenan were

studied for encapsulation of Flavourzyme. It was not possible to extrude chitosan

through the 300µm nozzle of the encapsulator even at a concentration of 0.1 % (w/v)

hence it was eliminated as a suitable biomaterial for enzyme encapsulation. Another

food grade polymer, -Carrageenan was also studied for Flavourzyme encapsulation

as -Carrageenan is known to form hard gels compared to weak gels from -

Carrageenan (Velde et al., 2002). Being a thermo reversible gel it was only possible

to extrude -Carrageenan through the encapsulator nozzle by maintaining a steady

temperature of 70°C as it formed gel easily upon contact with cooler surfaces of the

encapsulator even at a low concentration of 0.1 % (w/v). This high temperature for

long duration considerably reduces the activity of Flavourzyme (Flavourzyme

product sheet, 2004) hence -Carrageenan was also eliminated as a suitable polymer

for enzyme encapsulation using the Inotech® encapsulator. Alginate was found to be

a suitable polymer for enzyme encapsulation using the encapsulator; hence all

experiments in the current study were performed on alginate based encapsulation.

Sodium salt of alginic acid from Sigma (Sydney, Australia) and Protanal (Drammen,

Norway) were used to encapsulate Flavourzyme using the Inotech® encapsulator

followed by capsule gelling in 0.1M CaCl2, alginate from Sigma showed lower

viscosity compared to alginate from Protanal (data not shown) hence alginate from

Sigma was used for all further experiments. EE of Flavourzyme encapsulated in

alginate ranged from 16 to 18% between 1.4 and 2.0 % (w/v) alginate (Figure 3.2)

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 Enzyme encapsulation

with no significant increase (p > 0.05) in EE with increase in alginate concentration.

Low EE was due to leakage of Flavourzyme from capsules during encapsulation.

Pore size of alginate gels have been studied by various methods. Pore size

distribution in 3% alginate gel examined by transmission electron microscope was

found to be in the range 100-1000 with a distribution maxima at 250 (Smidsrod et

al., 1972). Klein et al. (1983) reported pore size of Ca-alginate gels to be about ~7 to

~17nm depending on the gel type. Fundueanu et al. (1999) estimated pores and

porous volume of micro particles in Ca-alginate to have a mean diameter of 220µm

when obtained by emulsion method and 1.2mm when obtained by dripping method.

Alginate gel beads produced using the Inotech® encapsulator have been reported to

allow molecules in the weight range of 1000kDa (Inotech® website, 2004). Based on

these reports it can be said that pores in alginate matrix is significantly large

compared to Flavourzyme molecules with molecular weight ~30kDa

(communication from Novozymes) and thus causing excessive leakage and poor EE.

Low EE can also be due to Flavourzyme loss as a result of syneresis during gel bead

formation. Flavourzyme being a water soluble enzyme was lost by diffusion into the

gelling cationic solution during gel bead formation. Water loss from gel beads due to

syneresis was found to be 41 to 37% for alginate concentration ranging from 1.4 to

2.0% (Table 3.1) in agreement with the findings of Pommersheim et al., (1994), who

reported 34% loss of enzyme by diffusion into solution during the formation of gel

beads and Dashevsky (1998) who reported 36% protein loss due to diffusion and

44% water leakage during cross linking of alginate and calcium cations. In the

current study, increase in alginate concentration lead to decreased water loss and in

turn better retention of enzyme in gel beads.

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 Enzyme encapsulation

Increased EE of folic acid on immobilization in a mixture of alginate-pectin

(Madziva et al., 2006) and increased cell encapsulation upon incorporation of Hi-

Maize starch during Ca-alginate encapsulation (Sultana et al., 2000) have been

reported. Similarly in the current study Flavourzyme was immobilised in alginate-

starch (Hi-Maize) and alginate-pectin mixtures in varying concentrations and

proportions followed by gelling in 0.1M CaCl2 solution. However, EE of

Flavourzyme was lower than that obtained when encapsulated in alginate alone, ~7-

12% for alginate-starch (Hi-Maize) and ~9% for alginate-pectin (Figure 3.3 & 3.4).

Though EE of Flavourzyme significantly increased (p < 0.05) with the increase in

alginate and starch (Hi-Maize) concentration, this polymer mixture could not be used

due to relatively high viscosity for extrusion through the encapsulator nozzle. Low

EE of Flavourzyme in alginate-starch (Hi-Maize) and alginate-pectin may be due to

insufficient blocking of the pores in alginate matrix thus causing excessive leakage

during encapsulation and gelling. Low EE can also be attributed to possible

interference of starch and pectin with the Flavourzyme assay leading to lower

activity, in agreement with the report of interaction of lipase with alginate leading to

poor EE (Betigeri and Neau, 2002). Based on screening results EE of Flavourzyme

was superior on encapsulation in alginate alone, hence alginate was chosen as the

preferred polymer.

Flavourzyme capsules with low EE was not an efficient means of enzyme

incorporation into cheese. Cationic solution was modified in an attempt to enhance

EE. Sezer and Akbuga (1999) reported increased drug loading capacity of alginate

capsules on addition of 0.3 % chitosan to the gelling solution. In the current study

addition of chitosan to cationic solution of 0.1M CaCl2 significantly increased (p <

0.05) the EE to 72.21, 76.28 and 84.0% for 0.1, 0.2 and 0.3% chitosan respectively

94
 Enzyme encapsulation

(Figure 3.5) for Flavourzyme immobilized in 2% alginate and gelling for 10 min.

Chitosan reacts with alginate as a cation with an anion, similar to CaCl2. As chitosan

is a larger molecule compared to calcium ions, it is more efficient in blocking the

pores in alginate matrix when combined with CaCl2 compared to CaCl2 alone.

Binding of chitosan to alginate is said to increase with decrease in degree of n-

acetylation and addition of calcium ions (Gaserod et al., 1998). Chitosan, minimum

85% deacetylated in 0.1M CaCl2 solution was used in this study, enhanced EE of

Flavourzyme upon treatment of cationic solution of 0.1M CaCl2 can be attributed to

increased binding of chitosan to alginate network thus blocking the pores. Therefore

by treating the capsules with chitosan it was possible to reduce the pore size and

consequently minimising leakage. Dashevsky (1998) reported weight loss of

capsules due to shrinkage and syneresis during cross linking of guluronate with

calcium cations, water loss decreased with increase in alginate concentration.

Similarly in the current study, capsule weight increased with increase in alginate

concentration from 1.4 to 2.0%. Water loss of ~37% was recorded for capsules

produced from 2% alginate when gel bead was formed in the cationic solution of

0.1M CaCl2 while water loss of 23, 15 and 12 % was recorded for capsules produced

from 2% alginate with gel bead formation in cationic solution of 0.1M CaCl2

containing 0.1, 0.2 and 0.3% chitosan respectively (Table 3.1). These results show

that gelling of capsules in 0.1M CaCl2 solution containing chitosan can minimise

bead shrinkage and water loss inturn leading to enhanced enzyme retention. Though

there was a significant (p < 0.001) yet small increase in the EE of Flavourzyme with

the increase in chitosan concentration, 0.1% (w/v) was chosen to be optimal as 0.2

and 0.3% (w/v) chitosan in gelling solution were too viscous for further application

95
 Enzyme encapsulation

causing clumping or surface aggregation of capsules during formation, which would

obstruct even distribution of capsules in the cheese matrix.

The next step in improving Flavourzyme encapsulation was selection of polymers for

efficient robust process. Alginate solutions of concentrations 1.4, 1.6, 1.8 and 2.0%

(w/v) were studied by immobilising Flavourzyme and gelling for 10 min in 0.1M

CaCl2 containing 0.1% (w/v) chitosan. There was a significant increase (p < 0.05) in

EE of Flavourzyme on increasing the concentration of alginate from 1.4 to 1.6 %,

however no significant increase (p > 0.05) in EE was observed upon increasing

alginate concentration beyond 1.6% (Figure 3.6), unlike the reports of Blandino et al.

(2000), who reported reduced glucose oxidase leakage on increasing the alginate

concentration. This disagreement in results may be due to the difference in the nature

of the enzymes and gelling solution employed in the two studies and also possibly

due to the constant size of the capsules produced from the same nozzle resulting in

similar surface area in capsules from different concentration and hence similar

enzyme retention capacity of the capsules. Consequently 1.6, 1.8 and 2.0% alginate

were used for further study.

Chitosan and PLL are the most widely used poly-cations for capsule production.

Coating of alginate capsules with polycations like PLL has shown to improve

mechanical stability of capsules (Lim and Moss, 1981; Liu and Krishnan, 1999,

Ouyang et al., 2004). Gugerli et al. (2002) reported < 150 kDa permeability of

capsules produced from 1.5% alginate coated with 0.05% (w/v) PLL (30-70kDa

molecular weight, from Sigma) suggesting that PLL-coating of alginate capsules can

minimise the porosity. Coating of calcium alginate capsules with chitosan-alginate

polyelectrolyte complex has been shown to reduce permeability (Peniche et al.,

2004). As the capsules used in the current study are formed in chitosan treated

96
 Enzyme encapsulation

cationic solution, alginate was considered as a suitable polymer for coating in

addition to PLL and both were used for coating of Flavourzyme capsules in an

attempt to minimise enzyme leakage and to improve mechanical stability of capsules.

However, initial Flavourzyme activity in capsules decreased from ~ 90 to 85 and 87

LAPU after 10 min of 0.15% alginate and 0.05% poly-L-lysine coating respectively

(Figure 3.7) indicating continued leakage of Flavourzyme from the capsules. Thus

there was no further improvement in capsule permeability upon coating. Poly-L-

lysine coated and uncoated capsules were used in further experiments to test stability,

as clumping was observed with alginate-coated capsules. Aggregation of alginate-

coated capsules could be due to interaction of residual chitosan on the surface of the

capsules with alginate, and this may cause uneven distribution of capsules in a

cheese matrix.

Duration of capsule gelling was optimised to obtain efficient enzyme micro-capsules.

Enzyme recovery significantly (p < 0.001) decreased with increase in gelling

duration of uncoated capsules from 10, 20 and 40 min (Figure 3.8a) where as in PLL-

coated capsules (Figure 3.8b), the enzyme retention in capsules allowed to gel for 10

min was significantly higher (p < 0.001) than those allowed to gel for 40 min while a

gelling time of 20 min had no significant effect (p > 0.05) on enzyme retention

compared to 10 and 40 min gelling duration. Decrease in enzyme retention in the

capsules upon increasing the gelling time may be due to continued diffusion/leakage

of Flavourzyme into the gelling solution.

In PLL-coated capsules (Figure 3.8b), as the gelling progressed from 10 to 40 min,

the recovery of Flavourzyme activity of capsules produced from 1.8 and 2 % alginate

was significantly higher (p < 0.001) than those produced from 1.6 % alginate

suggesting better interaction of PLL with alginate at higher concentrations of alginate

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 Enzyme encapsulation

and thus resulting in efficient blocking of pores and better retention of encapsulated

enzyme.

Complete in vitro release of encapsulated enzyme is essential for the measurement of

enzyme activity. Tri-sodium citrate was used for dissolution of capsules for in vitro

release of encapsulated enzyme from uncoated and PLL-coated capsules.

In uncoated capsules (Figure 3.9a) produced from 1.6 and 2% alginate, the release of

the encapsulated enzyme significantly increased (p < 0.001) with the increase in tri-

sodium citrate concentration from 0.5 to 1.0% however there was no significant

increase (p > 0.05) in enzyme release on increasing the tri-sodium citrate

concentration beyond 1%. whereas for the capsules produced from 1.8% alginate, the

release of encapsulated enzyme significantly increased (p < 0.001) with increase in

tri-sodium citrate concentration from 0.5 to 1.0 to 2.0% indicating that higher

concentrations of buffer is required for complete release of the encapsulated enzyme

from capsules made from higher concentrations of alginate. However, this trend

doesn’t continue beyond the alginate concentration of 1.8%, probably due to

relatively high concentration of alginate for dissolution by tri-sodium citrate.

In PLL-coated capsules (Figure 3.9b) produced from 1.6% alginate, there is no

significant difference (p > 0.05) in the release of encapsulated enzyme on increasing

the tri-sodium citrate concentration from 0.5 to 1.0% while an increase in tri-sodium

citrate concentration from 0.5 to 2.0% lead to a significant increase (p < 0.001) in the

release of encapsulated enzyme. Whereas for capsules produced from 1.8 and 2%

alginate there was no significant increase (p > 0.05) in the release of encapsulated

enzyme on increasing the tri-sodium citrate concentration beyond 1.0%. These

results suggest that at higher concentration of alginate, relatively lower concentration

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 Enzyme encapsulation

of tri-sodium citrate is sufficient for release of encapsulated enzymes from capsules

coated in PLL unlike uncoated capsules.

Uncoated and PLL-coated Flavourzyme capsules produced from 1.6, 1.8 and 2.0%

alginate were stored at 4°C after freeze-drying and air-drying and at -20°C after

freezing and the shelf-stability of the stored capsules was monitored over 10 weeks

by measuring the enzyme activity every week. In both uncoated and PLL-coated

capsules from all concentration of alginate stored frozen or freeze-dried retained over

80% of the initial enzyme activity at a steady rate over a 10-week period (Figure

3.10). Uncoated capsules stored after air-drying retained only about 50% of the

initial enzyme activity (Figure 3.10i, ii and iii) with most of the enzyme activity loss

occurring during the process of air-drying. A similar trend was observed in PLL-

coated air-dried capsules where only 25-34% of the initial enzyme activity was

retained over a 10-week storage period with majority of enzyme activity loss

occurring during air-drying (Figure 3.10iv, v and vi). It can be concluded that

Flavourzyme is highly sensitive to air-drying at room temperature and may lead to

irreversible loss of enzyme activity. In air-dried capsules loss of enzyme activity

increases with increase in alginate concentration, though more pronounced in PLL-

coated capsules this trend was also observed in uncoated capsules. This could be due

to the high surface tension during air-drying and also possibly due to the hydrophilic

nature of alginate as also reported by Betigeri and Neau (2002), who observed an

increase in leaching of lipase with increase in alginate concentration and

hypothesised that it could be due to the hydrophilic nature of the polymer.

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 Enzyme encapsulation

3.6 Conclusion

Alginate was found to be a suitable polymer for enzyme encapsulation using the

encapsulator. Enzyme encapsulation in alginate alone and also in polymer mixtures

of alginate-starch and alginate-pectin followed by gelling in 0.1M CaCl2 resulted in

poor encapsulation efficiency. Flavourzyme immobilization in alginate by gel bead

formation in cationic solution of 0.1M CaCl2 containing 0.1% (w/v) chitosan for 10

min is an efficient and novel means of enzyme encapsulation for acceleration of

cheese ripening. Alginate concentration ranging from 1.6 to 2.0% (w/v) is suitable

for Flavourzyme encapsulation. There was no enhancement in Flavourzyme retention

in capsules coated with alginate or poly-L-lysine. Flavourzyme microcapsules are

shelf stable for at least 10 weeks when stored after freezing or freeze-drying hence

suitable for large scale applications.

