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5-1959

A Method for Determining Proteinase Activity

Bhupendra V. Randeria
Utah State University

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A METHOD FOR DETERMINING PROTEINASE ACTIVITY
by
Bhupendra V. Randeria

A thesis submitted in partial fulfillment

of the requirements for the degree
of
KASTER OF SCIENCE
in
Dairy Manufacturing

UTAH STATE UNIVERSITY�

Logan, Utah
1959
 ACKNOWLEDGEMENT

I wish to express my appreciation and thanks to

Professor A . J . Morris and Professor P . B. Larsen for

their advice, aid , and encouragement throughout this

study .
B. V . Randeria
 TABLE OF CONTENTS

Page
Introduction

Review of literature 3

Experimental procedure 10

Fuld gross method 10

Method 10
Reagents 11
Determination . . 11
Calculat1on of proteinase unit 12

Curd tension method 13

Clotting method 14

Results and discussion . 16

Modified clotting method 18

Preparation of enzyme solution 18
Kinetics of the clotting procedure 18
Effect of variations of S.N.F. in
mil k 21
Mechanics of proteinase activity
on casein . . 21
Quantative formulation of the
kinetics of the two-phase re-
action in the case of prote-
inase during clotting 25

Effect of va 1 iations of different salts on

the animal proteinase activity 28

Activity of the plant proteinase . 32

Preparation of enzyme solution 32

Effect of various salts on the plant

proteinase activity 39
Effect of sodium carbonate on the
plant proteinase activity . 40
Effect of sodium citrate on the
plant pro teinase activity 41
Effect of pH 41
 TABLE OF CONTENTS (continued)

Page

Comparisons of bitterness of the curd

developed by animal and vegetable
proteinase 44
Animal proteinase 44
Vegetable proteinase 45
Nitrogen determination 45
Method 46
Formal titration method

Reaction during formal titration 47

Summary 48
Conclusion 50

Literature cited 51
 LIST OF TABLES

Table Page
1. The activation of intracellular enzymes 5
2. Properties of plant proteinases 7

3. Enzyme concentrations and the average

activity 15

4. Curd tension method: relationship between

enzyme concentrations and the clotting
time 17

5. Clotting method : relationship between enzyme

concentration and the clotting time 17
6. Effect of variation of animal proteinase
enzyme 19
7. Effect of variation of rennet extract on the
animal proteinase activity 21

8. Analysis of the milk sample 23

9. Effect of variation of solids-non-fat in milk
on animal proteinase activity . 24

10. Effect of variation of temperature on the

animal proteinase activity 27
11. Effect of variation of pH on the animal
proteinase activity 27
12. Effect of potassium phosphate on animal
proteinase activity 29
13. Effect of sodium carbonate on animal
proteinase activity 29

14. Effect of calcium chloride on the animal

proteinase activity 30

15. Effect of manganous carbonate on the animal

proteinase activity 30
 LIST OF TABLES (continued)

Page

16. Effect of ammonium sulfate on animal

proteinase activity 31

17. Effect of manganese chloride on animal

proteinase activity 31

18. Effect of variation of plant proteinase 36

19 . Effect of variation of rennet on plant

proteinase activity 36

20. Effect of variation of solids-not-fat on the

plant proteinase activity 37

21. Effect of variation of temperature on the

plant proteinase activity 37

22. Effect of potassium phosphate on the plant

proteinase activity 39

23. Effect of managanous carbonate on the plant

proteinase activity . 40

24. Effect of calcium chloride on the plant

proteinase activity . 40

25. Effect of ammonium sulfate on the plant

proteinase activity 41

26. Effect of pH on the plant proteinase activity 43

27. Animal proteinase activity by A.O.A.C. method 43

28. Vegetable proteinase activity by A.O.A.C.

method 44

29 . Analysis of nitrogen determination 46

 LIST OF FIGURES

Figures Page

1. Relationship between enzyme concentrations

and its activity (A.O.A.C. method) (animal
proteinase) 16

2. Relationship between clotting time (minutes)

and S.N.F. concentrations (animal proteinase) 22

3. Relationship between clotting time (minutes)

and different salts . (animal proteinase) . 33

4. Relationship between clotting time (reciprocal)

and the amount of enzyme 34
5. Relationship between clotting time (minutes)
and S.N.F. concentrations (vegetable
proteinase 35
6. Relationship between clotting time (minutes)
and temperature (OC) 38

7. Relationship between clotting time (minutes)

and different salts (vegetable proteinase . 42
 INTRODUCTION

Enzymes play a very important role during the ripening

process of the cheese. Enzymes are important in producing

the body, texture, and flavor of the cheese. The break-

down of carbohydrates, fats, and proteins by added enz ymes

into different e nd products during the ripening of the

cheese aids the process and may reduce t ime . The charac-

teristic flavors of different varieties of cheeses are

influenced by enzyme action. Some enzyme like proteinase s

can be used to improve the body, texture, and the flav or

of the cheddar cheese . Addition of the se enzymes during

cheddar cheese making may shorten the ripening period .

In order to use some specific amount of the enzyme to

bring about desirable changes, it is desirable to determine

the activity of that enzyme.

A variety of methods have been used to determine the

activity of proteolytic enzymes . The methods employ diff-

erent substrates , both natural and synthetic. Even with

the same substrate, the procedures for conducting the

test often vary . The A. O.A.C. (Fuld Gross Method), (1)

which employs Hammersten's casein as the substrate, has

been used quite extensively. In one case, hemoglobin is

used as the substrate (2 , 3). Colormetric apparatus can

be used to measure the partial hydrolysis of its protein .

 2

Activation of the enzyme apparently takes place during

the cheese making process and during the storage to produce
cheese with a softer and more elastic body .
 REVIEW OF LITERATURE

In cheese ripening the breakdown of casein represents

one of the most important changes. Ichiro, Ishibara (20)

and W. Keller (22) studied the breakdown of casein into

its different products. The breakdown has been attributed

at one time or another to the natural proteinase in milk,

to the proteinases in rennet or to the proteinases of

microbial origin. It was first suggested by Babcock and

Russell (4) and Babcock et al. (5) that an inherent milk

proteinase is the most important proteolytic agent in the

cheese ripening. Later work on the variation of rennet

levels in cheese making caused Babcock, et al. (5) to

revise their early views . The final conclusion was that

rennet is the most important proteinase source in cheese,

its protein digestive activity being due to the action of

pepsin present in the rennet as an impurity. Some workers

(6, 9, 18, 23, 33, 34, 49) studied the effect of rennin

on milk in relation to coagulation. Sherwood (35), however

reported that the use of pepsin instead of rennet in

cheddar cheese resulted in 40-50 percent less protein degra-

dation than took place in normal rennet control cheeses.