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4 APPLICATION OF ENCAPSULATED ENZYMES IN

CHEDDAR CHEESE MANUFACTURE

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 Application of enzyme capsules into cheese

4.1 Abstract

Flavourzyme-alginate hydrogel beads produced from 1.6, 1.8 and 2.0% (w/v)

alginate allowed to gel in chitosan treated CaCl2 solution were evaluated for stability

under simulated conditions of cheese manufacture prior to application in cheese for

acceleration of ripening. Flavourzyme leakage from capsules was tested in milk at

pH 5.0 & 6.0 and at temperatures 32 & 38°C for 10 min. At 38°C, Flavourzyme

retention in capsules was found to be significantly lower (p < 0.001) in capsules

produced from 1.6% alginate compared to those from 1.8 or 2.0% and significantly

lower (p < 0.001) at pH 6.0 compared to pH 5.0. While at 32°C, the concentration of

alginate had no effect on enzyme retention in the capsules. Flavourzyme capsules

tested under simulated cheese press showed considerable mechanical stability by

retaining over 70% of the encapsulated enzyme under a pressure of 0.79Ncm-2 for

16h. Leakage of encapsulated Flavourzyme increased with the duration of simulated

cheese press from 4 to 16h and was highest in capsules produced from 1.6% alginate.

Addition of enzyme capsules to milk prior to rennetting resulted in an even

distribution of the capsules compared to the aggregation of capsules, when added to

milled curd prior to salting. Cheddar cheese manufactured with alginate capsules

containing low, medium and high load of Flavourzyme and medium load of Palatase

and control cheese with no added enzymes showed higher levels of moisture and

lower levels of fat compared to standard Cheddar cheese. Cheeses (control and

experimental) produced for grading were tested for microbiological quality. Addition

of free or alginate-encapsulated Flavourzyme or Palatase had no effect on the

pathogen levels in cheese as there was no difference in numbers of coliforms, E.coli,

Salmonella, Listeria, coagulase positive staphylococci, Bacillus cereus, yeast and

moulds in control or experimental cheeses.

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 Application of enzyme capsules into cheese

4.2 Introduction

Cheese is one of the most widely consumed dairy products with Cheddar being the

most popular variety in Australia as natural Cheddar alone accounted for more than

54% of the total cheese supermarket sales in 2005/2006 (Anon, 2007d). Cheddar is a

semi-hard variety that is consumed after a period of ripening ranging from few

months to three years implying increased production costs due to refrigeration,

maintenance and low yield due to contamination and syneresis. Accelerated ripening

can potentially reduce the production costs and any attempt at reducing the ripening

time and yet maintaining the quality of cheese is appreciated and has attracted

attention of many researchers (Cliffe and Law, 1990).

Though several techniques such as elevation of ripening temperature (Folkertsma et

al., 1996; Hannon et al., 2005), high pressure processing (O’Reilly et al., 2003;

Garde et al., 2007), genetic modification of starters (Upadhyay et al., 2004b) and

addition of attenuated starters (Upadhyay et al., 2007) or adjunct cultures (Ong et al.,

2007a, 2007b) have resulted in increased proteolysis or lipolysis and enhanced

flavour formation, not many have been successful at accelerating cheese ripening at a

commercial scale. Exogenous addition of enzymes that catalyse the biochemical

reactions occurring during ripening appears to be a simple approach and a range of

enzymes have been tested for this purpose. Though exogenous addition of enzymes

caused increased proteolysis, enhanced flavour formation, reduction/elimination of

bitterness; premature extensive proteolysis, bitterness and development of off-

flavours were also reported by some groups in addition to whey contamination.

Addition of exogenous enzymes to milk during cheese manufacture is inefficient as

~90% of enzyme added to milk is lost at whey drainage (Law and Kirby, 1987) in

addition to premature extensive proteolysis and development of off-flavours (Skeie,

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 Application of enzyme capsules into cheese

1994). Addition of enzymes at dry-salting during cheese manufacture can also be

disadvantageous owing to the possibility of uneven distribution of the small amount

of enzymes in the large blocks of curd (Wilkinson and Kilcawley, 2005).

Enzyme encapsulation was the suitable alternative for delivery of enzymes into

cheese; it was first reported by Magee et al. (1981) who observed increased flavour

compounds in cheese due to addition of cell free extracts encapsulated in milk fat,

these capsules disrupted at 35°C. Later developments included capsules able to

withstand higher temperature (Braun and Olson, 1984) and capsules from alternative

materials such as hydrogels (Perols et al., 1997), food gums (Kailasapathy et al.,

1998) and liposomes (Law and King, 1985; Kirby et al., 1987; Skeie et al., 1995;

Picon et al., 1995, 1997; Laloy et al., 1998; Kheadr et al., 2000, 2003). Through

intense research by several groups, liposomes were developed for efficient enzyme

encapsulation (El Soda et al., 1989; Skeie, 1994; Dufour et al., 1996) for delivery

into cheese for acceleration of ripening. However, liposomes are expensive

ingredients and there are no known reports of commercial application of liposome-

encapsulated enzymes for acceleration of cheese ripening.

Though most studies report accelerated proteolysis, very few report enhancement and

acceleration of flavour development during ripening. Kheadr et al., (2000)

recommended addition of enzymes encapsulated in liposomes obtained from Pro-lipo

for acceleration of cheese ripening with the load of bacterial proteinase up to 1 X 10-5

AU/g cheese curd and fungal proteinase up to 1 X 10-6 AU/g cheese curd for

acceleration of flavour development in Cheddar cheese without any flavour or

textural defects. An increase in moisture content of cheeses due to the addition of

liposome (Kheadr et al., 2000) or food gum (Kailasapathy and Lam, 2005)

encapsulated enzymes have been reported.

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 Application of enzyme capsules into cheese

Addition of Flavourzyme an enzyme complex with endoprotease and exopeptidase

activity with Palatase a fungal lipase, both individually encapsulated in liposomes

during cheese manufacture lead to increased and balanced flavour development

without any bitterness, off flavours or rancidity (Kheadr et al., 2003).

Earlier studies on enzyme encapsulation for acceleration of cheese ripening involved

addition of encapsulated enzymes to milk during cheese manufacture. Picon et al.

(1997) reported 64-75 % retention of liposome encapsulated enzymes in cheese when

added to milk during manufacture while Kailasapathy and Lam (2005) reported over

90% retention of -Carrageenan and gellan encapsulated enzymes in cheese when

added to milk. Addition of neutral protease from Aspergillus oryzae to the curd at the

milling stage during Cheddar cheese manufacture resulted in increased proteolysis

(Fedrick et al., 1986). Kheadr et al. (2000, 2003) also reported addition of liposome

encapsulated enzymes to milk before cheese manufacture for acceleration of

ripening. Stage of addition of folic acid capsules into Cheddar cheese was evaluated

by Madziva et al. (2006). Addition of alginate-pectin encapsulated folic acid to milk

early during cheese manufacture resulted in even distribution while addition to

milled curd and injection into pressed cheese block lead to poor distribution with

capsule clusters.

Technique for microencapsulating flavour enhancing enzymes efficiently developed

earlier in section 3.0 was used to immobilise Flavourzyme and Palatase; flavour-

enhancing enzymes by Novozymes. This study was designed to test the suitability of

the enzyme microcapsules developed for application in Cheddar cheese for

acceleration of ripening.

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 Application of enzyme capsules into cheese

4.3 Materials and methods

Flavourzyme (Novozymes, Sydney, Australia) 2mL was dissolved in 8mL ZnCl2

(Sigma, Sydney, Australia) solution prepared with 0.15% (v/v) brij (Sigma, Sydney,

Australia) and mixed with 90mL alginate (Sigma, Sydney, Australia) solution to give

a final Flavourzyme concentration of 2% (v/v) and alginate concentration of 1.8%

(w/v). This mixture was extruded through a 300µm nozzle of the Inotech®

encapsulator IE 50R (Inotech, Dottikon, Switzerland) and the droplets were allowed

to gel in 0.1M CaCl2 (Sigma, Sydney, Australia) containing 0.1% (w/v) chitosan

(Sigma, Sydney, Australia) for 10 min. After gelling the enzyme microcapsules were

washed with Milli Q water (Massachusetts, USA) and used for experiments in this

section unless mentioned otherwise.

4.3.1 Leakage of encapsulated Flavourzyme in milk

Flavourzyme capsules (5g) from 1.6, 1.8 and 2.0% (w/v) alginate were suspended in

50mL milk at pH 5.0 and pH 6.0 and both held at 32°C and 38°C for 10 min in a

water bath to assess the loss of encapsulated Flavourzyme in milk during cheese

manufacture. Milk pH was adjusted using lactic acid (Sigma, Sydney, Australia). The

capsules were harvested from milk and washed with Milli Q water before 1g was

dissolved in 2.0% (w/v) tri-sodium citrate (Sigma, Sydney, Australia) and

Flavourzyme activity was measured as described in section 3.3.1. Initial and final

Flavourzyme activity was measured.

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 Application of enzyme capsules into cheese

4.3.2 Stability of encapsulated Flavourzyme under simulated cheese press

conditions

Flavourzyme capsules (30g) produced from 1.6, 1.8 and 2.0% alginate were

subjected to simulated cheese press pressure of 0.79Ncm-2 (a force similar to that

applied to cheese blocks during cheese press) for 4, 8 and 16h using the Texture

analyser TA-XT2 (Stable Micro Systems, Surrey, UK) to test the enzyme leakage

during simulated cheese press. As the capsules were compressed, the leaked enzyme

escaped into the space above the syringe plunger through the serrated edges allowing

the liquid while retaining the capsules. The leaked enzyme was separated from the

capsules by simply siphoning out the liquid above, and the capsules were collected

and 1g was dissolved in tri-sodium citrate to release and measure the activity of

Flavourzyme retained in the capsules. Flavourzyme activity before and after treating

to the simulated cheese press was measured.

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 Application of enzyme capsules into cheese

Figure 4.1 Application of simulated cheese press pressure on Flavourzyme

capsules

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 Application of enzyme capsules into cheese

4.3.3 Cheddar cheese manufacture with microencapsulated Flavourzyme and

Palatase

Cheddar cheese was manufactured in the Armfield cheese vat (Ringhood, England)

from 10L milk; 9L pasteurised skim milk combined with 1L fresh cream (35% milk

fat, unhomogenised) from the supermarket. Skim milk, 1L was combined with 1L

fresh cream and heated to 45-47°C in a water bath with constant stirring and blended

with a “House and Home” HHMM4 Multimixer (Sydney, Australia) for ~3 min and

mixed with 8L skim milk to yield ~3.5% fat calculated using the Pearson square

method. This 10L milk was heated to 31°C with constant stirring in the cheese vat

followed by addition of CaCl2 (50%; Cheeselinks, Victoria, Australia), 2mL diluted

to 20mL with sterile Milli Q water. Starter culture Delvo-Tec LL 50C direct set, deep

frozen cultures of Lc. lactis ssp cremoris and/or Lc. lactis ssp lactis (DSM Food

specialities, Sydney, Australia) was added at the rate of 1.85 units/1000L or 0.0185

units/10L without prior activation. Calf rennet 290 IU/mL (Cheeselinks, Victoria,

Australia), 2.5mL diluted to 25mL in sterile Milli Q water was added 10 min after

starter addition and the stirring was stopped after a min and the vat was left

undisturbed for 40 min. Curd was cut and left undisturbed for 10 min followed by

heating to 38°C over 55 min with gentle stirring until the pH reached 6.40 to 6.45,

followed by drainage of whey. The whey was filtered using a cheese cloth to collect

cheese curd and any capsules lost in the whey and added back to the vat. The curd

was banked up against the walls of the vat for 20 min allowed to settle and cut into

large blocks and turned every 20 min while maintaining the temperature of the curd.

Once the pH dropped to 5.40 to 5.45 the curd was cut into 1cm cubes and mixed well

with 25g salt and allowed to stand for 10min. followed by hooping and pressing

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 Application of enzyme capsules into cheese

overnight under 8kg weights. After about 16h of pressing the curd was sliced and

vacuum packed and allowed to ripen at 9°C.

Enzyme capsules were added either to the milk before rennetting or to the milled

curd before salting to determine the right stage of capsule addition.

Nine batches of Cheddar cheese were manufactured in triplicates. Low (0.5 LAPU/g

milk protein), medium (0.75 LAPU/g milk protein) and high (1.0 LAPU/g milk

protein) levels of Flavourzyme with Palatase (0.5LU/g milk fat) were added to milk

before rennetting, in free and encapsulated form, these included;

1) Cheese with no added enzymes-control, C

2) Cheese with added free Flavourzyme (medium), D

3) Cheese with added encapsulated Flavourzyme (low), F50

4) Cheese with added encapsulated Flavourzyme (medium), F75

5) Cheese with added encapsulated Flavourzyme (high), F100

6) Cheese with added encapsulated Flavourzyme (low) and encapsulated

Palatase, PF50

7) Cheese with added encapsulated Flavourzyme (medium) and encapsulated

Palatase, PF75

8) Cheese with added encapsulated Flavourzyme (high) and encapsulated

Palatase, PF100

9) Cheese with added encapsulated Palatase, P

A, D, F75, PF75 and PF100 were again manufactured in duplicates at Dairy Farmers

Innovation center, Sydney for the purpose of grading.

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 Application of enzyme capsules into cheese

Figure 4.2 Flow process chart of Cheddar cheese manufacture (adapted from
Madziva, 2006)

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 Application of enzyme capsules into cheese

4.3.4 Effect of stage of capsule addition during cheese manufacture on the

distribution of enzyme capsules in cheese matrix

Flavourzyme, 2mL was mixed with alginate-starch solution to give a final

concentration of 1.8:1.0 (alginate:starch) and gel beads were produced by extruding

from 300µm nozzle of the encapsulator into 0.1M CaCl2 solution containing 0.1%

chitosan and allowed to gel for 10 min. Flavourzyme capsules were incorporated into

cheese at two stages of manufacture:

1) Flavourzyme capsules (25g) were added to 10L milk prior to rennetting and

the cheese block was cut into 50g cubes and vacuum packed.

2) Flavourzyme capsules (25g) were added to milled curd prior to salting and

the cheese block was cut into 50g cubes and vacuum packed.

The distribution of the capsules in Cheddar cheese was established by staining the

starch containing capsules with iodine. Olympus-SZH-ILK fitted with a digital

camera (Olympus Optical Co., Tokyo, Japan) and a Fibreoptic Illuminator-15001

(Fibreoptic Lightguides, Melbourne, Australia) was used to visualise the stained

capsules. The number of capsules counted were represented as the number of

particles and the area occupied by stained capsules on the surface of cheese slice was

established in terms of pixels using the software Image Pro Plus and the ratio of

number of pixels to the number of particles was also used to estimate the distribution

of capsules in addition to visual observation.

4.3.5 Analysis of Cheddar cheese

Control and experimental Cheddar cheeses manufactured were subjected to

following chemical analysis.

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 Application of enzyme capsules into cheese

4.3.5.1 Moisture

Moisture in cheese was determined by the oven drying method (AOAC 1990,

Method 926.08). A clean dry metallic container was filled 1/3rd with acid-washed

sand and weighed (m0). About 5g grated cheese was mixed with the sand and

weighed (m1) again and the mixture was moistened and heated on a hot plate and

transferred to an oven at 104°C for drying for about 20h. The container was cooled in

a desiccator and weighed (m2). Percentage moisture was calculated using the

following equation

Moisture (%) = X 100

4.3.5.2 Fat

Fat in cheese was determined by Babcock method (AOAC 1995, Method 989.04 and

989.10). Hot water (9mL) was added to 9g grated cheese followed by 0.5mL

Zephiran and mixed well. Concentrated H2SO4 was slowly added drop wise with

constant mixing until all the lumps were dissolved and the solution turned into a deep

brown/purple colour which was transferred into a Babcock flask and centrifuged for

5 min followed by addition of hot water till the neck of the flask and 2 min

centrifugation. Again hot water was added in order to read fat in the graduated

section of the flask and centrifuged for another min and the percentage of fat was

read from the fat corresponding to the graduations on the Babcock flask.

4.3.5.3 pH

Cheese block was pierced at least 1cm deep with a pointed pH probe and the pH was

read using the Inolab pH meter (Weilheim, Germany).

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 Application of enzyme capsules into cheese

4.3.5.4 Microbiological status of cheese samples

Cheese samples C, D, F75, PF75 and PF100 were manufactured in duplicates for

grading and were tested by Silliker Microtech (Sydney, Australia) for following

pathogens as per the corresponding Australian StandardTM (Anon, 2007a) or AOAC

methods;

Coagulase positive Staphylococci - AS 1766.2.4 - 1994

Coliforms AS 1766 - 1992

E. coli AS 1766 - 1992

Salmonella AOAC 996.08

Listeria AOAC 999.06

Bacillus cereus AS 1766.2.6 - 1991

Yeasts and moulds AS 1766.2.2 - 1997

4.3.5.5 Statistical analysis

All data presented are a mean of 6 replicates unless otherwise mentioned and

expressed with standard error of mean or standard deviation as mentioned. Data were

statistically analysed by SPSS® for WindowsTM Version 14 (SPSS Inc. 2005) for one

(two) factorial, general linear analysis of variance (ANOVA). The assumption of

normal distribution was checked using P-P plot and homogeneity of variance using

Levene’s test of equality of error variances. Treatments were tested against each

other using Ryan-Einot-Gabriel-Welsch Q test if the assumption of equality of

variance was met or Dunnett’s T-test if the assumption was not met. Standard

deviation was calculated using Microsoft Excel.

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4.4 Results

4.4.1 Stability of encapsulated Flavourzyme

Flavourzyme capsules produced from 1.6, 1.8 and 2.0% (w/v) alginate by gelling in

0.1M CaCl2 containing 0.1% (w/v) chitosan were assessed for stability in milk by

measuring the leakage of encapsulated Flavourzyme from capsules suspended in

acidified milk at pH 5.0 and 6.0 at 32°C (Figure 4.3a) and 38°C (Figure 4.3b),

conditions similar to that during certain stages of Cheddar cheese manufacture.

Flavourzyme activity in capsules was measured before and after treatment in

acidified milk and the difference in the activity is the estimated loss of activity due to

leakage.

At 38°C, the retention of Flavourzyme activity in capsules produced from 1.8 and

2.0% alginate was significantly higher (F 2, 44 = 27.378, p < 0.01) than that retained

in capsules produced from 1.6% alginate. Initial activity of Flavourzyme in the

capsules prior to treating to acidified milk was significantly higher (F 2, 44 = 210.397,

p < 0.01) than those treated to pH 5 which in turn was significantly higher than those

treated to pH 6 (Figure 4.3b).

At 32°C, there was no significant difference (p > 0.05) in the enzyme retention of

capsules produced from 1.6, 1.8 and 2.0% alginate. There was also no significant

difference (p > 0.05) in the enzyme retention in capsules treated to pH 5 or 6,

however the retention of Flavourzyme activity in capsules treated to acidified milk

was significantly lower (F 2, 44 = 7.386, p < 0.01) than the initial activity before

subjecting to acidified milk (Figure 4.3a).

Mechanical stability of Flavourzyme capsules produced from 1.6, 1.8 and 2.0% (w/v)

alginate by gelling in 0.1M CaCl2 containing 0.1% (w/v) chitosan was estimated by

subjecting the capsules to a pressure similar to that applied to the cheese curd before

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 Application of enzyme capsules into cheese

packaging. The duration of simulated press tested were 4h as in commercial

application (5h high pressure press), 8h and 16h similar to the traditional

manufacturing process where the cheese is pressed overnight for at least 16h. The

difference in enzyme activity in the capsules before and after subjecting to simulated

cheese press pressure was used to estimate the leakage of encapsulated enzyme under

simulated cheese press pressure (Figure 4.4). There was significant interaction (F 4, 45

= 14.197, p < 0.01) between the concentration of alginate and the duration of cheese

press hence the differences between the concentrations of alginate was tested for

individual simulated cheese press duration separately. There was no significant

difference (p > 0.05) in the leakage of Flavourzyme from capsules produced from

1.6, 1.8 and 2% alginate when the capsules were pressed for 4h however, when

pressed for 8h the leakage of Flavourzyme from capsules produced from 2% alginate

was significantly lower than (F 2, 15 = 47.866, p < 0.01) those produced from 1.8 and

1.6% alginate. After 16h simulated cheese press, the leakage of encapsulated

Flavourzyme produced from 2% alginate capsules was significantly lower than those

produced by 1.8% alginate, which in turn was significantly lower than those

produced by 1.6% alginate. Thus at 16h simulated cheese press the leakage of

encapsulated Flavourzyme was significantly lower (F 2, 15 = 197.292, p < 0.01) in the

order of increasing alginate concentration; 2a < 1.8b < 1.6c %, values with different

superscript letter are significantly different.

4.4.2 Determination of stage of capsule addition during cheese manufacture

Starch was incorporated into Flavourzyme microcapsules during encapsulation with

1.8% (w/v) alginate and the capsules were added at two stages of manufacture to

determine the ideal stage for addition of enzyme capsules. Distribution of stained

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 Application of enzyme capsules into cheese

capsules in cheese was observed for both the methods of capsule addition; before

rennetting (Figure 4.5a) and before salting (Figure 4.5b). Stained capsules appeared

as dark objects and were counted using the software Image pro plus. The area

occupied by dark objects (capsules) was recorded as the number of pixels for a fixed

size of cheese slice. The ratio of number of pixels to the number of particles was also

used to establish the pattern of distribution between the treatments (Table 4.1).

4.4.3 Composition of cheese

All cheeses were tested for moisture, fat and final pH and there was no significant

difference (p > 0.05) in the fat and final pH of control or experimental cheeses.

However, the moisture content of control cheeses C was only significantly lower (p <

0.05) than cheeses D with free Flavourzyme, F75 with encapsulated Flavourzyme at

a concentration of 0.75 LAPU/g milk protein and PF50 with encapsulated

Flavourzyme at a concentration of 0.5 LAPU/g milk protein and encapsulated

Palatase at a concentration of 0.5 LU/g milk fat (Table 4.2).

4.4.4 Microbiological status of cheese

Cheese samples manufactured for grading were tested for microbial pathogens as the

pathogen level above the regulatory limits are not permitted for sale or preparation of

food for sale (Standard 1.6.1, Australian StandardTM). All the pathogens were below

the regulatory limits of the Australian standard for microbiological limits in

commercial cheese. Though the yeast and mould count was high, there was no

difference in the counts of control or experimental cheeses (Table 4.3).

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 Application of enzyme capsules into cheese

110 110

Flavourzyme retention (LAPU/mL)

Flavourzyme retention (LAPU/mL)

100 100

90 90

80 80

70 70

60 60

50 50

40 40
1.6 1.8 2.0 1.6 1.8 2.0
Alginate concentration (% w/v) Alginate concentration (% w/v)

Initial pH 5 pH 6 Initial pH 5 pH 6

(a) (b)

Figure 4.3 Retention of Flavourzyme activity in capsules produced from 1.6, 1.8 and 2.0% (W/V) alginate and gelling in 0.1M CaCl2
containing 0.1% (w/v) chitosan suspended in milk at pH 5.0 and 6.0 at 32°C (a) and 38°C (b) for 10 min. The Flavourzyme activity in the
capsules prior to suspension in acidified milk is labelled initial and the difference in the activity initially and at different pH and
temperature treatments is equivalent to the loss of activity as a result of leakage of encapsulated Flavourzyme in milk. Error bars
indicate standard error of mean.

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 Application of enzyme capsules into cheese

120

Flavourzyme retention (LAPU/mL)

100

80

60

40

20

0
1.6 1.8 2
Alginate concentration (% w/v)

4 8 16

Figure 4.4 Retention of Flavourzyme activity in the capsules produced from 1.6,
1.8 and 2.0% (w/v) alginate and gelling in 0.1M CaCl2 containing 0.1% (w/v)
chitosan subjected to simulated cheese press pressure in a texture analyser for 4,
8 and 16h. The Flavourzyme activity was measured before and after subjecting
to the simulated cheese press pressure and the difference in the initial and final
Flavourzyme activity is presented as the retained activity after simulated cheese
press. Error bars indicate standard error of mean.
Note: The initial enzyme activity is different for capsules from different
concentrations of alginate.

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 Application of enzyme capsules into cheese

(a) (b)

Figure 4.5 Distribution of stained Flavourzyme capsules in Cheddar cheese, (a) capsule addition to milk prior to rennetting and (b)
capsule addition to curd prior to salting

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Table 4.1 Capsule distribution represented by ratio of number of pixels to

number of particles, * mean of 30 fields ± standard deviation

Stage of capsule addition Ratio of number of pixels to number of

particles*

To milk prior to rennetting 45.6 ± 10.96

To curd prior to salting 104.5 ± 101.67

Note: Aggregated capsules will be counted as smaller number of particles with

higher pixels corresponding to the area occupied hence more aggregated the capsules

are, greater is the pixels to particle ratio.

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Table 4.2 Fat, moisture and final pH of cheeses with and without flavour-
enhancing enzymes * mean of 3 samples ± standard error of mean

Sample Fat* Moisture* Final pH*

C 29.9 ± 0.26 35.86 ± 0.28a 5.36 ± 0.005

D 29.9 ± 0.23 39.13 ± 0.68c 5.35 ± 0.005

F50 29.95 ± 0.12 37.33 ± 0.24ab 5.35 ± 0.021

F75 30.16 ± 0.14 37.96 ± 0.20b 5.35 ± 0.008

F100 29.9 ± 0.11 37.3 ± 0.46ab 5.35 ± 0.020

PF50 30.23 ± 0.12 37.83 ± 0.46b 5.34 ± 0.017

PF75 30.16 ± 0.06 37.0 ± 0.17ab 5.32 ± 0.006

PF100 30.06 ± 0.13 37.1 ± 0.15ab 5.35 ± 0.012

P 29.8 ± 0.05 37.26 ± 0.31ab 5.36 ± 0.008

abc
means followed by different superscript letter are significantly different (p < 0.05)

Note: Cheese composition measured on the day of packing.

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Table 4.3 Pathogens tested in control and experimental cheeses produced for grading (acceptable microbiological level in a cheese
sample (AS), other dairy products (M) as per the Australian Standard 1.6.1)

Sample Total Yeast and Coagulase positive Coliform E. coli per g Salmonella Listeria per Bacillus cereus
mould staph per g per g per 25 g 25 g CFU per g
C(1) 160,000 yeast (est) Not detected 4 Not detected Not detected Not detected <100
<100 mould
C(2) 140,000 yeast (est) Not detected 4 Not detected Not detected Not detected <100
<100 mould
D(1) 10,000 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
D(2) 46,000 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
F75(1) 320,000 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
F75(2) 160,000 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
PF75(1) 28,000 yeast (est) Not detected 3 Not detected Not detected Not detected <100
<100 mould
PF75(2) 8,900 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
PF100(1) 9,100 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
PF100(2) 21,000 yeast (est) Not detected <3 Not detected Not detected Not detected <100
<100 mould
AS - - - 10 0 0 -
M - 10 10 3 0 0 -

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4.5 Discussion

Flavourzyme capsules were subjected to pH 5 and 6 at temperatures 32°C and 38°C

to assess the leakage of encapsulated Flavourzyme during cheese manufacture. The

difference in the initial and final Flavourzyme activity in the capsules suspended in

acidified milk was used to calculate the loss of enzyme activity as a result of leakage.

At 32°C (Figure 4.3a), the activity of Flavourzyme in capsules treated to acidified

milk was significantly lower (F 2, 44 = 7.386, p < 0.01) than the initial activity before

subjecting to acidified milk however the variation in alginate concentration or milk

pH had no significant effect (p > 0.05) on the enzyme retention of capsules. However

at 38°C (Figure 4.3b), leakage of encapsulated Flavourzyme significantly increased

(p < 0.01) with decrease in the alginate concentration and increase in the milk pH

from 5 to 6. These results indicate that during early stages of Cheddar cheese

manufacture, when the temperature is ~32°C and the pH ~6.6, concentration of

alginate or the pH of the milk has no effect on the leakage of encapsulated

Flavourzyme, but as the cheese manufacture progresses towards lower pH and higher

temperature, leakage of encapsulated enzymes is higher in capsules produced from

lower concentration of alginate. Improved retention of Flavourzyme activity in

capsules produced from 2% alginate suggests better cross linking of the polymer and

hence better retention of encapsulated enzymes at higher concentrations of alginate.

Based on these results, highly porous or compact matrix for capsules can be designed

for desired release of encapsulated enzymes.

Flavourzyme activity in capsules subjected to simulated cheese press pressure was

measured before and after cheese press and the leakage of encapsulated Flavourzyme

increased with the duration of cheese press (Figure 4.4). Alginate concentration had

no significant effect (p > 0.05) on leakage of encapsulated Flavourzyme when

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 Application of enzyme capsules into cheese

pressed for 4h, however, when the cheese press duration was prolonged, the leakage

of encapsulated Flavourzyme increased significantly (p < 0.01) with the decrease in

alginate concentration indicating continued leakage under simulated cheese press

pressure. These results suggest that capsules produced from higher concentrations of

alginate are less porous.

Porosity of the alginate capsules is essential for sustained release of encapsulated

enzymes in cheese during ripening and at the same time the capsules are not

excessively porous as over 70% of the encapsulated enzyme activity was retained in

the capsules even after 16h of pressing indicating that the capsules are elastic and

fairly stable under simulated cheese press conditions and are suitable for sustained

and prolonged release of encapsulated enzymes in cheese. By altering the

concentration of alginate in the capsules and the duration of cheese press it is

possible to regulate the initial release of encapsulated enzymes, and these results

show that these capsules are suitable for controlled and sustained release of

encapsulated enzymes in cheese.

Microscopic observation of stained capsules on the surface of cheese slice shows that

capsule addition to milk leads to even distribution in the cheese matrix (Figure 4.5a)

which can be attributed to the stirring of capsules in a liquid medium such as milk

leading to separation of capsules and thus even distribution in curd. Aggregation of

capsules was observed when added to curd before salting (Figure 4.5b). As the

enzyme microcapsules were added as a large aggregate, it was difficult to attain even

distribution when mixing with cheese curd. Aggregation of enzyme microcapsules in

cheese imply localised release of encapsulated enzymes leading to pockets of fast

ripened zones (“hot spots”) in the cheese blocks which can be undesirable. These

results are in agreement with the findings of Madziva et al. (2006) who reported even

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 Application of enzyme capsules into cheese

distribution of alginate-pectin encapsulated folic acid in Cheddar cheese when added

to milk compared to aggregation of capsules when added to milled curd or injected in

to the pressed cheese block.

Starch incorporated Flavourzyme capsules were produced from 1.8% alginate and

from earlier tests it was found that these capsules range from 500 to 530µm and only

the particles in this size range were programmed to be counted by the software Image

Pro plus. Due to a small variation in the capsule size, the area occupied by capsules

will also vary only to a small extent if distributed evenly. But if the capsules are

aggregated together, then will be counted as just one particle but the number of

pixels will be higher. The ratio of pixels to particles was calculated to be 45.6 ±

10.96 and 104.5 ± 101.67 for capsule addition to milk and curd respectively (Table

4.1) showing higher value for pixels when added to curd, when same amount of

capsules was added in both treatments. The high value of standard deviation, 101.67

obtained for capsule addition to curd show large variation in distribution (dense or

sparse) of capsules in cheese. The results for distribution estimated by calculating the

ratio of number of pixels to particles support the visual assessment of capsule

distribution. These results are in agreement with the proposition of Wilkinson and

Kilcawley (2005) that addition of enzymes at dry salting stage would be

disadvantageous due to uneven distribution of the small amount of enzyme among

large blocks of curd. The whey drained was filtered and the capsules lost in the whey

were collected and added back into the curd thus improving the retention of capsules

in the curd.