Several workers (8, 13, 14, 16, 19, 21, 36, 37, 40, 43,

46) were able to isolate proteinase from bacteria and able

to study some of their effects on different cheeses.

 4

Peterson, Johnson, and Price (31, 32) were able to determine

the proteinase content of the cheddar cheese during making

and ripening . A . E. Axetrod (3) and C . J. Martin (26)

studied the proteolytic enz yme system of skin in relation

to its purification and its activity. Several workers

(ll, 15, 17, 24, 38, 46) were able to study plant prote-

inases and their activity in the crystalline stage. W. J .

Ellis (15) J. R. Kimmel and E. L. Smith were able to pre-

pare crystalline papain of more purity . While A. Walri

(46) was able to prepare crystalline ficin from the fig

latex. Zettle (50) studied the effect of some salts on

milk clotting by heat.

During the past several years, one of the major pro-

blems to be dealt with in the dairy industry has been con-

cerned with the ripening of the cheddar cheese. In an

effort to attack this problem from enzymatic view point ,

it was first necessary to determine the proteinase activity

and then use the enzymes in the cheddar cheese manufacture .

Addition of these enzymes during the cheese making may

shorten the ripening period and improve the body texture

and flavor of the cheddar cheese.

It is believed that the activity of the proteinase

enzyme depends upon the structural configeration of that

particular enzyme (28) specificity and activity also depend

upon the constitutional properties common to a group of

homospecific enxymes that might account for the similarity

of their specificity requirements. At present, such

questions cannot be answered since our present knowledge

 5

of the constitutional details of proteolytic enzyme is

almost nil . There is only one constitutional property

that we know to be common to many proteolytic enzymes,

and that has frequently been discussed in connection with

their classification. It is a fact that many proteolytic

enzymes are active only after they have been activated by

HCN or sulfydryl compounds. Others are active when

activated by metals . The activation by HCN and sulfydryl

compounds is frequently and, according to Nord and Work-

man, (28) erroneously regarded as a reduction of the

general type described schematically in table l.

Table l . The activation of intracellular enzymes

Oxidation-Reduction Theory

2 (Enz . SH) Oxidation Enz . S-S Enz .

Proteolytically Reduction Proteolytically

On the other hand , a group of enzyme chemists have advanced

the hypothesis that all proteolytic enzymes have a du a listic

nature in that they consist of a colloidal protein acting

as a carrier and a specific active part of unknown chemical

nature. It is known from the experiments of Johnson (20)

that the activity of the intestinal enzyme the hydrolyses

L-Leucylglycine increases considerably on the addition of

manganese or magnesium salts. The enzyme is also known to

be inhibited by HCN or H2s. Nord and Workman (28) pointed

out that, these properties may be taken as indications

 6

that the active enzyme is a dissociable metal protein com-

pound. Indeed, it has recently been observed that on
dialysis of the active enzyme preparation is obtained which
is completely inactive towards L-Leucylglycine but which
regains a high activity on addition of manganese or mag-
nesium salts. This reactivation is the time reaction and
the degree of the final activity depends upon the con-
centration of the metal ions added.
The activation of the intracellular enzymes of plants
and animals by HCN and sulfhydryl compounds is complicated
by the fact that these enzymes are almost always accompanied
by sulphur containing compounds which are sometimes referred
to as natural activators (43). Recently papain and cathe-
psin preparations have been obtained that contain no natural
activators (24). Such purified papain preparation was found
to be completely inactive towards the substrate benzoylar-
ginine amide and it remained inactive after HCN had been
added. There are some enzymes like purified papain trypsi-
nase which is inactive and is not activated by HCN and
which is known as papain trypsinase may be activated by
H2S (28). When the H2S is subsequently removed in vacuo,
a second inactive form papain B trypsinase is obtained.
B. trypsinase can be activated by HCN.
The reaction can be represented as follows:
~ papain Reduction B papain H2S H2S-B papain
trypsinase trypsinase trypsinase
( i nac t U'e) "'o<=x'"'ir-:ar:a'"'t'"'lr:o=n (inactive) 'Evacuation (active)
According to Colowick and Kaplan (10) the plant proteinases
are broadly classified in table 2.
 Table 2. Properties of plant proteinases (lO)

Enzyme Common Genus and Source of Crystal- Isoelectric Stability Heat pH of

name of species material line form PH in acid Stability optimum
plant or alkali

Papain Papaya Carica Latex of Needles 9 Unstable Half life 7- 7.5

Papaya green Hexagonal below pH 56 min.
fruit plates 2 . 5 and at 75° C
above 12

Chyme- Papaya Carica Latex of Needles - Stable at - 7

papain Papya green plates pH 2
fruit

Ficin Fig Ficus Carica Latex Hexagonal 5 Stable at - 7

Glabrata plates pH 2
Coliaria

Mexicain Cuagua-Pileus Leaves Lanceolate - Stable at

yote Mexicanus fruit plates pH 8

Asclepain Milk- Asciepias Latex Rectangular 3.1 Unstable Asclepain 7 - 7 . 5

weed Species a roots plates in acid 78 min.
Mexicana or alkali at 60°
Cyriaca

Bromelin Pine- Anana Satira Fruit ----- - Unstable 21-5 min. 6 - 7

apple leaves at PH 2.3 at 60°

Pingui- Maya Bromelia Fruit ----- - - - 3

nain Pinguin

Taberna Taberna- Sap ----- - - - 5 - 6

Mountanin montana fruit
Grandiflora
 Table 2. (Continued)

Enzyme Common Genus and Source of Crystal- isoelectric Stability Heat pH of

name of species material line form pH in acid stability optimum
plant or alkali

Soy in Soya
bean
Soya
Hispidus
Germ in-
ated beans
- - - - 6 - 8

Solana in Horse- Solanum Fruit - - Stable in 51 min. 8.5 weak

nettle Elaeagni- diluted at 750 8
folium alkali

Euphor- Caper Euphorbia Latex - - - - 6 Strong

bain Spurge Lathyris

Hurain Jabillo Huracrepi- Sap - - 4-5 stable - 5 Weak

tans in diluted
alkali

Pomiferin Osage Mac lura Fruit - - - - 6.5

Orange Pomifera

Arachain Peanut Arachis Seed - - - Destroys 0 6- 7.5

Hypagen above 40
 9

The structure of the activated enzyme agrees with the

theory of the dualistic nature of proteolytic enzymes.

However, specificity studies do not support the claim that

the protein part of the dualistic enzyme is merely a col-

loidal carrier for another and supposed to be active.