Composition of the control and experimental cheeses were compared in terms of fat,

moisture, pH and microbiological level as these are the most important indicators of

cheese quality early in ripening. Moisture, fat and pH of the cheese samples were

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 Application of enzyme capsules into cheese

analysed on the day of vacuum packaging (the day following cheese manufacture),

while microbiological level was assessed prior to cheese grading.

High moisture content in all cheeses can be attributed to the variation in the Cheddar

cheese manufacture protocol. High moisture in all experimental cheeses can be

attributed to addition of Flavourzyme during cheese manufacture, probably

interfering with the curd syneresis leading to increased moisture retention in curd

during manufacture or the increased breakdown of proteins in cheese decrease

protein solvation and increase the levels of moisture in cheeses manufactured with

proteolytic enzymes; Flavourzyme in the current study. Higher moisture could also

be due to excess moisture from the enzyme capsules. Alginate capsules are hydrogels

with bound and free water, which may have diffused into the cheese upon pressing

and also due to breakdown of hydrogel beads in cheese during ripening. Hydrogels

beads may have absorbed water during cheese manufacture and hence may be

responsible for increased moisture in cheese. These results agree with the reports of

increased moisture levels in cheeses with liposome (Kheadr et al, 2000) or food gum

(Kailasapathy and Lam, 2005) encapsulated enzymes. The moisture level in all

cheeses was higher than the standard commercial Cheddar which is 34.5% (Anon,

2007c) hence these cheeses may be called as Cheddar-type cheeses. Moisture content

of control cheeses was significantly lower (p < 0.05) than cheeses D with free

Flavourzyme, cheese PF75 with encapsulated Flavourzyme at a concentration of 0.75

LAPU/g milk protein, and PF50 with encapsulated Flavourzyme at a concentration

of 0.5 LAPU/g milk protein and encapsulated Palatase at a concentration of 0.5 LU/g

milk fat (Table 4.2). Variation in the moisture content among different cheese can be

attributed to the variation in cheese manufacturing process.

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 Application of enzyme capsules into cheese

The cheese milk was standardised for 3.5% fat, which in theory must yield 35% fat

in Cheddar cheese but the fat content in all cheeses was 29.8 to 30.2% (Table 4.2),

lower than the standard Cheddar cheese fat content of 33.3% (Anon, 2007c). Higher

moisture in cheese would be expected to cause relative reduction in other cheese

components such as fat content. There was no significant difference (p > 0.05) in the

fat content of control or experimental cheese. Similarly no significant difference (p >

0.05) in final pH was noted for experimental or control cheese (Table 4.2). This

indicates that addition of free or encapsulated enzymes during cheese manufacture

had no effect on the total fat content or final pH of the cheese.

Cheeses manufactured for grading were tested for pathogens to assess the

microbiological quality of cheese. Cheeses with pathogen levels above the regulatory

limits are considered unsafe and hence are not permitted for sale or in preparation of

food for sale (Australian Standard 1.6.1). There was no difference in the numbers of

coliforms, E.coli, Salmonella, Listeria, coagulase positive staphylococci, Bacillus

cereus, yeast and moulds in control or experimental cheeses; hence addition of free

or encapsulated enzymes had no effect on the pathogen numbers. All cheeses had

pathogen numbers under the regulatory limits except for yeast and mould count

(Table 4.3). High levels of yeast and mould in both control and experimental cheeses

could be due to contamination of the vat or cheese making equipment. As there is no

difference in the yeast and mould count of control or experimental cheeses;

Flavourzyme, Palatase or the encapsulation materials such as alginate and chitosan

cannot be the sources of contamination. These results show that both Flavourzyme

and Palatase are of good microbiological quality and can be used for commercial

application.

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4.6 Conclusion

Enzyme capsules produced from alginate by gelling in chitosan treated cationic

solution are stable in acidified milk and also under simulated cheese press pressure.

The Flavourzyme capsules are capable of prolonged and sustained release of

encapsulated enzymes during cheese press and also during ripening. By altering the

concentration of alginate and the duration of cheese press, it is possible to regulate

initial release of encapsulated enzymes into cheese which is advantageous in

controlled release of the encapsulated enzymes. Addition of capsules to milk during

cheese manufacture is found to yield even distribution, and addition of encapsulated

enzymes do not affect the fat content or pH of the cheese, hence are suitable for

incorporation into Cheddar cheese for acceleration of ripening.

129
5 MONITORING AND EVALUATION OF CHEESE

RIPENING

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 Monitoring & evaluation of cheese ripening

5.1 Abstract

Ripening of cheeses manufactured with and without free/encapsulated enzymes was

monitored by analysing peptides, amino acids and free fatty acids. Analysis of water-

soluble peptides from control and experimental cheeses showed increased and

prolonged proteolysis in cheeses incorporated with encapsulated Flavourzyme while

rapid proteolysis was observed in cheese with free Flavourzyme. Consequently an

increased accumulation of hydrophobic and/or high molecular weight peptides was

observed in cheeses with free Flavourzyme followed by encapsulated Flavourzyme

and control cheese with no added enzymes. Increase in the concentration of water-

soluble peptides was noted with the increase in the concentration of encapsulated

Flavourzyme in cheese. Peptide analysis also showed higher concentrations of water-

insoluble peptides from 60 to 180 days in control cheese than in cheeses with

encapsulated Flavourzyme. Free amino acid analysis showed that the concentration

of most amino acids was over 3 times greater in cheese PF75 with encapsulated

Flavourzyme than in control cheese C after 30 days of ripening and over 7 times

greater after 90 days ripening. Total amino acid concentration increased with the

ripening duration and showed rapid increase after 90 days of ripening. Profiles of

fatty acid methyl esters showed increased number and concentration of fatty acids in

cheeses incorporated with encapsulated Palatase than control cheese with no added

Palatase. During grading, cheeses incorporated with free Flavourzyme scored high

initially about 30 days ripening while cheese with encapsulated Flavourzyme scored

high about 56 and 86 days ripening with a good overall flavour score till about 86

days. Though the flavour of control cheese was scored lower initially, compared to

experimental cheese, the flavour improved as the ripening progressed and these

cheeses received high overall flavour score. The overall score of experimental

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 Monitoring & evaluation of cheese ripening

cheeses was lower than control for colour, condition, body and texture during the

entire period of ripening. Cheeses with encapsulated Palatase scored lowest for

flavour with rancid and strong unpleasant lipolytic flavour.

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5.2 Introduction

In long-ripened cheeses a range of biochemical processes leads to development of

characteristic flavour, aroma and texture over time and proteolysis, lipolysis and

glycolysis are the main biochemical processes occurring in cheese during ripening

(McSweeney, 2004). Glycolysis is mainly due to the action of cheese microflora but

is greatly influenced by the chemical composition of the cheese. Proteolysis refers to

the hydrolysis of milk proteins to peptides and amino acids and their conversion to

smaller aroma and flavour compounds while lipolysis refers to the hydrolysis of milk

fat to fatty acids and glycerol and their conversion to smaller flavour and aroma

compounds (McSweeney, 2004). It is possible to achieve accelerated cheese ripening

with an understanding of the biochemical processes occurring in cheese during

ripening and this subject has been extensively reviewed (Wilkinson, 1993, Law,

2001; Azarnia et al., 2006).

Exogenous addition of enzymes is a simple and specific approach for accelerating

the rate of biochemical reactions occurring during ripening thus accelerating the

formation of flavour compounds and reduction of ripening duration. Proteolysis is

the main biochemical event occurring in Cheddar cheese (McSweeney, 2004) and

can be accelerated by addition of proteolytic enzymes specially exopeptidases known

to reduce bitterness as it is one of the major flavour defects in Cheddar cheese.

Though lipolytic flavours are not dominant in Cheddar cheese, lipolysis of milk fat

contributes to the overall flavour in mature Cheddar (Fox and Stepaniak, 1993).

Encapsulated enzymes can minimise the problems associated with addition of free

enzymes.

Several techniques have been developed for the assessment and characterisation of

cheese ripening by qualitative and quantitative determination of chemical compounds

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 Monitoring & evaluation of cheese ripening

formed during cheese ripening. Chromatography is one of the most widely used

methods; RP-HPLC is commonly used for analysis of peptides (Aguilar, 2004) and

amino acids (Cohen, 2001) while GC is used for fatty acid analysis (Metcalfe and

Wang, 1981) although HPLC can also be used for this purpose. Cheese grading is

also an important tool for evaluation of ripening. It involves judging cheeses against

specified defects on standardised scorecards and is widely used for sensory

evaluation in the cheese industry (Delahunty and Drake, 2004).

In the current study, Flavourzyme with exopeptidase and endoprotease activity in

combination with Palatase, (a lipase) were efficiently encapsulated in alginate and

incorporated into Cheddar-type cheese for the evaluation of acceleration of ripening.

Monitoring of proteolysis and lipolysis by HPLC and GC respectively in addition to

grading was used to evaluate cheese ripening.

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5.3 Materials and methods

Milli Q (Millipore, Massachusetts, USA) water was used for all water-based

preparations in this study unless otherwise mentioned.

5.3.1 Monitoring of proteolysis in cheese

Proteolytic pattern in experimental and control cheeses was established by analysing

the water-soluble and water-insoluble peptides and free amino acids by Reverse

Phase High Performance Liquid Chromatography (RP-HPLC) and total amino acids

by spectrophotometer.

5.3.1.1 Extraction of water-soluble peptides

All cheeses; control and experimental, were grated and 10g was homogenised with

30g water followed by pH adjustment to 4.4.-4.6. This homogenate was held at 40°C

for 1h before centrifuging for 30 min at 5°C and 4800rpm. After centrifugation, fat

layer was removed and the supernatant was diluted to 100ml with water and filtered

through a No 42 Whatman filter paper. This filtrate was stored at -20°C until RP-

HPLC analysis (Verdini et al, 2004).

5.3.1.2 Analysis of water-soluble peptides

Water-soluble extract was filtered through a 0.2µm filter (Sartorius, Melbourne,

Australia) and 100µL was injected into Shimadzu LA20 Prominence HPLC system

with CBM 20A system controller (Shimadzu Corporation, Kyoto, Japan) fitted with

an Alltech Altima C18 reverse phase column of length 250mm and 4.6mm internal

diameter (Deerfield, USA). Detection was at 220nm using the SPD M20A Photo

Diode Array detector and the gradient elution was with solvent A (0.1% trifluoro

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 Monitoring & evaluation of cheese ripening

acetic acid (TFA) in water) and solvent B (0.1% TFA in acetonitrile) with a flow rate

of 1.0mL/min.

Gradient: Initially 0% B, isocratic step at 0% B for 5 min then linear step to 50% B

in 30 min, isocratic step at 50% B for 5 min (Verdini et al., 2004).

5.3.1.3 Extraction of water-insoluble peptides

The precipitate obtained after centrifugation of grated cheese (section 5.3.1.1) was

dissolved in 7M urea (Sigma, Sydney, Australia) and dialysed against distilled water

for 48h using a MWCO 6000-8000 membrane then freeze-dried in an Alpha-1-4

freeze drier with controller LDC-1M (CHRIST® Gefriertrocknungsanlagen, Osterode

am Harz, Germany) and stored at -20°C until RP-HPLC analysis (Verdini et al.,

2004).

5.3.1.4 Analysis of water-insoluble peptides

Freeze dried water-insoluble extract (20mg) obtained in section 5.3.1.3 was dissolved

in 0.01M imidazole (Fluka, Sydney, Australia) pH 7.0, 0.01M dithioerytritol (Sigma,

Sydney, Australia) and 6.6M urea (Sigma, Sydney, Australia), total volume 1.5mL

and filtered through a 200µm filter (Sartorius, Melbourne, Australia) and 100µL of

this solution was injected into Shimadzu LA20 Prominence HPLC system with CBM

20A system controller fitted with an Alltech Altima C18 reverse phase column of

length 250mm and 4.6mm internal diameter. Detection was at 220nm using the SPD

M20A Photo Diode Array detector and the gradient elution was with solvent A

(0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) with a flow rate of

1.0mL/min.

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 Monitoring & evaluation of cheese ripening

Gradient: Initial composition 0% B, isocratic step at 0%B for 5 min, linear step to

25% B in 5 min, linear step to 35% B in 30 min, linear step to 50% B in 10 min,

isocratic step at 50% B for 10 min (Verdini et al., 2004).

5.3.1.5 Extraction of amino acids

To 10mL of the water-soluble extract of cheese obtained in section 5.3.1.1, 2mL 15%

sulphosalicylic acid (SSA) (Sigma, Sydney, Australia) was added and centrifuged for

30 min at 20°C and 4800rpm. Supernatant was collected and the pH was adjusted to

4.0. This SSA-soluble extract was stored at -20°C until reverse phase ultra

performance liquid chromatography (RP-UPLC) analysis (Verdini et al., 2002).

5.3.1.6 Derivatization of free amino acids and analysis by RP-UPLC

Sulpho salicylic acid-soluble extract obtained in section 5.3.1.5 was analysed for free

amino acids at the Australian Proteomic Analysis Facility (APAF, Macquarie

University, Sydney, Australia). Free amino acids were analysed using ACQUITY

Ultra Performance LC system (Waters Corporation, Milford, MA, USA) with Waters

Empower Pro control and analysis software. Waters AccQTag Ultra column (BEH

C18, 2.1 X 100mm, 1.7µm) was used for all separations. Free amino acid

concentrations were determined using pre-column derivatization with 6-

aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) followed by separation of

the derivatives and quantification by RP-UPLC (Cohen and Michaud, 1993; Cohen,

2001). 5µL of the sample was dried in a Savant Speed Vac centrifugal concentrator,

model SC210A (Thermo Savant, Holbrook, NY, USA) with 10µL of internal

standard ( amino butyric acid – AABA). The dried sample was redissolved in 80µL

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 Monitoring & evaluation of cheese ripening

of borate buffer followed by 20µL derivatizing reagent (Waters AccQTag Ultra

reagent). The reaction mixture was incubated at 50°C for 10 min in a heating block.

Derivatised amino acid samples were transferred to the auto-sampler. The column

and auto-sampler temperatures were maintained at 60°C and 20°C respectively. The

sample injection volume and flow rate were 1.0µL and 0.7mL/min respectively. The

mobile phase A consisted of AccQTag Ultra eluent A concentrate (Waters) diluted 1

in 20 with milli Q water. The mobile phase B consisted of AccQTag eluent B

(Waters). The wavelength of the UV detection was set at 260nm. Analysis time was

10.2 min per sample. Each sample was analysed in duplicate and the results

averaged.

5.3.1.7 Determination of total amino acids by spectrophotometer

Water-soluble extract of cheese (100µL) was diluted to 1mL and 2mL of Cadmium-

ninhydrin reagent was added and this mixture was incubated at 84°C for 5 min,

cooled and the absorbance was read at 507nm in a Helios Gamma spectrophotometer

(Thermo electron corporation, England) (Folkertsma and Fox, 1992).

5.3.1.8 Preparation of cadmium-ninhydrin reagent for the determination of

total amino acid concentration by spectrophotometer

Ninhydrin (0.8g, Aldrich, Sigma, Sydney, Australia) was dissolved in 80mL absolute

ethanol (Aldrich, Sigma, Sydney, Australia) and to this 10mL glacial acetic acid

(Sigma-Aldrich, Sydney, Australia) was added followed by 1g cadmium chloride

(Aldrich, Sigma, Sydney, Australia) dissolved in 1mL water (Folkertsma and Fox,

1992).