There are many methods available which can be used

for determining proteinase activity. The methods like

Van Slyke reaction method, formal titration. Ninhydrin

colorimetric test can be used in determining proteinase

activity. It is the purpose of this study to compare some

methods like the A.O.A.C. (Fuld Gross Method) (1), Curd

Tension Method, Clotting Method, which are now available

and then modify a suitable one. The A.O.A.C . specifies

the Fuld Gross Method which is quite elaborate and time

consuming. The method is based on determining the rate

of protein hydrolysis.
 EXPERIMENTAL PROCEDURE

In this project two different types of enzymes,

animal proteinase, and vegetable proteinase were used.

An inactive animal enzyme the so called proenzyme or

zymogen lot No. D. 870-156-l was obtained from the "' Armour

Laboratories" and mixase C an active enzyme which con-

tained mixture of enxymes Lot No. 530-295-l obtained from

the same laboratories. The proenzyme or zymogen was

activated by the addition of small amount of an active

enxyme mixase C.

The vegetable proteinase "p" which was an active

enzyme Lot No. 20208 obtained from " Paul Lewis Laboratories. "

Since the vegetable proteinase was active it was used di-

rectly. The source of the vegetable proteinas was from

the latex of the fruit of papaya (Carica papaya).

The following three methods were compared for enzyme

activity and then the suitable one was modified for practical
purposes.

1. Fuld Gross Method.

2. Curd Tension Method.

3. Clotting Method.

Fuld Gross Method

Method

Fuld Gross Method outlined by A.O.A.C. is as follows:

 ll

Inactivated. If the enzyme preparation is solid,

finely divide it by grinding to a smooth paste in a small

morter with a little freshly boiled cold water. Then

suspend in cold boiled water. After 5-10 minutes centri-

fuge and discard sediment .

Activated. Proceed as directed in (a) but use half

standard H2S- H20 instead of boiled water. After centri-

fuging, incubate enzyme solution at 40° C. for one hour

to complete activation.

Reagents

Casein solution. Make 6 percent solution of Hammer-

stein's casein solution using 60 grams of casein with a

little water in morter and gradually adding 60 ml. or

lNNaoH and water until volume totals l liter. Heat

viscous solution for 30 minutes in the bath of boiling

water; cool and filter if necessary.

Buffer solution. Prepare 0.2 M. monosodium citrate

solution by partial neutralization of citric acid with

NACH.

Titrating solution: 0 . 1 N. alcoholic KOH.

Indicator. 1 percent alcoholic thymopthalein solution.

Determination

Place 10 ml. of the casein solution and small charge

of 4 mm. diameter glass beads in each of several 125 ml.

glass-stoppered bottles and bring bottles and contents to

40° C. Add desired volume of prepared enzyme solution

 12

but do not use more than 4 ml. If the quantity is

insufficient, prepare a more concentrated solution of

enzyme. Add immediately exactly 3 ml. of buffer solution

in constant-temperature water bath at 40° C. Incubate

mixture 20 minutes at 40° C., counting time from adding

of buffer. Add 1 ml. of the indicator and begin titrating

with the titrating solution. As soon as deep blue color

appears, shake the bottle until the color is discharged

or ppt. is completely dissolved. (It is usually best to

add the alkali in doses of ca. 0.5 ml. at a time.) When

all ppted. casein has been brought into solution, transfer

contents of bottle to 400-500 ml. flask and rinse out

bottle 2-3 times with alcohol, using total of 25 ml. for

this purpose. Add a sufficient amount of the KOH

solution to restore blue color, then add 175 ml. of

boiling alcohol . Carefully add more KOH solution until

pale but distant blue color persists in solution. Make

control titration exactly as described, but do it

immediately after addition of the buffer and therefore

without any incubation time. Differences between

titration of undigested sample and that of digested

sample is a measure of proteolytic activation of the

enzyme.

Calculation of proteinase unit

For smaller quantities of enzyme extent of hydrolysis

determined by titration described is a straight line

fraction of quantities of papain used. For accurate work

 13

determination, draw a straight line by making several

titrations with different quantities of enzyme. If

quantities of papain used are too large, the straight

line relationship will no longer hold. If they are too

small, determinations will be inaccurate. Quantities of

enzymes giving titration differences of 0.6- 1 . 2 ml of

0.1 N. KOH are recommended. A unit of papain may be

considered to be quantity of enzyme that produces under

conditions outlined. Titration differences of l ml. of

0.1 N. KOH are determined either graphically or arith-

metically . The value of the original preparation is then

expressed in units/ mg. as mg. of papain necessary to make

one unit.

Curd Tension Method

One hundred cc. of milk of good quality was placed

in glass jars as used for curd tension measurements . Samples

were placed in a constant temperature water bath at 96° F .

To 100 cc. of milk, l cc. of solution of enzyme to

be tested was added. This was followed by addition of

l cc. of rennet solution made by mixing 9 cc. of rennet

extract with 91 cc. of cold water.

After 20 minutes, the curd was tested with a curd-

meter. Three g of active proteinase along with 1.5 g of

inactive proteinase were dissolved in 50 cc. of distilled

water and the solution was then centrifuged at 1000 rpm

for 10 minutes and supernatent solution was used for the

test.
 14

Clotting Method

Although the milk clotting action of the proteinase

has been recognized for many years, this property as the

enzyme has received little quantitative attention.

The method was based upon the principle of c l otting

the ca-caseinate in a certain interval of time. Add a

suitable amount of enzyme to be tested in 200 ml. milk.

The milk containing the enzyme was placed in a 250 cc.

graduated flask with a hole near the bottom in which was

inserted a capillary glass tubing which is bent in such

a manner as to prevent the milk flowing through the tube

to drip slowly on a piece of black bakelite so the clot

could be easily detected and the time was recorded.

The enzyme solution was added to the milk immediately

before the addition of rennet. On the other band, when

fully activated enzyme was added to the milk, the rate of

digestion or breakdown of the curd is sometimes so rapid

that much of the casein is lost in the whey . Futhermore ,

an active enxyme produced a curd which was soft and

flocculent .
 RESULTS AND DISCUSSION

Different concentrations of animal proteinase was

used and the average activity in terms of units / mg was

measured by A.O.A.C. method. It was found that increase

i n concentration of the enzyme increases the average

activity in terms of units / mg. Figure 1 of enzyme con-

centration was plotted against activity units / mg was a

straight line indicating enzyme activity increases with

increase in enzyme concentration.

Table 3. Enzyme concentrations and the average activity .

(A.O.A . C. Method)

Concentration of enzyme Average activity

(ml.) Units/ mg .