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 Monitoring & evaluation of cheese ripening

5.3.2 Monitoring of lipolysis in cheese

Free fatty acids from cheese was extracted and derivatized to fatty acid methyl esters

(FAME) according to the method of Metcalfe and Wang (1981) modified by Martin-

Hernandez et al. (1988) and Chavarri et al. (1997) and analysed by gas

chromatograph (Poveda et al., 1999).

5.3.2.1 Extraction and esterification of fatty acids from cheese for Gas

Chromatography analysis

To 10g grated cheese, 5mL water, 0.5mL 5.5M H2SO4 (adjust pH 0.7-1.3) and 15mL

diethyl ether (Sigma-Aldrich) was added and centrifuged at 3000 rpm for 5 min at

0°C. Upper organic layer was transferred to a screw capped bottle containing 1g

anhydrous sodium sulphate (Sigma-Aldrich). After 5 min, 3mL was transferred to a

screw capped vial and 0.2mL 20% Tetra methyl ammonium hydroxide (TMAH) in

methanol (Aldrich, Sigma, Sydney, Australia) was added followed by shaking for 1-

2 min. Upper layer with FAMEs from glycerides was injected into a Shimadzu Class

10 Gas Chromatograph. Short/medium chain free fatty acids settled as TMAH soaps

were extracted and neutralized to pH 9.0 with thimol blue indicator and 2M

methanol-HCl before injecting into a Shimadzu Class 10 GC (Poveda et al., 1999).

5.3.2.2 Analysis of fatty acid methyl esters by GC

Fatty acid methyl esters from cheese (1µl) was injected into a Shimadzu Class 10

Gas Chromatograph with a flame ionization detector and an OmegawaxTM 250 fused

silica capillary column of the dimension 30mX0.25mm and 0.25µm film thickness

(Supelco, Sydney, Australia). Hydrogen was the carrier gas while hydrogen-nitrogen

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 Monitoring & evaluation of cheese ripening

was the make up gas and hydrogen-air was the detector gas (Martin-Hernandez et al.,

1988, Chavarri et al., 1997 and Poveda et al., 1999).

Time program for separation of FAME by GC:

Injector 280°C

Column 60°C for 3 min

8°C/min till 230°C and hold for 8 min

Detector 280°C

Split ratio 1:80

Hydrogen flow rate 1.39mL/min

Linear velocity 38.8cm/sec

Stop time 38 min

5.3.3 Cheese grading

Experimental and control cheeses were graded by a master cheese grader at Dairy

Farmers Innovation centre (Sydney, Australia). Cheeses were allowed to warm to 15-

22°C before grading for a final score of 100 points; 50 points for flavour, 30 points

for body and texture and 20 points for colour and condition. Score was deducted for

following defects in each category. Small cubes of cheese was used for grading and

expectorated after evaluation and the palate was cleansed with water after each

sample.

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 Monitoring & evaluation of cheese ripening

Table 5.1 Defects in Cheddar cheese considered while grading

Flavour and aroma Body and texture Colour and condition

Bitter Pasty Uneven

Stale Dry / crumbly Mottled

Sour / high acid Mealy Surface creasing

Salty Greasy Seamy

Flat / lacks flavour Gas openness Free fat

Fermented Mechanical openness Misshapen

Rancid / lipolytic Slits Free moisture / serum

Chemical / metallic Short Lactate

Weedy

Feedy

Cowy / barny

Mouldy

Scorched

Sweet

Harsh

Fruity

Uncharacteristic

Brothy /umami

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 Monitoring & evaluation of cheese ripening

5.4 Results

5.4.1 Assessment of proteolysis

In Cheddar and related cheese varieties proteolysis is an important factor responsible

for development of characteristic flavour and texture (Forde and Fitzgerald, 2000;

Upadhyay et al., 2004a). In the current study Flavourzyme, a protease peptidase

complex was encapsulated and added during Cheddar-type cheese manufacture and

its effect on proteolysis was assessed by monitoring water-soluble and water-

insoluble peptides, free amino acids and total amino acids formed during ripening by

HPLC. All cheeses were vacuum packed and allowed to ripen at 9°C for 270 days

and sampled at intervals of 30 days.

5.4.1.1 Peptide profile: water-soluble and water-insoluble

Comparison of HPLC chromatograms of water-soluble peptides of three cheeses;

control C with no added enzyme, cheese D with free Flavourzyme at a concentration

of 0.75 LAPU/g milk protein and cheese F75 with encapsulated Flavourzyme at a

concentration of 0.75 LAPU/g milk protein showed higher area for peptides

represented by peaks 1, 4, 10, 11, 13, 14, 18, 19, 21 and 22 in cheeses D and F75

compared to C in addition to formation of new peptides represented by peaks 3, 5, 6,

8, 9, 12, 16, 17, 20 and 23 in cheeses D and F75 which were absent in cheese C. At

30 days ripening, peptides represented by peaks 2, 7 and 15 were of similar

concentration in all cheeses; C, D and F75 (Figure 5.1). At this stage of ripening,

peptides 1, 2, 5, 8, 10, 11, 18 and 19 are found in relatively higher concentrations in

cheese D compared to cheese F75 (Figure 5.1).

As the ripening progressed to 90 days, the concentration of peptides represented by

peaks 1, 2, 6, 11, 12, 14, 19 and 20 was higher in cheeses D and F75 compared to C,

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 Monitoring & evaluation of cheese ripening

while peptides marked by peaks 4, 10 and 15 were at higher concentration in cheese

C compared to D and F75. Peptides marked by peaks 3 5, 7, 8, 9, 16, 17, 18, and 21

were found only in cheeses D and F75 but absent in C while peptide 13 was found

only in cheese C and absent in experimental cheeses D and F75 (Figure 5.2).

Comparison of water-soluble peptide profile of cheeses D and F75 shows that the

peptides represented by peaks 1, 5 and 12 are at higher concentration in cheese F75

compared to D while peptides 4, 10, 14, 16, 18, 19, 20 and 21 are at higher

concentration in cheese D compared to F75 while all other peptides appear to be of

relatively similar concentration in cheese D and F75 after 90 days ripening (Figure

5.2).

After 180 days ripening, peptides represented by peaks 1, 2, 3, 4, 10, 11, 13, 14, 18,

19 and 20 are in higher concentration in cheeses D and F75 compared to C while

peptides represented by peaks 6, 9 and 12 are in higher concentration in cheese C

compared to D and F75. Peptides represented by peaks 5, 8, 15, 16 and 17 are found

only in cheeses with free and encapsulated Flavourzyme (D and F75) and absent in C

while peptide 7 is found only in control cheese C while absent in cheeses D and F75.

Comparison of water-soluble peptides of cheeses D and F75 show that the peptides 1

and 14 are at higher concentration in cheese F75 compared to D while peptides 17,

18, 19 and 20 are at higher concentration in cheese D compared to F75 while all

other peptides are at relatively similar concentrations after 180 days ripening (Figure

5.3).

Effect of variation in Flavourzyme activity on proteolysis in cheese was assessed by

monitoring water-soluble peptides in cheeses with increasing level of encapsulated

Flavourzyme activity.

F50 – with encapsulated Flavourzyme at a concentration of 0.5 LAPU/g milk protein

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 Monitoring & evaluation of cheese ripening

F75 – with encapsulated Flavourzyme at a concentration of 0.75 LAPU/g milk

protein

F100 – with encapsulated Flavourzyme at a concentration of 1.0 LAPU/g milk

protein

After 60 days ripening, the concentration of peptides represented by peaks 2, 4 and 5

was highest in cheese F100 followed by F75 and F50 while the concentration of

peptides 1 and 3 was highest in cheese F100 but at similar concentration in cheeses

F50 and F75. All other peptides were of relatively similar concentration in all three

cheeses (Figure 5.4).

Water-insoluble peptide profile of control cheese C with no added enzymes was

compared with that of cheese F50 with encapsulated Flavourzyme at a concentration

of 0.5 LAPU/g milk protein. Area of the peaks from cheese F50 are relatively

smaller compared to cheese C and the comparison of the water-insoluble peptide

profile with - and -casein indicates that the water insoluble peptides in cheeses C

and F50 are mainly - and -caseins, however -casein could not be identified due to

overlapping of the peaks (Figure 5.5).

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Figure 5.1 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 30 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.2 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 90 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.3 Chromatograms of the water-soluble peptides from control cheese

(C), cheese with free Flavourzyme (D) and cheese with encapsulated
Flavourzyme (F75) extracted after 180 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.4 Chromatograms of the water-soluble peptides from cheeses

manufactured with encapsulated Flavourzyme at concentrations of 0.5 (F50),
0.75 (F75) and 1.0 LAPU/g milk protein (F100) after 60 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.5 Chromatograms of the water-insoluble peptides from cheese F50

with encapsulated Flavourzyme at a concentration of 0.5 LAPU/g milk protein,
cheese C with no added enzymes extracted after 180 days of ripening, -casein
and -casein standards at concentration of 15mg/mL

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 Monitoring & evaluation of cheese ripening

5.4.1.2 Free and total amino acid composition

Amino acids are largely responsible for the characteristic flavour of cheese

(McSweeney and Sousa, 2000; Yvon and Rijnen, 2001), some may contribute

positively while an imbalance in amino acid composition may lead to off-flavours

(Habibi-Najafi and Lee, 1996). Comparison of 20 standard amino acid composition

of control cheese C and cheese F75 with encapsulated Flavourzyme at a

concentration of 0.75LAPU/g milk protein at 30 days ripening showed that the

concentration of all amino acids in F75 was higher than in cheese C; concentrations

of serine (Ser), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), threonine

(Thr), methionine (Met), valine (Val), isoleucine (Ile) and leucine (Leu) in cheese

F75 were about 3 times that in cheese C while arginine (Arg) was detected only in

cheese F75 (Figure 5.6a). After 90 days ripening, concentration of all amino acids

from cheese F75 was ~7-8 times greater than that found in cheese C; asparagine

(Asn), serine (Ser), glutamine (Gln), glycine (Gly), glutamic acid (Glu), threonine

(Thr), methionine (Met), valine (Val), isoleucine (Ile) and leucine (Leu) from cheese

F75 were over 10 times that in cheese C while arginine (Arg) and cystine (Cys) were

detected only in cheese F75 (Figure 5.6b).

Total amino acid concentration in all cheeses increased with the ripening duration

and was highest in cheese F100, with highest level of encapsulated Flavourzyme at a

concentration of 1.0 LAPU/g milk protein. Rapid increase in total amino acid

concentration was noted after 90 days of ripening (Figure 5.7).

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 Monitoring & evaluation of cheese ripening

10 35

9
30
8
Concentration (mg/g cheese)

Concentration (mg/g cheese)

7 25

6
20
5
15
4

3 10

2
5
1

0 0

Gln
Gly

Glu

Val
Gln
Gly

Glu

Ile
Val

Leu
Ile
Leu

His

Ser

Thr

Lys
Tyr

Cys
Ala
Pro

Phe
Trp
Ser

Thr

Tyr
His

Lys

Cys
Ala

Phe

Met
Pro

Trp
Met

Arg
Arg

Asn

Asp
Asn

Asp

Amino acids Amino acids

C 30 F75 30 C 90 F75 90

(a) (b)

Figure 5.6 Composition of free amino acids from control cheese C with no added enzymes and cheese F75 with encapsulated
Flavourzyme. Free amino acid composition in cheeses C and F75 after (a) 30 days ripening (b) 90 days ripening

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 Monitoring & evaluation of cheese ripening

3.500

3.000
C
2.500
Absorbance (507nm)
D
F50
2.000 F75
F100
1.500
PF50
PF75
1.000
PF100
0.500 P

0.000
0 30 60 90 180 270
Age of cheese (days)

Figure 5.7 Total amino acid concentration measured by spectrophotometer in

the following cheeses; C, D, F50, F75, F100, PF50, PF75, PF100 & P
C – control with no added enzymes
D – free Flavourzyme at a concentration of 0.75 LAPU/g milk protein
F50 – encapsulated Flavourzyme at a concentration of 0.5 LAPU/g milk protein
F75 – encapsulated Flavourzyme at a concentration of 0.75 LAPU/g milk
protein
F100 - encapsulated Flavourzyme at a concentration of 1.0 LAPU/g milk protein
PF50 - encapsulated Flavourzyme at a concentration of 0.5 LAPU/g milk
protein + encapsulated Palatase at a concentration of 0.5 LU/g milk fat
PF75 - encapsulated Flavourzyme at a concentration of 0.75 LAPU/g milk
protein + encapsulated Palatase at a concentration of 0.5 LU/g milk fat
PF100 - encapsulated Flavourzyme at a concentration of 1.0 LAPU/g milk
protein + encapsulated Palatase at a concentration of 0.5 LU/g milk fat
P - encapsulated Palatase at a concentration of 0.5 LU/g milk fat

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 Monitoring & evaluation of cheese ripening

5.4.2 Assessment of lipolysis

Lipolysis is also known to contribute to Cheddar cheese flavour (Fox and Stepaniak,

1993; Arora et al., 1995) though at a subtle level (Collins et al., 2004). Contribution

of Palatase, a lipase to Cheddar-like flavour was assessed by incorporation of

encapsulated Palatase at a concentration of 0.5LU/g milk fat with and without

encapsulated Flavourzyme during cheese manufacture and ripened at 9°C for 270

days. All cheeses were sampled at 30 day intervals and the evolution of free fatty

acids (FFAs) was studied by esterification to fatty acid methyl esters (FAME) using

TMAH in methanol followed by neutralising the THMA soaps and monitoring by

gas chromatograph (GC).

5.4.2.1 Fatty acid methyl ester profile of Cheddar-type cheese

Fatty acids from the following cheeses were extracted and esterified to fatty acid

methyl esters (FAME) using tetra methyl ammonium hydroxide (TMAH).

C – control with no added enzymes

F75 – with encapsulated Flavourzyme

PF75 – with individually encapsulated Flavourzyme and Palatase

P – with encapsulated Palatase

Comparison of FAME profile of cheeses C and P extracted after 90 days ripening

shows that the number and concentration of FAME is greater in cheese P than C

(Figure 5.8). Similarly comparison of FAME of cheese F75 and PF75 shows that

after 90 days ripening the number and concentration of FAME from cheese PF75 is

greater than cheese F75 (Figure 5.9). Comparison of FAME of cheeses PF75 and P

show similar concentrations and numbers of FAME in both cheeses, though a few

more new FAMEs appear to be present in cheese PF75 (Figure 5.10). Comparison of

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 Monitoring & evaluation of cheese ripening

standard FAME with that of cheese P suggests that most of the free fatty acids

extracted from the TMAH soaps fraction of cheese P appear to be in the chain length

C11 to C23 (Figure 5.11). However it was not possible to identify all the compounds

due to the lack of appropriate standards.

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 Monitoring & evaluation of cheese ripening

Figure 5.8 Profile of fatty acid methyl esters of control cheese C (KAA90B
with no added enzymes) and cheese P (KAI90B, with encapsulated Palatase)
extracted after 90 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.9 Profile of fatty acid methyl esters of cheeses with encapsulated
Flavourzyme (F75; KAD90B ) and with encapsulated Flavourzyme and
Palatase (PF75; KAG90B ) extracted after 90 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.10 Profile of fatty acid methyl esters of cheeses; PF75 with
encapsulated Flavourzyme and Palatase (KAG90B ) P and with encapsulated
Palatase (KAI90B ) extracted after 90 days of ripening

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 Monitoring & evaluation of cheese ripening

Figure 5.11 Profile of Standard fatty acid methyl esters (KAFAME7 ) and
FAME from cheese P with encapsulated Palatase (KAI90B ) extracted after
90 days of ripening

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 Monitoring & evaluation of cheese ripening

5.4.3 Grading of control and accelerated ripened Cheddar-type cheeses

Cheese grading is an essential tool for characterising flavour and for maintenance of

consistent quality (Delahunty and Drake, 2004) and involves judgement of cheese on

the basis of whether it has properties characteristic of its variety (Lawrence et al.,

2004). Following cheeses were graded by judging against specified defects and

awarding scores based on flavour, body and texture, colour and condition and a

collective final score.