0.1 0.21
0.2 0 . 45
0 .3 0 . 60
0 .4 0 . 80
0.5 0.95
0.6 1.20
0.7 1.32
0 .8 1.50
0.9 1.70
1.0 1.85
 16

18
/

16

14
I
12
bl)
s
'rn
:j.O
c
~

»
.........
~8
.....
0
<
6

/
4 I
I
2 I

I 1 2 3 4
- .,.-- ~-

5
~ --

6
,. --
7 8 9 10
Enzyme concentration (ml) v

Figure 1. Relationship between enzyme concentrations

and its activity (A . O. A.C. Method) (Animal
proteinase)
 17

Table 4. Curd tension method: relationship between enzyme

concentration and the clotting time

Enzyme concentration in ml . Time of clotting in min.

0.1 2.30
0.2 2.17
0.3 1.38
0.4 1.30
0.5 1.13
0.6 0 . 95
0.7 0.80
0 .8 0.65
0.9 0.57
1.0 0.47

Different concentrations of proteinase was used for

clotting the milk, but the curd was soft so that the curd-

meter did not give any significant results.

Tab l e 5. Clotting method : relationship between enzyme

concentration and the clotting time

Enzyme concentration in ml. Time of clotting in min.

- .1 2.30
0 .2 2.17
0.3 1.38
0.4 1 . 30
0.5 1.13
0 .6 0.95
0 .7 0.80
0.8 0.65
0.9 0.57
1.0 0 .4 7

Different concentrations of proteinase was used and the

time of clotting was recorded. It gave some significant

results . Then it was thought to modify the clotting method .

 18

Modi fi ed Cl o ttin g Method

Different combinations of inactive on active enzymes

were tried . The best results were obtained. The following

combination was used :

0.7 grms. of unactive enzyme

0 . 3 grms of Mixase C (active enzyme)

The above enzymes were dissolved in 100 cc. of distilled

water. Lower concentrations than this of active enzyme

increased the clotting time; while if the higher concen-

trations than the above were used , then too soft curd and

more proteolysis took place .

Preparation of enzyme solution

0 . 7 grms . of unactive proteinase DH 70-150-1 was

accurately weighed out and it was then dissolved in 100

cc . of distilled water. Then 0.3 grms. of mixase C, an

active proteinase, was dissolved in the same solut i on .

The solution was then centrifuged at 1000 rpm for 15

minutes, and the sediment was removed. The supernatant

enzyme solution was taken for the test.

The milk was heated to 37° C. before using the enzyme .

It seems that at the concentration of 4- 5 mg ., it gives

a good curd . The body and texture of the curd is also

quite g ood.

Kinetics of the clotting process

Except for very small concentrations of proteinase,

the time required for clotting is inversely proportional

 19

to the amount of proteinase present .

1
Clotting time Amount of proteinase present

The relationship between the clotting time and the enzyme

concentration is therefore a straight line as shown in

figure l .

Table 6 . Effect of variation of animal proteinase enzyme

Amount of Amount of Time of Type of E = K/ T

mil k enzyme clotting curd or
in min. E = ET
10 cc. 15 mg . 1.3 too soft 19.5
10 cc. 12 mg . 1.15 too soft 13.80
10 cc. 10 mg . 1.37 too soft 13 . 70
10 cc. 9 mg . l. 50 soft curd 13.50
10 cc. 8 mg. l. 54 soft curd 12.32
10 cc. 7 mg. 2 .0 soft curd 14 . 00
10 cc. 6 mg. 2.15 soft curd 12.9
10 cc. 5 mg. 2 . 20 good curd 11.00
10 cc. 4 mg . 2 . 25 good c urd 9.00
10 cc . 3 mg . 2 . 50 semi-hard 7.20
10 cc. 2 mg. 3. 15 semi-hard 6 . 30
10 cc. l mg. 4.10 semi-hard 4.10

Where E is the weight of the enzyme in milligrams and

and T is the time in minutes and K is constant

E • KI T - - - - - - - - (l)

It follows from the equation (l) that :

E = K when T = l. therefore the activity per mg.

l l
(~ ) is K.
At low concentrations of proteinase, however, the

relationship no longer holds. The time required for co-

agulation by a small dose of enzyme is much longer than

 20

would be expected from the foregoing and the system behaves

as though a part of the enzyme did not take part in the

clotting reaction . Experiments have shown, however, that

the amount of proteinase too small to clot the milk within

a reasonable time, nevertheless, has the same effect on

the system because such treatment of milk reduces the

clotting time observed when an adequate quantity of enzyme

is subsequently added .

If C denotes the amount of enzyme removed from the

action during clotting, then available enzyme is E - C.

C can be determined by drawing an intercept on the E axis

1
when T is plotted against E, and the previous expression

(equation 1) becomes

(E - C)T =K
This accurately describes the relation between the time

and the enzyme concentrations over the range of our

experiments.

While the clotting with rennin is represented by the

expression

E (T - X) • K,
X is probably the time lag of clotting after proteolysis

has reached the requisite stage.

Too low concentration of rennin in presence of

proteinase gave semi-hard curd, as in table 7, while too

high concentrations of rennin the the presence of prote-

inase gave very soft curd and the separation of whey. The

optimum concentration of rennin is between 0.3- 0.4 ml.

 21

Table 7. Effect of variation of rennet extract on the

animal proteinase activity&

Amount Amount of Amount of Time of Type of

of milk rennet enzyme clotting curd
in ml. solution (in min.)

10 ml . 0.1 ml. 5 mg. 1.58 semi-hard

10 ml. 0.2 ml. 5 mg. 1 . 52 semi-hard
10 ml. 0.3 ml. 5 mg. 1.43 good curd
10 ml. 0.4 ml. 5 mg. 1.37 good curd
10 ml. 0.5 ml. 5 mg. 1.20 soft curd
10 ml. 0.6 ml. 5 mg. 1.17 soft curd
10 ml. 0.7 ml. 5 mg. 1. 70 very soft
10 ml. 0 .8 ml. 5 mg. 0 . 7l very soft
10 ml. 0.9 ml. 5 mg. 0 . 68 very soft
10 ml. 1.0 ml. 5 mg. 0.52 very soft
and sepa-
ration of
whey

a9 cc. of the rennet extract was dissolved in 91 cc. of

the distilled water.

Effect of variation of S. N. F. in milk

It is also interesting to observe the effect of diff-

erent concentrations of solids non-fat on the proteinase

activity. The milk samples were first analysed by the

Majonnier method.

From the table 9 and the figure 3 it concluded that

increased concentrations of S . N.F. decreases the clotting

time and hence increases the proteinase activity . At very

high concentrations of S.N.F., the activity of the prote-

inase decreases.

Mechanism of proteinase action ~ casein

Concerning the mechanism of the reaction between the

 22

'T
1.6 ~

1. s·

1. 4-
I

Ill 1.3-
...
Q)

:I
...s
d

E-< 1 . 2-

1.1-

t I t -- - t
9 10 11 12 13 14
S.N.F. concentrations
Figure 2. Relationship between clotting time
(minutes) and S.N.F . concentrations .
(animal proteinase)
 Table 8. Analysis of the milk sample

Determination of total solids Determination of fat Determination of S.N.F.

Dish T solids 17.4248 17.9296 Dish -t- fat 36.6475 36.8878 II

Wt. of dish 17.1832 17 . 6892 Wt. of dish 36.2952 36.5366 8.557 8.508

Wt. of solids 0.2416 0.2404 Wt. of fat 0.3523 0.3512 Ave. S.N.F. = 8.5302%

Wt. of sample 2 grms. 2 grms. Wt. of sample 10 grms.lO grms.

% T . S. 12.08 12.02 % fat 3.523 3.512

""w
 24

Table 9 . Effect of variation of Solids-non-fat in milk

on animal proteinase activity.a

% S.N.F. Amount of Time of Type of K • ET (for

in mil k S.N.F. in Clotting curd proteinase)
25 ml. of (min.)
milk

8.5 1.58 soft curd 7.90

9.0 0.25 grms 1.51 soft curd 7.55
9.5 0.375 grms. 1.47 soft curd 7.35
10.0 0.50 grms. 1.35 soft curd 6.75
10.5 0.625 grms. 1.20 soft curd 6.00
11 . 0 0.750 grms. 1.15 semi-hard 5.75
11 . 5 0 . 875 grms. 1.10 semi-hard 5.50
12.0 1.00 grms. 0.67 semi-hard 3.35
12.5 1.125 grms. 0.88 semi-hard 4.40
13.0 1.250 grms. 0.90 hard 4.50
13 . 5 1.375 grms. 1 . 00 hard 5.00
14 .0 1 . 500 grms. 1.06 hard 5.30

aSamples were taken with 10 cc. of milk, 5 mg. of enzyme,

and 0 . 3 ml. of rennet.

proteinase and casein, different authors reached similar

views in different ways .

.s...,.
E+S "'iC2" E-S E + p

It is assumed that each casein molecule, before it

is decomposed, first combines with the proteinase enzyme.

Later, after a definite time interval, the substrate is

split and throws off its products. In the case of prate-

inase and casein, different products like proteoses,

peptones, polypeptides, and aminocide can be formed. After

the products are ejected, the place of combination on the

enzyme molecule is left vacant until another casein molecule

makes contact.
 25

Combination and decomposition are repeated. The time

required for a single cylce is the sum of the time required

for another casein molecule to hit the proteinase on the

combining point plus the time the proteinase then takes to

split the casein molecule and eject its products. The

more abundant the casein molecules are about the proteinase

enzyme, the shorter will be the probable path of the next

casein molecule to the combining point on the proteinase,

and hence the shorter will be the average time interval

during which the proteinase is left uncombined and there-

fore inactive, if the casein concentration is great enough.

This was done by increasing the concentrations of S.N.F.

in the milk; then the inactive interval becomes negligible

compared with the interval required for decomposition, when

the casein concentrations are at or above the level. The

proteinase works at the full speed, because its unused

intervals are negligible and further increase of casein

concentrations cannot push the reaction rate any faster.

Quantitative formulation of the kinetics of the two-phase

reaction in the case of proteinase during clotting

The time required for the cycle can be expressed as

the sum of the intervals required for the two consecutive

phases.

Time required for one cycle - 1 -t- K0 - - - (l)

Q
S = concentration of the casein

Kc = velocity constant for combination of

proteinase and casein
 26

Ko = velocity constant for decomposition of the

combined caseinate

-ds = velocity of casein or proteolysis

---a£
-ds K
---a£ = time required for 1 cycle

or

-ds = 1 - - - - - (2)
Cit I I
Q+xo
When the concentrations of casein are large enough so

that 1 (i.e . , when the combination of proteinase and

K~

casein is practically instantaneous in comparison with the

decomposition phase) the above velocity equation simplifies

to

---ds = K0 - - - - - (3)
at
Hence, Ko can be determined very simply as the rate of

proteolysis in sufficient concentration of casein.

Now, integration of equation (2) gives as the time

curve of a single reaction

T - 1 log 1 s
Kc x-:.s + XD
where A is the initial casein concentration and S is the

concentration after the proteolysis bas taken for T minutes.

It is obvious that S is so small that S/ Ko is negligible

and the equation simplifies to the monomolecular reaction

T = 1 log 1
Kc r=-s
 27

Table 10. Effect of variation of temperature on the

animal proteinase activity . a

Temp. Amount of Amount of Clotting Type of K:ET

in o C. enzyme rennet time in curd
minutes

lO~C. 5 mg . 0 .3 ml . 2.55 too soft 12.75

20 c. 5 mg . 0 .3 ml. 2.50 too soft 12 . 50
25°C. 5 mg. 0 .3 ml. 2.50 too soft 12.50
30°C. 5 mg. 0.3 ml. 1.80 soft 9.00
35°C. 5 mg. 0 .3 ml. 1.68 good 8.40
37°C. 5 mg . 0 .3 ml. 1.60 good 8 . 00
40°C. 5 mg. 0 .3 ml. 1. 51 good 7 . 55
45°C. 5 mg. 0 .3 ml. 1.30 slty. stiff 6.50
50°C 5 mg . 0.3 ml. 0.86 semi-hard 4 . 30
55°c: 5 mg . 0.3 ml. 1 . 70 semi-hard 3 . 50
60°C. 5 mg . 0 .3 ml. 0.65 hard-wheyed 3.25
off
65°C. 5 mg . 0 . 3 ml. 0.61 3 . 05
70°C. 5 mg. 0 . 3 ml. 0 . 50 2.50

~he optimum temp. seems to be at 37°C. At lower temper-

atures the curd was soft while at temperatures the curd
was rubbery.