C - control with no added enzymes

D - with free Flavourzyme at a concentration of 0.75LAPU/g milk protein

F75 - with encapsulated Flavourzyme at a concentration of 0.75LAPU/g milk protein

PF75 - with individually encapsulated Flavourzyme (0.75LAPU/g milk protein) and

Palatase (0.5LU/g milk fat)

PF100 - with individually encapsulated Flavourzyme (1.0 LAPU/g milk protein) and

Palatase (0.5LU/g milk fat)

Comparison of colour and condition scores of cheeses C, D and F75 showed that

cheese C consistently scored better than D and F75, while score of D was similar to

C from 50 to 72 days ripening and that of F75 improved around 56 days of ripening

but later was scored low due to mechanical holes and mottled surface (Figure 5.12a).

Colour and condition scores of cheese F75 and PF75 were similar for the entire

period of grading as the appearance was similar (Figure 5.12b). In comparison the

appearance of cheese PF100 was similar to PF75 initially but was scored lower later

during ripening due to mottled surface and accumulation of free serum (Figure

5.12c).

Comparison of body and texture scores of cheeses C, D and F75 shows that the body

and texture of cheese C improved with the ripening duration and was constant during

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 Monitoring & evaluation of cheese ripening

the whole grading period, while the overall body and texture score of D and F75 was

lower than C due to crumbly and pasty texture (Figure 5.13a) while that of cheeses

F75 and PF75 were similar until about 56 days ripening when the body and texture

score of F75 increased while that of PF75 decreased due to pastier and crumbly

texture with accumulation of excessive serum compared to F75 (Figure 5.13b). Body

and texture score of cheese PF100 was lower than PF75 for the most part of the

ripening time due to extremely pasty and crumbly texture in addition to pale surface

(Figure 5.13c). Enzyme beads in the cheese were not detected during grading by the

master cheese grader.

Grading of cheeses C, D and F75 based on flavour showed that initially at about 30

days ripening, cheese D scored higher for flavour compared to C and F75 as cheeses

C and F75 were scored lower due to lack of flavour (Figure 5.14a). After about 56

days ripening, flavour score of F75 was highest due to development of savoury

umami flavour, followed by D and C. The Flavour of F75 scored lowest about 71

days ripening due to development of bitterness and again improved after 86days

ripening while slight unclean flavour was noted in cheese F75 after about 90 days

ripening hence downgrading cheese F75 after 90 days (Figure 5.14a). Flavour score

of cheese PF75 was lower than F75 for the whole ripening duration due to strong

unclean lipolytic flavour (Figure 5.14b) and it was also lower than PF100 for most of

the ripening duration due to strong unpleasant pungent flavour compared to the

unclean savoury umami flavour with after taste in cheese PF100 (Figure 5.14c).

Final grading score is an aggregate of the scores for flavour, body and texture, colour

and condition. Comparison of final scores of cheeses C, D and F75 shows that

control cheese C scored consistently during the entire period of ripening while

cheese D scored higher initially, the overall score was lower than C (Figure 5.15a).

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 Monitoring & evaluation of cheese ripening

The overall final score of F75 was constantly lower than C and D (Figure 5.15a) and

higher than PF75 (Figure 5.15b) for most of the ripening duration while that of

PF100 was higher than PF75 (Figure 5.15c).

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 Monitoring & evaluation of cheese ripening

Colour and condition Colour and condition Colour and condition

20.5 20.5 20.5

20 20 20

19.5 19.5 19.5

Score/20

Score/20
Score/20

19 19 19

C
18.5 18.5 18.5

18 18 18

17.5 17.5 17.5

0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120
Grading days Grading days Grading days

C D F75 F75 PF75 PF75 PF100

(a) (b) (c)

Figure 5.12 Comparison of grading score of cheeses (a) C, D and F75, (b) F75 and PF75, (c) PF75 and PF100 based on colour and
condition on a scale of 1-20. C – no added enzymes, D - free Flavourzyme at a concentration of 0.75LAPU/g milk protein, F75 –
encapsulated Flavourzyme at a concentration of 0.75LAPU/g milk protein, PF75 – encapsulated Flavourzyme at 0.75LAPU/g milk
protein and encapsulated Palatase at 0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at 1.0LAPU/g milk protein and
encapsulated Palatase at 0.5LU/g milk fat.

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 Monitoring & evaluation of cheese ripening

Body and Texture Body and Texture Body and texture

30 30 30

29.5 29.5 29.5

29 29 29
Score/30

Score/30
Score/30
28.5 28.5 28.5

28 28 28

27.5 27.5 27.5

27 27 27
0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120
Grading days Grading days Grading days

C D F75 F75 PF75 PF75 PF100

(a) (b) (c)

Figure 5.13 Comparison of grading score of cheeses (a) C, D and F75, (b) F75 and PF75, (c) PF75 and PF100 based on body and texture
on a scale of 1-30. C – no added enzymes, D - free Flavourzyme at a concentration of 0.75LAPU/g milk protein, F75 – encapsulated
Flavourzyme at a concentration of 0.75LAPU/g milk protein, PF75 – encapsulated Flavourzyme at 0.75LAPU/g milk protein and
encapsulated Palatase at 0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at 1.0LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat.

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 Monitoring & evaluation of cheese ripening

Flavour Flavour Flavour

44 44 44

43 43 43

42 42 42

41 41 41

Score/50
Score/50
Score/50

40 40 40

39 39 39

38 38 38

37 37 37
0 20 40 60 80 100 120 0 20 40 60 80 100 120 0 20 40 60 80 100 120
Grading days Grading days Grading days

C D F75 F75 PF75 PF75 PF100

(a) (b) (c)

Figure 5.14 Comparison of grading score of cheeses (a) C, D and F75, (b) F75 and PF75, (c) PF75 and PF100 based on flavour on a scale
of 1-50. C – no added enzymes, D - free Flavourzyme at a concentration of 0.75LAPU/g milk protein, F75 – encapsulated Flavourzyme at
a concentration of 0.75LAPU/g milk protein, PF75 – encapsulated Flavourzyme at 0.75LAPU/g milk protein and encapsulated Palatase
at 0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at 1.0LAPU/g milk protein and encapsulated Palatase at 0.5LU/g milk fat.

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 Monitoring & evaluation of cheese ripening

Final score Final Score Final score

93 93 93

92 92 92

91 91 91
90 90 90
89 89 89
Score/100

Score/100
Score/100
88 88
88
87 87
87
86 86
86
85 85
85
84 84
84
83
83
0 20 40 60 80 100 120 83
0 20 40 60 80 100 120
0 20 40 60 80 100 120
Grading days
Grading days
C D F75 F75 PF75 Grading days PF75 PF100

(a) (b) (c)

Figure 5.15 Comparison of the final grading score of cheeses (a) C, D and F75, (b) F75 and PF75, (c) PF75 and PF100 on a scale of 1-100.
C – no added enzymes, D - free Flavourzyme at a concentration of 0.75LAPU/g milk protein, F75 – encapsulated Flavourzyme at a
concentration of 0.75LAPU/g milk protein, PF75 – encapsulated Flavourzyme at 0.75LAPU/g milk protein and encapsulated Palatase at
0.5LU/g milk fat and PF100 - encapsulated Flavourzyme at 1.0LAPU/g milk protein and encapsulated Palatase at 0.5LU/g milk fat.

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 Monitoring & evaluation of cheese ripening

5.5 Discussion

The comparison of ripening shows clear differences between cheeses with and

without added enzymes and also between free and encapsulated enzymes.

Comparison of water-soluble peptide profile of cheeses C, D and F75 after 30 days

ripening show that the concentration of peptides in cheeses D and F75 are higher

compared to cheese C also with development of a larger number of peptides in

cheeses D and F75 but absent in C (Figure 5.1), this can be attributed to the action of

Flavourzyme on caseins leading to faster development of peptides, which would take

longer when occurring naturally as seen in cheese C. These results are similar to the

results reported by Bergamini et al. (2006) who found increased concentration of

several peptides by visual matching of chromatograms of water-soluble extract of a

semi-hard cheese with probiotic lyophilised culture compared to control cheese.

Higher concentration of peptides represented by peaks 1, 4, 10, 11, 13, 14, 18, 19, 21

and 22 in cheeses D and F75 compared to C (Figure 5.1) may possibly be due to

faster proteolysis in cheeses D and F75 with free and encapsulated Flavourzyme

respectively. Theses results are in agreement with reports of increased size-exclusion

peak areas of astringent fractions of Cheddar cheeses with liposome-encapsulated

Flavourzyme added at concentrations of 0.5, 0.75 and 1.0 LAPU/g milk protein at 30

days ripening (Kheadr et al., 2003). In the current study, presence of peptides 3, 5, 6,

8, 9, 12, 16, 17, 20 and 23 in cheeses D and F75 but absence in C (Figure 5.1)

suggests that these peptides may be the products of hydrolysis of caseins by

Flavourzyme in these cheeses. Higher concentration of the peptides 1, 2, 5, 8, 10, 11,

18 and 19 in cheese D compared to F75 (Figure 5.1) suggests that at 30 days

ripening, these peptides may be the products of rapid hydrolysis of caseins by free

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 Monitoring & evaluation of cheese ripening

Flavourzyme in cheese D but due to delayed release of encapsulated Flavourzyme in

cheese F75, these peptides appear to be at a lower concentration.

After 90 days ripening, most of the free Flavourzyme added to milk during

manufacture of cheese D is likely to be lost during whey drainage, but the presence

of peptides 1, 2, 6, 11, 12, 14, 19 and 20 at higher concentrations in D and F75

compared to C (Figure 5.2) suggests that these peptides are possibly the result of

immediate hydrolysis of cheese proteins and resistance to further hydrolysis by

Flavourzyme and hence continue to be present in higher concentrations even after 90

days ripening in cheese D with free Flavourzyme. These results are in agreement

with the reports of higher concentration of size-exclusion peak areas of astringent

fractions of Cheddar cheeses with liposome-encapsulated Flavourzyme added at

concentrations of 0.5, 0.75 and 1.0 LAPU/g milk protein after 90 days ripening

(Kheard et al., 2003). In the current study, presence of peptides marked by peaks 4,

10 and 15 at higher concentrations in cheese C compared to D and F75 (Figure 5.2)

suggests that these peptides are sensitive to the action of Flavourzyme and

hydrolysed by free and encapsulated Flavourzyme in cheese D and F75 and thus

found in relatively lower concentrations in these cheeses. Presence of peptides

marked by peaks 3 5, 7, 8, 9, 16, 17, 18, and 21 only in cheeses D and F75 but

absence in C (Figure 5.2) indicates that these peptides may be the products of

hydrolysis of caseins by Flavourzyme, which is absent in C. Presence of the peptide

represented by peak 13 only in cheese C and absence in cheeses D and F75 (Figure

5.2) suggests that this peptide may be highly susceptible to the action of

Flavourzyme and may be hydrolysed in cheeses D and F75 hence found only in C.

Concentration of peptides represented by peaks 1, 5 and 12 at higher concentration in

cheese F75 compared to D (Figure 5.2) suggests that these peptides are products of

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 Monitoring & evaluation of cheese ripening

sustained action of Flavourzyme released from the capsules in cheese F75 whereas in

cheese D, most of the free Flavourzyme would be lost in whey as it is water soluble.

Higher concentration of peptides 4, 10, 14, 16, 18, 19, 20 and 21 in cheese D

compared to F75 (Figure 5.2) suggests that these peptides may be the products of

early hydrolysis by Flavourzyme may possess the site Phe105-Met106 or other

cleavage sites for Flavourzyme hence appear to be present in lower concentration in

cheese F75.

Presence of peptides 1, 2, 3, 4, 10, 11, 13, 14, 18, 19 and 20 at higher concentration

in cheeses D and F75 compared to C (Figure 5.3) is indicative of the prolonged effect

of Flavourzyme on cheese proteolysis. Higher concentration of peptides 6, 9 and 12

in cheese C compared to D and F75 and presence of peptide 7 in C but absent in D

and F75 suggests that these peptides are sensitive to the action of Flavourzyme and

may possess the Flavourzyme cleavage site Phe105-Met106. Presence of peptides 5, 8,

15, 16 and 17 only in cheese D and F75 but absence in C (Figure 5.3) indicates that

these peptides are products of the action of Flavourzyme on cheese proteins. Highest

concentration of peptides 1 and 14 in cheese F75 after 180 days ripening (Figure 5.3)

suggests that these peptides are constantly released due to the prolonged action of

encapsulated Flavourzyme on cheese proteins and tend to accumulate during

ripening. Presence of peptides 17, 18, 19 and 20 in cheese D at higher concentration

compared to F75 suggests that these peptides are produced as early as the day of

manufacture and accumulated due to the diminishing effect of free Flavourzyme,

(expected to be lost during whey drainage) in cheese D even after 180 days ripening

(Figure 5.3).

Reverse-phase high-performance liquid chromatography (RP-HPLC) involves

separation of molecules based on hydrophobicity; solutes are eluted in the order of

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 Monitoring & evaluation of cheese ripening

increasing molecular hydrophobicity (Cliffe and Law, 1990; Aguilar, 2004). Peptides

20, 21, 22 and 23 (Figure 5.1) appear to be relatively hydrophobic or high molecular

weight compared to other peptides as they were eluted after other peptides. These

peptides start to accumulate early during ripening and are at high concentration in

cheese with free and encapsulated Flavourzyme compared to control C with no

added enzyme and these peptides continue to accumulate during ripening and do not

appear to be sensitive to the action of Flavourzyme even after 180 days ripening

(peaks 17, 18, 19 and 20, Figure 5.3). In a similar study, Ardo et al. (2007) reported

that large hydrophobic peptides that elute late in RP-HPLC in a narrow group (83-89

min) and accumulate in the semi-hard cheese Herrgard during ripening and suggested

that these peptides are products of early hydrolysis of -casein by plasmin. In the

current study a similar trend in accumulation of hydrophobic or high molecular

weight peptides was observed may be the products of -casein hydrolysis and

accumulate during ripening. However in the current study, the retention time of these

peaks was found to be 37-40 min and the variation in the retention time of the peaks

reported in the current study and that reported by Ardo et al. (2007) may be due to

the variation in the type of cheese, extraction, purification and RP-HPLC analysis.

Cheeses F50, F75 and F100 with the increasing levels of encapsulated Flavourzyme

at concentrations of 0.5, 0.75 and 1.0 LAPU/g milk protein respectively were

analysed for water-soluble peptides to determine the suitable enzyme load for

optimal proteolysis. Although most peptides appear to be of similar concentration at

60 days ripening, peptides 1, 2, 3, 4 and 5 were detected at highest concentration in

cheese F100 compared to F50 and F75 indicating greater extent of proteolysis in

F100 with higher concentration of Flavourzyme. Concentration of peptides 4 and 5

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 Monitoring & evaluation of cheese ripening

increased with the increase in Flavourzyme concentration (Figure 5.4), indicating

varying levels of enzyme activity.

Water-insoluble peptides in cheese mainly consist of caseins, after 180 days of

ripening, concentration of water-insoluble peptides from cheese C was found to be

higher than those from cheese F50 with encapsulated Flavourzyme (Figure 5.5)

suggesting rapid break down of caseins in cheese F50 by encapsulated Flavourzyme

resulting in lower concentrations of water-insoluble peptides. Comparison of the

chromatograms of -casein and -casein standard, shows that these caseins are one

of the major components of water-insoluble peptides from cheeses C and F50 (Figure

5.5).

Amino acids are produced as a result of peptide hydrolysis and are known to

contribute to the flavour of Cheddar cheese directly and also indirectly by acting as

precursors for the production of flavour compounds (Yvon and Rijnen, 2001). From

the water-soluble and water-insoluble peptide profiles it was found that the cheeses

with free and encapsulated Flavourzyme showed increased proteolysis compared to

control cheese; with no added enzymes. Free amino acid composition of control

cheese C and cheese F75; with encapsulated Flavourzyme was compared at 30 and

90 days ripening to assess the exopeptidase activity of Flavourzyme. At both

ripening times, levels of all amino acids were at elevated levels in cheese F75

compared to cheese C. The concentration of most free amino acids from cheese F75

were found to be ~1-6 times greater compared to cheese C after 30 days ripening

(Figure 5.6a) and ~7-30 times greater after 90 days ripening (Figure 5.6b).

Higher concentration of amino acids in cheese F75 suggests extensive proteolysis

due to the action of Flavourzyme. Presence of most amino acids at very high

concentration after 90 days of ripening suggests that a large number of amino acids

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 Monitoring & evaluation of cheese ripening

are released around that time, as the amino acids released earlier would be

catabolised to smaller compounds by the cheese microflora, but the presence of most

amino acids at very high concentration after around 90 days ripening also suggests

that these amino acids may be released in high concentrations at this stage of

ripening possibly as a result of low numbers of SLAB and NSLAB reported to be

present in Cheddar cheese at this stage of ripening (Fox and McSweeney, 2004). The

lower microbial activity would result in less catabolism and thus accumulation of

amino acids in the cheese. Release of large number of amino acids especially after 90

days ripening may also be due to the time involved in break down of peptides and

subsequent release of amino acids. This observation was also supported by the

measurement of total amino acids; the concentration of which increased rapidly after

90 days ripening (Figure 5.7) and was highest in cheese F100 with the highest level

of encapsulated Flavourzyme at a concentration of 1.0 LAPU/g milk protein.

After 30 days ripening, serine (Ser), glutamine (Gln), aspartic acid (Asp), glutamic

acid (Glu), threonine (Thr), methionine (Met), valine (Val), isoleucine (Ile) and

leucine (Leu) were about 3 times greater in cheese F75 than in cheese C, while

arginine (Arg) was detected only in cheese F75 (Figure 5.6a). Most of these amino

acids are hydrophilic and the release of these hydrophilic amino acids indicates the

tendency of hydrophilic cleavage of Flavourzyme. Similarly after 90 days ripening,

asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), glutamic acid (Glu),

threonine (Thr), methionine (Met), valine (Val), isoleucine (Ile) and leucine (Leu)

were over 10 times that in cheese C, while arginine (Arg) and cystine (Cys) were

detected only in cheese F75 (Figure 5.6b). Most amino acids in C were 10-30%

lower compared to F75; these results indicate the exopeptidase activity of

Flavourzyme. At 90 days ripening, Leu, Phe and Lys are the most abundant amino

171
 Monitoring & evaluation of cheese ripening

acids in cheese C, where as in cheese F75, Leu, Phe and Glu are the most abundant

amino acids. These results agree with Aston and Creamer (1986), McSweeney and

Fox (1993) and De Wit et al. (2005) who reported that Leu, Phe and Glu are the most

abundant FAA in mature Cheddar cheese. Hence release of free amino acids in

cheese F75 appears to be similar to standard Cheddar cheese.

While some amino acids in cheese C decreased marginally after 90 days ripening,

some were found at similar concentration. In contrast most amino acids in F75

increased 3-6 folds after 90 days ripening suggesting sustained release of

encapsulated Flavourzyme and its effect on peptide hydrolysis. De Wit et al. (2005)

also reported a significant increase in free amino acid content of yeast-inoculated

Cheddar cheese from 60-120 days.

Lipolysis in Cheddar cheese is known to contribute to the overall flavour only to a

small extent and there are very few reports of studies on the effect of lipolysis on

Cheddar cheese flavour (Collins et al., 2004). Palatase, a lipolytic enzyme was

encapsulated and incorporated into Cheddar-type cheese to study its effect on

lipolysis and the overall flavour of Cheddar-type cheeses. Free fatty acids from the 4

cheeses; control cheese with no added enzymes (C), cheese with encapsulated

Flavourzyme at a concentration of 0.75LAPU/g milk protein (F75), cheese with

individually encapsulated Flavourzyme (0.75LAPU/gmilk protein) and Palatase

(0.5LU/g milk fat) (PF75) and the cheese with encapsulated Palatase at a

concentration of 0.5LU/g milk fat (P) was extracted and esterified to fatty acid

methyl esters (FAME) and analysed by GC.

Comparison of FAME profile of cheese C and P extracted after 90 days ripening

shows smaller number and lower concentration of fatty acids in control cheese C

compared to cheese P with encapsulated Palatase (Figure 5.8). Increased numbers

172
 Monitoring & evaluation of cheese ripening

and concentration of free fatty acids in cheese P can be attributed to the hydrolysis of

cheese fat by encapsulated Palatase. Similarly, higher number and concentration of

FAME was observed in cheese PF75 compared to F75 (Figure 5.9), demonstrating

that the presence of a large number of free fatty acids in cheese PF75 is possibly due

to the action of encapsulated Palatase which was absent in cheese F75. These results

suggest that encapsulated Palatase, like Flavourzyme is efficiently released during

Cheddar-type cheese ripening. Cheeses PF75 and P were manufactured with identical

load of encapsulated Palatase and the free fatty acids from these cheeses are expected

to be identical. FAME profile of cheeses PF75 and P extracted after 90 days ripening

are similar (Figure 5.10) suggesting that the release and activity of encapsulated

Palatase was not affected by the presence of encapsulated Flavourzyme. Individual

encapsulation of Palatase and Flavourzyme in alginate appears to be an efficient

means for isolation of Palatase, which may also be a target for hydrolysis by

Flavourzyme. Comparison of FAME profile of cheese P with standard indicate that

most of the FAME in cheese P are in the chain length range of C11 and C23 (Figure

5.11) suggesting that most of the free fatty acids extracted after 90 days ripening

were of intermediate length. These results agree with the findings of Yilmaz et al.

(2005) who reported increase in concentration of long chain free fatty acids in Tulum

cheese made with microbial lipase with palmitic (C16:0), oleic (C18:1) and myristic

(C14:0) found to be the most abundant FFA and Fernandez-Garcia et al. (1988) who

reported increased levels of (C16:0), (C18:0) and (C14:0) in addition to C18:1 in Spanish

hard cheese manufactured with Palatase.

Cheeses C with no added enzymes, D with free Flavourzyme at a concentration of

0.75LAPU/g milk protein, F75 with encapsulated Flavourzyme at a concentration of

0.75LAPU/g milk protein, PF75 with individually encapsulated Flavourzyme

173
 Monitoring & evaluation of cheese ripening

(0.75LAPU/g milk protein) and Palatase (0.5LU/g milk fat) and PF100 with

individually encapsulated Flavourzyme (1.0 LAPU/g milk protein) and Palatase

(0.5LU/g milk fat) were graded by a master cheese grader during ripening. These

cheeses were judged for flavour, colour, condition, body and texture against

specified defects (Table 5.1) for about 100 days. Grading scores based on colour and

condition show that cheeses F75, PF75 and PF100 with encapsulated enzymes scored

lower than C and D, but after about 65 days ripening cheese D also scored lower than

the control cheese C for colour and condition (Figure 5.12a, b and c) due to mottled

surface and accumulation of serum which may be caused by excessive proteolysis.

Increased proteolysis leading to extensive breakdown of casein network in cheese is

known to decrease the water available for protein solvation resulting in easily

deformed cheese (Lawrence et al., 2004). Similarly in the current study, extensive

proteolysis may be the cause for liquid expulsion as result of reduced water-binding

capacity of the protein network

Grading of cheeses based on body and texture showed that cheese D initially scored

better than C but deteriorated thereafter due to crumbly texture, while the body and

texture score of cheese F75 was consistently lower than D and C during the entire

grading period due to crumbly and pasty texture (Figure 5.13a). Texture of cheese C

improved as the ripening progressed but that of D and F75 improved only till about

86 days ripening and deteriorated there after (Figure 5.13a). Body and texture of only

cheese C improved with ripening while that of other cheeses deteriorated with time.

Crumbly and pasty texture of cheeses D, F75, PF75 and PF100 can be attributed to

higher levels of proteolytic enzymes leading to rapid and extensive breakdown of

protein network which in turn lowers the water-binding ability of cheese thus

resulting in expulsion of water leading accumulation of serum and a pasty and

174
 Monitoring & evaluation of cheese ripening

crumbly texture (Lawrence et al., 2004). Though the body and texture of cheese

PF75 was similar to F75 initially, but declined as the ripening progressed (Figure

5.13b). Poor texture of PF75 compared to F75 may be due to extensive lipolysis in

PF75 due to the presence of encapsulated Palatase, which was absent in F75. These

results suggest that fat is also an important factor affecting cheese texture and

extensive lipolysis like proteolysis may lead to texture defects in cheese. Poor texture

of cheese PF100 compared to PF75 (Figure 5.13c) suggests extensive protein

breakdown in PF100 due to higher level of encapsulated Flavourzyme. These results

agree with the reports of brittle texture in Cheddar cheeses with liposome (Kheadr et

al., 2000) and food gum (Kailasapathy and Lam, 2005) encapsulated Flavourzyme.

All control cheeses and those with Flavourzyme scored high for pleasant, high

intensity acceptable flavour and those with Palatase scored low for unpleasant

unacceptable flavour. Flavour scores of cheeses varied with the ripening time, with

moderate variation in flavour score was observed for cheese C, more variation was

observed in D and F75 (Figure 5.14a). This variation in flavour profile may be due to

the formation of new peptides and amino acids some of which may contribute to a

pleasant flavour while some to an unpleasant flavour (Raksakulthai and Haard,

2003). Higher flavour score of cheese D at 30 days ripening indicate immediate

proteolysis in cheese D resulting from free Flavourzyme leading to rapid

development of flavour compared to slower proteolysis in cheese F75 with delayed

release of encapsulated Flavourzyme. These results also suggest that the leakage of

encapsulated Flavourzyme during cheese press is not responsible for immediate

proteolysis as observed in cheese F75.

Cheese F75 scored highest for flavour around 56 days ripening due to development

of savoury umami flavour but later scored lower than C and D due to mild bitterness

175
 Monitoring & evaluation of cheese ripening

and later scored higher due to elimination of bitterness and formation of salty

savoury flavour which may be due to the presence of certain peptides and/or amino

acids which are absent in C as evidenced by the development of higher number of

peptides in cheeses D and F75 (Figure 5.1, 5.2, 5.3 and 5.6) and increased levels of

free amino acids in cheese F75 compared to C (Figure 5.6a and b). Better flavour

score of cheese F75 may also be due to the formation of the sulphur containing

amino acid cysteine in F75 but absent in C and presence of methionine; another

sulphur containing amino acid in high concentration in cheese F75. These amino

acids are known to impart savoury flavour to cheese and may be released as a result

of increased level of proteolysis in F75 as result of sustained release of encapsulated

Flavourzyme. Cheeses D and F75 scored better than C for flavour after about 50 days

ripening due to elimination of bitterness which was persistent in cheese C with the

development of savoury umami flavour. Elimination of bitterness in D and F75 with

free and encapsulated Flavourzyme respectively, as early as 50 days ripening

suggests that Flavourzyme can be used for debittering cheese. In a similar study

bitter and astringent peptides accumulated in experimental cheeses with liposome-

encapsulated bacterial and fungal proteinases but were no longer detected after 3

months ripening (Kheadr et al., 2000). However in the current study the flavour of

cheese F75 deteriorated after about 86 days ripening with development of bitterness

and unclean flavour which did not improve during next grading hence cheese grading

was not continued after about 100 days ripening. Arginine was found only in cheese

F75 and is thought to be responsible for bitterness (Barcina et al., 1995). Izco et al.

(2000) reported arginine concentration of 0.1-3.1mg/g cheese in Ossau-Iraty cheese

while Zaki and Salem (1992) reported arginine concentration in the range 2.2-

9.3mg/g Edam cheese treated with Neutrase and the bitterness in these cheeses were

176
 Monitoring & evaluation of cheese ripening

attributed to higher concentrations of arginine. Similarly in the current study, higher

levels of arginine in the range 0.55 and 0.65mg/g cheese was noted after 30 and 90

days ripening respectively. Development of mild bitterness in cheese F75 after 50

days ripening may be due to increased concentration of arginine at 90 days ripening.

Concentration of the amino acid isoleucine in cheese F75 was about 30 times greater

than C after 90 days ripening, the hydrophobicity value of Ile is high (Habibi-Najafi

and Lee, 1996) and hydrophobic amino acids are know to impart bitterness in cheese

(Cliffe and Law, 1990). High concentration of Ile in F75 may also be responsible for

the poor flavour developing with longer aging. The concentration of many branched

chain amino acids such as threonine, glutamine, glutamate and leucine was higher in

cheese F75, these amino acids are also known to impart bitter taste (Raksakulthai and

Haard, 2003) and this could also be the reason for development of mild bitterness in

cheese F75 after 86 days ripening. Some amino acids such as phenylalanine and

some branched amino acids in excess can cause unclean flavour defects in Cheddar

cheese (Dunn and Lindsay, 1985). In the current study, Phe, an aromatic amino acid

and Glu a branched amino acid were found to be the most abundant FAA after 90

days ripening (Figure 5.6b) and may also be responsible for the unclean flavour of

cheese F75 after 86 days ripening.

Though the level of Flavourzyme in cheeses F75 and PF75 were identical, the overall

flavour score of F75 was higher than PF75 (Figure 5.14b) as PF75 was downgraded

due to development of strong unclean lipolytic flavours possibly due to liberation of

free fatty acids released as result of hydrolysis of fat in cheese by encapsulated

Palatase. Poor score of PF75 compared to F75 suggests that strong lipolytic flavours

are unacceptable in Cheddar-type cheeses. Initially cheese PF100 scored higher than

all cheeses for flavour (Figure 5.14c) suggesting that around 27 days ripening, only a

177
 Monitoring & evaluation of cheese ripening

small amount of encapsulated Palatase and Flavourzyme would have been released

but sufficient to enhance the flavour. Higher flavour score of PF100 at 26 days

ripening suggests that mild lipolysis may contribute positively to the overall Cheddar

cheese flavour. However the flavour score of cheese PF100 deteriorated with the

progress in the ripening time due to the development of unpleasant unclean flavour.

Strong lipolytic flavours are a result of production of large number of FFAs and their

catabolism to smaller compounds and these can contribute to flavour either positively

or lead to rancid defect depending on the concentration and perception threshold

(Molimard and Spinnler, 1996).

Though cheese PF100 had higher level of encapsulated Flavourzyme and similar

level of encapsulated Palatase compared to PF75, the overall flavour score of PF75

was lower than PF100 due to strong unpleasant pungent flavour compared to the

unclean savoury umami flavour with after taste in cheese PF100 (Figure 5.14c).

Though lipolysis is undesirable in young Cheddar cheese, extra mature Cheddar

shows considerable lipolysis and the lipolytic flavour impact is balanced by the

products of proteolysis (Fox and Stepaniak, 1993). Similarly in the current study,

better flavour of PF100 compared to PF75 may be due to masking of strong

unpleasant lipolytic flavours in PF100 by generation of more proteolytic compounds

as a result of higher rate of proteolysis from higher levels of Flavourzyme.