Table 11. Effect of variation of pH on the animal

proteinase activity. a

Amount pH Amount Amount Clotting Type of K • ET

of milk of enzyme of rennet time in curd
minutes

10 ml. 1.5 5 mg. 0 .3 ml. none

10 ml. 2.0 5 mg . 0 .3 ml. none
10 ml. 7. 0 5 mg. 0.3 ml. 1.87 soft 9.35
10 ml. 7.5 5 mg. 0 .3 ml. 1.81 soft 9,08
10 ml. 8 .0 5 mg . 0.3 ml. 1. 79 good 8.95
10 ml. 8 .5 5 mg . 0.3 ml, 1.68 good 8 . 40
10 ml. 9.0 5 mg . 0 .3 ml. none
10 ml. 9. 5 5 mg. 0.3 ml. none

~he pH of the milk can be changed by using different

buffers. The pH was changed by using O. ln NaoH on the
alkaline side and using pure lactic acid on the acid side .
Milk of different pH was tried and observed the effect of
animal proteinase on clotting.
 28

It was found that when pH 1 . 5 or 2 . 0 were used , no

clotting took place even after a long time . The effect of

pH 2-6 could not be observed because of the isoelectric

point of casein. Whi l e on the alkaline side up to pH 8 . 5

it g ave fairly significant results . The milk did not clot

at the pH9 or above. It seems that proteinase activity

c an be best measured at pH between 8-8 . 5.

Effect of Variations of Different Salts on the

Animal Proteinase Activity

In determining the activity of the proteinase, it

was nec e ssary to find the effect of different salts , as

some s alts activate and other salts inhibit the proteinase

activity. One percent solution of the following salts

were pr e pared in distilled water and used in 10 ml . of

milk samples :

(1) Potassium Phosphate KH 2 P0 4

(2) Sodium carbonate Na2 co 3

(3) Calcium chloride Cacl2

(4) Sodium citrate Na 3 CsH 5 0 7 - 2H 2 o

(5) Ammonium sulphate (NH 4 ) 2 so 4

(6) Manganese chloride MnC12

 29

Table 12. Effect of potassium phosphate on animal

proteinase activity. a

Amount of Amount of Amount of Time of Type of K = ET

potassium enzyme rennet clotting curd
phosphate (min.)

1 mg. 5 mg. 0.3 ml. 1.81 soft 9.05

2 mg. 5 mg. 0.3 ml. l. 72 soft 8.60
3 mg. 5 mg. 0.3 ml. 1.20 soft 6.00
4 mg. 5 mg. 0.3 ml. 0.71 good 3.55
5 mg. 5 mg. 0.3 ml. 0.50 good 2.50
6 mg. 5 mg. 0.3 ml. 0.47 semi-hard 2.35
7 mg. 5 mg. 0.3 ml. 0.41 semi-hard 2.05
8 mg. 5 mg. 0.3 ml. 0.37 hard 1.85
9 mg. 5 mg. 0.3 ml. 0.25 hard 1.25
10 mg. 5 mg. 0.3 ml. 0.20 hard 1.00

aPotassium phosphate increased the proteinase activity.

The optimum concentration of potassium phosphate was
between 4-5 mg.

Table 13. Effect of sodium carbonate on animal

proteinase activity.a

Amount of Amount of Amount of Time of Type of K = ET

sodium enzyme rennet clotting curd
carbonate (min.)

1 mg. 5 mg. 0.3 ml. 0.55 very slight 2.75

clotting
2 mg. 5 mg. 0.3 ml. 0.75 very slight 3.75
3 mg. 5 mg. 0.3 ml. 1.10 very slight 5.50
4 mg. 5 mg. 0.3 ml. none
5 mg. 5 mg. 0.3 ml. none
6 mg. 5 mg. 0.3 ml. none
7 mg. 5 mg. 0.3 ml. none
8 mg. 5 mg. 0.3 ml. none
9 mg. 5 mg. 0.3 ml. none
10 mg. 5 mg. 0.3 ml. none

~he amount of Na2C03 had a direct effect on the proteinase

activity. As the concentration of Na2c0 3 was increased the
clotting time was increased and, when the concentration
reached 6 mg. or above, there was no clotting of milk.
 30

Table 14. Effect of calcium chloride on the animal

proteinase activity.a

Amount of Amount of Amount of Time of Type of K • ET

calcium enzyme rennet clotting curd
(min . )

1 mg. 5 mg. 0.3 ml. 0.78 soft 3 . 90

2 mg. 5 mg . 0.3 ml. 0.62 soft 3.10
3 mg . 5 mg . 0 .3 ml. 0 . 58 good 2.90
4 mg . 5 mg . 0.3 ml. 0 . 42 good 2.10
5 mg . 5 mg. 0.3 ml. 0.42 good 2.10
6 mg . 5 mg. 0 .3 ml. 0 . 40 firm 2.00
7 mg. 5 mg. 0 .3 ml. 0.38 firm 1.90
8 mg . 5 mg. 0 .3 ml. 0.31 hard 1.55
9 mg. 5 mg. 0 .3 ml. 0.28 hard 1.40
10 mg . 5 mg. 0 .3 ml. 0 . 21 hard 1.05

~he increase in CaCl2 concentration increased the prote-

inase activity. The increased calcium ions made the curd
firmer . After certain concentration of the CaCl2, the
curd became hard .

Table 15. Effect of manganous carbonate on the animal

proteinase activity . a

Amount of Amount of Amount Clotting Type of K ET

mang anous enzyme of time curd
carbonate rennet (min . )

l mg . 5 mg . 0.3 ml. 0 / 57 semi-liquid 2.85

2 mg. 5 mg. 0 .3 ml. 0.51 semi-liquid 2.55
3 mg. 5 mg . 0 .3 ml. 0.49 semi-liquid 2.45
4 ' mg. 5 mg. 0.3 ml. 0.49 semi-liquid 2 . 45
5 mg. 5 mg . 0 .3 ml. 0.38 semi-liquid 1.90
6 mg. 5 mg . 0 .3 ml. 0.29 soft 1.45
7 mg. 5 mg. 0.3 ml. 0.26 soft 1.30
8 mg. 5 mg. 0 .3 ml. 0 . 20 soft 1.00
9 mg. 5 mg. 0.3 ml. 0.18 soft 0.90
10 mg. 5 mg. 0 .3 ml. 0.12 soft 0.60

aT he increased concentrations of MnC03 increased the prote-

inase activity when it was used less than 5 mg . It did clot
the milk.
 31

When different concentrations of Na-Citrate were

used in milk samples, the clotting was prevented. In other

words, the proteinase activity was inhibited.

Table 16 . Effect of ammonium sulfate on animal

proteinase activity.a

Amo unt of Amount of Amount of Clotting Type of K • ET

(NH 4 )so 4 enzyme rennet time curd
(min . )

1 mg. 5 mg . 0 .3 ml . l. 75 semi-soft 8.74

2 mg. 5 mg . 0 .3 ml. 1. 62 semi-soft 8 . 10
3 mg. 5 mg . 0 .3 ml. 1.60 semi-soft 8.00
4 mg. 5 mg . 0.3 ml . 1.57 semi-soft 7 . 85
5 mg . 5 mg. 0.3 ml. 1.55 soft 7.75
6 mg. 5 mg. 0 .3 ml. 1.48 soft 7.40
7 mg. 5 mg. 0 .3 ml. 1.30 soft 6.50
8 mg. 5 mg. 0.3 ml. 1.21 soft 6.05
9 mg . 5 mg. 0.3 ml. 1.15 firm 5 . 75
10 mg . 5 mg . 0.3 ml. 1.10 firm 5.50
aThe increase in concentration of ammonium sulfate de-
creased the clotting time and hence increased the proteinase
activity .

Table 17. Effect of manganese chloride on animal

proteinase activity.a

Amount of Amount of Amount of Clotting Type of K • ET

Mncl 2 enzyme rennet time curd
(min.)
1 mg. 5 mg. 0 .3 ml . 0 . 73 soft 3.65
2 mg. 5 mg . 0 .3 ml. 0 . 68 soft 3.40
3 mg. 5 mg. 0.3 ml. 0.62 soft 3.10
4 mg. 5 mg . 0.3 ml. 0.56 semi-hard 2 . 80
5 mg. 5 mg . 0 .3 ml. 0.51 semi-hard 2.55
6 mg . 5 mg. 0.3 ml. 0.46 semi-hard 2.30
7 mg. 5 mg . 0 .3 ml. 0.39 hard 1.95
8 mg. 5 mg. 0.3 ml. 0.30 hard 1.50
9 mg. 5 mg. 0.3 ml. 0.22 hard 1.10
10 mg. 5 mg. 0.3 ml. 0 . 18 hard 0 . 90

~he increase ill concentration of Mncl2 decreased the clotting

time and hence increased the proteinase activity. The curd
was comparatively harder than other curds.
 32

It was determined from the above tables and figure 3,

salts like potassium phosphate, CaCl2, MnCo3, ammonium

sulfate, decreased the clotting time and hence they in-

creased the proteinase activity; while salts like Na2Co3,

Na-Citrate increased the clotting time, that is, they

decreased the proteinase activity.

Activity of the Plant Proteinase

The effect of various concentrations of the plant

proteinase was used to determine its activity. It was

found that when l gram of the proteinase was dissolved in

100 cc. of distilled water it gave the best results. When

2 percent enzyme solution was used, the curd obtained was

too soft and when less than 1 percent proteinase concen-

tration was used the clotting time was prolonged.

Preparation of enzyme solution

One gram of active plant proteinase was dissolved in

100 cc. of distilled water, and the solution was centri-

fuged at 1000 rpm for 15 minutes and the sediment was

removed. The supernatant enzyme solution was used for

the tests.
 33

2 .0 I 1
1. 75 ~
1.50
\
\
1 . 25

...::>~1.0
.....c
a
Eo<
0.75

0.5

0 . 25 .

1 2 3 4 5 6 7 8 9 10
Amount of salts (mg)

Figure 3. Relationship between clotting time

(minutes) and different salts.
(Animal proteinase)
 34

1.00

I
0 . 75

I
,/ I
I
I
.
'
I
I
/

I II I
I

I
0
I
0.50 I

I
I

I
.I
I
'I
I
I
0 . 25 I

0
2 4 6 8 10 12 14 16 18
E mg
Figure 4 . Relation between clotting time
(reciprocal) and the amount of
enzyme. Curve I represents vege-
table proteinase. Curve II re-
presents animal proteinase .
 35

5.5

5.0

4.0

3.0
Ul
II)
+"
:l
....c:
~-
s
E-o

2 0 -
0

1.0 -

L. 8 9 10 11 12 13 14
S.N . F . concentrations
Fi g ure 5. Relationship between clotting time
(minutes) and S . N. F . concentrations.
(vegetable proteinase)
 36

Table 18. Effect of variation of plant proteinase. a

Amount of Amount of Clotting Type of K ET

milk enzyme time curd
(min.)

10 ml. l mg. 2.4 soft 2.4

10 ml. 2 mg. 2.0 soft 4.0
10 m1. 3 mg. 1.74 soft 5.22
10 ml. 4 mg. 1.60 soft 6.40
10 ml. 5 mg. 1.56 soft 7.80
10 ml. 6 mg. 1.48 soft 8.88
10 ml. 7 mg. 1.38 good 9.66
10 ml. 8 mg. 1.20 good 9.60
10 m1. 9 mg. 1.10 slight bard 9.90
10 ml. 10 mg. 1.07 slight hard 10.7

alt gave good curd at the concentrations between 7-8 mg.

Lower concentrations than these gave very soft curd and
higher concentrations gave hard curd.

Table 19. Effect of variation of rennet on plant

proteinase activity

Amount of
enzyme
Amount of
rennet
Clotting
time
Type of
curd
K = ET
(min.)

7 mg. 0.1 ml. 1.55 good 10.85

7 mg. 0.2 ml. 1 .30 good 9.10
7 mg. 0.3 ml. 1.20 semi-bard 8.40
7 mg. 0.4 ml. 1.17 semi-bard 8.19
7 mg. 0.5 ml. 1.10 semi-bard 7.70
7 mg. 0.6 ml. 1.05 hard 7.35
7 mg. 0.7 ml. 1.03 bard 7.21
7 mg. 0.8 ml. 0.45 bard 3.15
7 mg. 0.9 ml. 0.43 hard 3.01
7 mg. 1.0 ml. 0.30 hard 2.10

aTbe rennet at the concentration of 0.1 gave higher K

value and it also gave good curd. The optimum rennet
concentration was taken as 0.1.
 37

Table 20. Effect of variation of solids-not-fat on the

plant proteinase activity.a

% S.N.F. Amount of SNF Clotting Type of K ET

in milk added in 25 Time curd
ml. of milk (Min.)

8.5 0 . 25 grms. 5.58 soft 39.06

9.0 0.375 grms. 5.47 soft 38.29
9 .5 0 . 50 grms 5 . 20 soft 36.40
10.0 0 .625 grms. 4.81 soft 33.67
10.4 0.750 grms. 4.61 soft 32 .2 7
11.0 0.875 grms. 3.48 semi-hard 24.36
12.0 1.00 grms. 3 15
0 semi-hard 22.05
12.5 1.125 grms. 3.0 hard 21.00
13.0 1.250 grms. 2.48 hard 17.36
13.5 1.375 grms. 2.45 hard 17 .15
13.5 1.500 grms. 2.31 hard 16 17
0

14.0 2.11 hard 14.77

aSamp1es were taken with 10 ml. of milk, 7 mg. of enzyme,

and 0.1 ml. of rennet. It was determined that as the
S.N.F. in the milk was increased, the clotting time de-
creased, and hence proteinase activity was increased.