In a similar study by Kheadr et al. (2003), addition of individually encapsulated

Flavourzyme and Palatase in liposomes added to milk during Cheddar cheese

manufacture resulted in development of well balanced flavour. It was concluded that

addition of Flavourzyme at a concentration of 0.75LAPU/g milk protein and Palatase

at a concentration of 0.2 LU/g milk fat was responsible for development of well

balanced flavour without textural defects or bitterness. In the current study although

178
 Monitoring & evaluation of cheese ripening

addition of alginate encapsulated Flavourzyme at 0.75LAPU/g milk protein (cheese

F75) resulted in increased proteolysis and faster development of flavour as early as

26 days ripening, extensive proteolysis and off-flavours were also observed. The

variation in the proteolysis resulting from the same level of Flavourzyme in similar

cheeses suggests that the enzymes encapsulated in alginate are released completely

compared to those encapsulated in liposomes. In a similar study, Cheddar cheese

manufactured with Flavourzyme encapsulated in food gums such as -Carrageenan

and gellan resulted in bitterness (Kailasapathy et al, 1998) while in the current study,

control Cheddar-type cheeses were found to be bitterer than the cheeses with alginate

encapsulated Flavourzyme until about 71 days ripening and the development of mild

bitterness in cheese F75 with encapsulated Flavourzyme may be due to accumulation

of hydrophobic peptides or amino acids as a result of extensive proteolysis. These

variations in findings of the current study and that of Kailasapathy et al. (1998) and

Kheadr et al. (2003) suggests that alginate is an efficient alternative material for

encapsulation of Flavourzyme and Palatase leading to sustained release of

encapsulated enzymes. However, development of textural and flavour defects in

cheeses with encapsulated and free Flavourzyme suggests that the level of enzyme

added was very high. The optimum temperature range of Flavourzyme is 30-50°C

which is well above the ripening conditions of Cheddar cheese and it was assumed

that the activity of encapsulated Flavourzyme in the cheese would be reduced during

ripening due to low temperature, however extensive proteolysis in all the cheeses

with free and encapsulated Flavourzyme suggest that the activity of Flavourzyme is

not drastically reduced even at Cheddar cheese ripening conditions and the

encapsulated enzymes are efficiently released.

179
 Monitoring & evaluation of cheese ripening

5.6 Conclusion

Incorporation of Flavourzyme into Cheddar-type cheese resulted in increased

proteolysis. Immediate proteolysis was observed in cheese with free Flavourzyme

with the development of flavour as early as 26 days ripening while control cheese at

this stage of ripening lacked flavour with pronounced bitterness. Bitterness in

cheeses with encapsulated Flavourzyme was eliminated as early as 56 days ripening

but persisted in control cheeses with no enzymes. Development of uncharacteristic

yet pleasant savoury umami flavour was also observed in cheeses with encapsulated

Flavourzyme and this flavour was dominant until about 86 days ripening. However

the level of enzymes studied was very high as extensive proteolysis lead to flavour

and textural defects in all cheeses with Flavourzyme and unpleasant lipolytic

flavours noted in cheeses with encapsulated Palatase. Release and activity of

encapsulated Flavourzyme and Palatase was not affected by cheese ripening

conditions and individual encapsulation of these two enzymes in alginate is suitable

for combined application. Incorporation of encapsulated Flavourzyme in moderation

with trace amounts of encapsulated Palatase can potentially lead to enhanced flavour

development in Cheddar cheese.

180
6 CONCLUDING SUMMARY

181
 Concluding summary

A range of food grade polymer hydrogels were screened and the most suitable

polymer for microencapsulation of Flavourzyme was selected and the encapsulation

parameters were optimized for maximum encapsulation efficiency and stability.

Flavourzyme microcapsules were tested under simulated conditions of Cheddar

cheese manufacture. Flavourzyme and Palatase microcapsules were added to the

milk during the manufacture of Cheddar-type cheese for even distribution. Cheese

ripening was assessed by analysis of water-soluble peptides, water-insoluble

peptides, amino acids and fatty acids and thus monitoring proteolysis and lipolysis.

Flavour and texture of accelerated ripened cheese was evaluated by grading.

Cheddar is a widely consumed cheese variety requiring long ripening for the

development of mature flavour (Wilkinson and Kilcawley, 2005). Extended

ripening of this variety is a financial burden for the manufacturer due to increased

costs of refrigeration and maintenance in addition to loss of yield due to

contamination and syneresis, thus increasing the overall cost (Cliffe and Law, 1990;

Picon et al., 1997; Wilkinson and Kilcawley, 2005). Biochemical reactions

occurring in cheese during ripening, leading to the breakdown of proteins, fat and

sugars with the formation of smaller compounds are responsible for the

development of flavour and texture characteristic of mature cheese (Forde and

Fitzgerald, 2000; McSweeny, 2004). As Cheddar is ripened between 8-12°C, the

rate of these biochemical reactions is low thus extending the ripening duration (Fox

et al., 1993; Fox and McSweeney, 1996; Fox and Wallace, 1997). Though several

techniques have been developed to accelerate cheese ripening, development of

balanced flavour still remains a challenge (Wilkinson and Kilcawley, 2005).

Exogenous addition of enzymes can be used to accelerate cheese ripening.

182
 Concluding summary

Microencapsulation of enzymes can minimise the problems associated with addition

of free enzymes (Wilkinson and Kilcawley, 2005).

In the current study alginate, chitosan and κ-Carrageenan were studied for

microencapsulation of Flavourzyme using the Inotech® encapsulator. Owing to the

high viscosity, chitosan and κ-Carrageenan were eliminated as suitable polymers for

enzyme encapsulation and alginate was selected for enzyme encapsulation. Gelling

of alginate-Flavourzyme mixture in 0.1M CaCl2 resulted in poor encapsulation

efficiency possibly due to high porosity of the capsules and diffusion of

Flavourzyme from capsules during formation (Pommersheim et al. 1994;

Dashevsky, 1998). Incorporation of starch or pectin into alginate-enzyme matrix as

filler material did not improve enzyme retention in capsules unlike reports by

Madziva et al. (2006) and Sultana et al. (2000) who reported increased folic acid

and cell encapsulation upon addition of pectin and starch to alginate matrix

respectively. Low encapsulation efficiency of Flavourzyme in alginate-starch and

alginate-pectin could be due to insufficient blocking of the pores in alginate matrix

or interference of added starch and pectin with the Flavourzyme assay.

Flavourzyme-alginate gelling in 0.1M CaCl2 resulted in poor encapsulation

efficiency due to increased porosity or diffusion of Flavourzyme from capsules.

Treatment of cationic gelling solution with chitosan resulted in an improved

encapsulation efficiency. This could be due to blocking of pores in alginate matrix

by possible interaction of chitosan with alginate similar to the interaction of CaCl2

with alginate; gelling of capsules in chitosan treated gelling solution can minimize

bead shrinkage and water loss thus minimizing diffusion and leading to enhanced

enzyme retention. Though 0.3% (w/v) chitosan in the gelling solution resulted in

higher encapsulation efficiency, 0.1% (w/v) was chosen for further work as higher

183
 Concluding summary

concentrations of chitosan in gelling solution resulted in aggregation of capsules

during formation.

There was no enhancement in Flavourzyme retention in capsules coated with

alginate or poly-L-lysine, hence uncoated capsules were used for further

experiments also poly-L-lysine is an expensive ingredient and hence not suitable for

commercial application.

Frozen or freeze-dried Flavourzyme capsules are suitable for industrial application

as they retain about 80 % of the enzyme activity over 10 weeks when stored

appropriately.

Retention of Flavourzyme activity was marginally better (p < 0.01) in capsules

produced from 2.0% alginate. As there was no significant difference (p > 0.05) in

the enzyme retention of capsules produced from 1.6, 1.8 or 2.0% (w/v) alginate, any

of these concentrations of alginate are suitable for delivery of Flavourzyme into

cheese. Retention of over 70% of the initial enzyme activity even after 16h

simulated cheese press (section 4.4.1), indicates only about 30% of the enzyme was

released during cheese press and the rest released in cheese during ripening possibly

as a result of breakdown of alginate matrix or through diffusion of the encapsulated

enzymes from capsules. There is also the possibility that the surface layer of the

beads may show a greater binding of Ca2+ and chitosan with alginate and this may

form a barrier to the flow of enzymes. Hence the capsules are mechanically stable

and are capable of sustained release of encapsulated enzyme thus preventing

immature extensive proteolysis. Leakage of encapsulated enzyme increased with the

decrease in alginate concentration when subjected to simulated cheese press over 8h

indicating that the enzyme microcapsules produced from alginate can be designed

184
 Concluding summary

for suitable application in a wide variety of cheeses with varying cheese press

duration.

Retention of Flavourzyme activity was marginally better when added to milk at pH

5.0, but similar pH is achieved only after milling, addition of capsules at this stage

can be disadvantageous due to the resultant uneven distribution of capsules in

cheese. Though the leakage of encapsulated Flavourzyme is higher at milk pH it is

recommended to add capsules to milk for even distribution.

Increased moisture, yield, and lower fat content compared to standard Cheddar

cheese can be attributed to the variation in cheese manufacture and can be

eliminated with the adoption of appropriate manufacturing protocol. There was no

difference in fat, moisture, final pH and pathogen levels between control or

experimental cheeses, thus addition of encapsulated Flavourzyme does not alter

cheese composition and hence suitable for commercial application.

Addition of encapsulated Flavourzyme and Palatase lead to accelerated proteolysis

and lipolysis in Cheddar-type cheeses as evidenced by the profiles of water-soluble

and water-insoluble peptides, amino acids and fatty acid methyl esters development

in these cheeses during ripening. Cheeses with encapsulated Flavourzyme showed

reduced bitterness as early as 56 days ripening while bitterness persisted in control

cheeses with no added enzymes. Cheese F75 with medium level of encapsulated

Flavourzyme scored highest at about 56 and 86 days ripening due to the

development of uncharacteristic yet pleasant acceptable savoury umami flavour.

Cheese D with free Flavourzyme scored highest initially about 30 days ripening due

to immediate and increased proteolysis, but scored lower as the ripening progressed

due to development of unpleasant flavour as a result of extensive proteolysis.

Flavour of cheeses with encapsulated Flavourzyme deteriorated after about 86 days

185
 Concluding summary

ripening possibly due to formation of hydrophobic peptides or amino acids as a

result of extensive proteolysis. Texture of all experimental cheeses was crumbly and

pasty with excessive serum also due to extensive proteolysis. These flavour and

texture defects can be controlled by addition of lower levels of enzymes in the range

0.2LAPU/g milk protein. Hydrophobic or high molecular weight peptides can cause

bitterness and accumulation of these peptides can be minimised by addition of much

lower levels of enzymes as these are robust enzymes, active even at low ripening

temperatures.

The salt content in all cheeses were found to be lower due to increased moisture.

Cheeses manufactured with encapsulated Flavourzyme gave salty taste after about

56 days of ripening indicating that the salty taste was due to certain amino acids or

peptides. This can be a very useful application in salty cheeses such as Haloumi.

Though cheese is consumed in moderation increased salt/ NaCl concentration can

have undesirable effects on health such as hypertension and osteoporosis (Fox and

Cogan. 2004). Use of encapsulated Flavourzyme to develop salty taste can be

advantageous for eliminating unwanted excessive salt from cheese which is an

essential part of daily diet for many individuals.

Growth in the demand for diversified distinctive food-flavours has lead to increased

interest in varied cheese products. Addition of encapsulated Flavourzyme led to

development of uncharacteristic Cheddar yet acceptable flavours in addition to

accelerated ripening. Encapsulated Flavourzyme can thus be used for development

of new flavours in cheeses or cheese products offering a competitive edge

commercially. Rancid or lipolytic flavours resulting from addition of encapsulated

Palatase were unsuitable in young Cheddar cheese (Fox and Stepaniak, 1993) but

mild lipolysis is acceptable in vintage Cheddar cheese (Fox and Stepaniak, 1993;

186
 Concluding summary

Collins et al., 2004). Highest levels of lipolysis are observed in mould ripened

cheeses (Gripon, 1993; Spinnler and Gripon, 2004) and strong lipolytic flavours are

characteristic of many Italian varieties of cheese (Bosset and Gauch, 1993), and

hence alginate encapsulated Palatase can be applicable for flavour development in

these varieties. It can be concluded that alginate encapsulated Flavourzyme or

Palatase can be applied for acceleration of cheese ripening and also for production

of low-cost high-intensity cheese flavours for various applications.

187
7 FURTURE DIRECTIONS

188
 Future directions

In the current study, alginate-Flavourzyme capsules were allowed to gel in chitosan

treated CaCl2 and found to be considerably stable under simulated cheese press

pressure, retaining over 70% of the initial enzyme activity. Though prolonged

proteolysis was observed in cheeses with encapsulated Flavourzyme, extensive

proteolysis leading to formation of unacceptable off-flavours was also a concern.

This may be due to immediate breakdown of capsules in cheese matrix. However in

the current study it was not possible to study the breakdown of the enzyme

microcapsules and understand the release mechanism of encapsulated enzymes in

cheese. Though accelerated proteolysis and lipolysis leading to rapid development of

flavour in cheese has been reported, mechanism of release of encapsulated enzymes

is yet to be understood. In the current study Cheddar-type cheese was manufactured

with Fluorescein-iso-thio-cyanate (FITC) labelled Flavourzyme in order to track

encapsulated Flavourzyme in cheese, to understand the release mechanism of

encapsulated enzymes in cheese matrix during ripening. However, this approach was

not successful as the surface of cheese also fluoresced with capsules and it was not

possible to differentiate capsules from cheese. Even though FITC-labelled

Flavourzyme was treated to resist further binding, fluorescence of the entire surface

of cheese suggests that the FITC was bound to cheese proteins also thus causing

fluorescence of cheese upon observation at the recommended wavelength ( max).

Development of probes/markers to track the encapsulated enzymes in cheese to

understand the release mechanisms of encapsulated enzymes is a challenge and is

strongly recommended for successful direction of accelerated ripening.

Accelerated ripening due to addition of encapsulated enzymes resulted in

development of new peptides absent in ripened control cheese with no added

enzymes; these peptides may confer positive or negative effect to the flavour and

189
 Future directions

texture. It is advantageous to isolate and characterise these peptides using amino acid

analyser and sequencers to better understand the effects of the encapsulated enzymes

on cheese.

Formation of new peptides and amino acids and increased concentration of amino

acids can potentially alter cheese composition thus altering the nutrients and inturn

leading to change in the microbial composition of cheese. Altered microflora can

potentially modify the final flavour and texture of cheese. It is recommended to

monitor the SLAB and NSLAB in cheese during ripening.

In the current study, Flavourzyme and Palatase were delivered into Cheddar-type

cheese by encapsulating in 1.8% alginate and gelling in 0.1M CaCl2 containing 0.1%

chitosan for 10 min. It is known that by altering the polymer concentrations and

encapsulation parameters it is possible to obtain various kinds of capsules such as

highly porous or very compact and durable capsules. It is recommended to

incorporate enzymes encapsulated in various capsules aimed at early, intermediate

and delayed release of encapsulated enzymes in cheese for continuous yet moderate

proteolysis.

In the current study only 3 polymers; alginate, chitosan and -Carrageenan were

screened, author recommends screening of more polymers such as pectin, xanthan,

agar for enzyme encapsulation for targeted and controlled release of enzymes in

cheese.

190
8 REFERENCES

191
 References

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