Table 21. Effect of variation of temperature on the plant

proteinase activity.

Temp. Amount of Amount of Clotting Type of K = ET

in ° C. enzyme rennet time curd
(min . )

l0°C. 7 mg. 0.1 ml. 8.30 too soft 58.10

15°C. 7 mg . 0.1 ml. 7.45 too soft 52.15
20°C 0 7 mg. 0.1 ml. 7.30 too soft 51.10
25°C 0 7 mg. 0.1 m1. 6.48 too soft 45.36
30°C. 7 mg . 0.1 ml. 5.58 soft 39.36
35°c . 7 mg. 0.1 ml. 5.45 soft 39.25
3 7°C. 7 mg. 0.1 ml. 4.42 soft 36.94
40°C. 7 mg . 0.1 ml. 4.18 good 29 .26
45°C. 7 mg. 0 0l ml. 3.50 slight stiff 24.50
50°C 0 7 mg. 0.1 ml. 3 010 slight stiff 21.70
55°C 0 7 mg. 0.1 m1. 3.05 hard 21.35
60°C. 7 mg. 0.1 ml. 2.65 bard 18.55
65°C. 7 mg. 0.1 ml. 2.15 bard 15.05
70°C. 7 mg. 0 .1 ml. 1.35 bard 9.45
 38

11.0

1.0

9 .0 -

8 .0 -

7.0 -
Ill
6.0
....:IQ) II
.....r::
a 5.0
E-o

4 .0

3 .0 .J

-·- I
----
2.0 '-
., ),

1.0 ' '

· --~ -._ -.I
'o

0 10 20 30 40 50 60 70
Temperature oc

Figure 6 . Relationship between clotting time

(minutes) and temperature (OC).
Curve I represents animal proteinase.
Curve II represents vegetable prote-
inase .
 39

Various temperatures from 10° C to 700 C were tried.

The optimum temperature of the plant proteinase activity

was at 40° C. At lower temperatures it gave soft curd

and the time of clotting was delayed. When the higher

temperatures were used the plant proteinase gave bard

curd but there was too much separation of whey. The clot-

tin g time at higher temperatures was decreased .

Effect of Various Salts on the Plant Proteinase Activity

In determining the plant proteinase activity it was

necessary to find the effect of various salts , as some

salts increase the proteinase activity while some decrease

the proteinase activity. One percent solution of the

following salts were prepared in distilled water and their

effect on the plant proteinase activity was determined.

Table 22 . Effect of potassium phosphate on the plant

proteinase activity.a

Amount of Amount of Amount of Clotting Type of K -ET

phosphate enzyme rennet time curd
(min.)

l mg. 7 mg. 0.1 ml . 4.48 soft 31.36

2 mg. 7 mg. 0.1 ml. 3.05 soft 21.35
3 mg. 7 mg. 0.1 ml. 3.00 soft 21.00
4 mg. 7 mg. 0.1 ml. 2.35 soft 16.45
5 mg. 7 mg. 0.1 ml. 2.30 soft 16.10
6 mg. 7 mg. 0.1 ml. 2.15 soft 15.05
7 mg. 7 mg. 0.1 ml. 2.00 soft 14.00
8 mg. 7 mg. 0.1 ml. 1.55 soft 10.95
9 mg. 7 mg. 0 1
0 ml. 1.48 soft 10.36
10 mg. 7 mg. 0 .1 ml. 1.30 bard 9.10

aAs the concentrations of KH2P04 was increased the time of

clotting was decreased and hence the plant proteinase
activity was increased.
 40

Effect of sodium carbonate ~ the plant proteinase activity

Various concentrations of sodium carbonate were tried

but there was no clotting and hence the Na 2 co 3 prevents

the proteinase activity.

Table 23. Effect of managanous carbonate on the plant

proteinase activity. a

Amount of Amount of Amount of Clotting Type K = ET

MnCo3 enzyme rennet time of
(min.) curd

1 mg. 7 mg. 0.1 ml. 4.15 soft 29.05

2 mg. 7 mg. 0.1 ml. 4.10 soft 28.70
3 mg. 7 mg . 0.1 ml. 4.00 soft 28 ". 00
4 mg. 7 mg. 0. 1 ml. 3. 58 soft 25.06
5 mg. 7 mg. 0.1 ml. 3.52 soft 24.64
6 mg. 7 mg. 0.1 ml. 3.00 soft 21.00
7 mg. 7 mg. 0.1 ml. 2.57 soft 17.99
8 mg. 7 mg. 0.1 ml. 2.41 soft 16.87
9 mg. 7 mg. 0.1 ml. 2.38 soft 16.66
10 mg. 7 mg. 0.1 ml. 2.23 soft 15.61

alncrease in concentrations of MnCo 3 decreased the clotting

time and hence increased the plant proteinase activity.

Table 24. Effect of calcium chloride on the plant

proteinase activity.a

Amount of Amount of Amount of Clotting Type of K=ET

Cac12 enzyme rennet time curd
(min.)
1 mg. 7 mg. 0.1 ml. 4.45 soft 31.15
2 mg. 7 mg. 0.1 m1. 4.31 soft 30.17
3 mg. 7 mg. 0.1 ml. 4 . 26 soft 29.82
4 mg. 7 mg. 0.1 ml. 3.57 soft 24.99
5 mg. 7 mg. 0.1 ml. 3.48 soft 24.36
6 mg. 7 mg . 0. l ml. 3.38 semi-hard 23.66
7 rug. 7 mg. 0.1 ml. 2.55 semi-hard 17.85
8 rug. 7 mg. 0.1 ml. 2.30 semi-hard 16.10
9 mg. 7 mg. 0.1 ml. 2.25 hard 15.75
10 mg. 7 mg. 0.1 m1. 2.21 hard 15.47
awhen the calcium chloride concentration was increased, there
was a decrease in the clotting time, hence the proteinase
activity was increased.
 41

Effect of sodium citrate~ the plant proteinase activity

Different concentrations of Na Citrate were used,

but there was no clotting of the milk . This indicates

that Na Citrate inhibits the proteinase activity.

Table 25. Effect of ammonium sulfate on the plant

proteinase activity

Amount of Amount Amount of Clotting Type of K • ET

ammonium of rennet time curd
sulfate enzyme (min.)

1 mg. 7 mg. 0.1 ml. 6 . 31 soft 44.17

2 mg. 7 mg. 0.1 ml. 6.17 soft 43.19
3 mg. 7 mg. 0.1 ml. 6.03 soft 42 . 21
4 mg. 7 mg. 0 .1 ml . 5.59 soft 39 . 13
5 mg. 7 mg. 0.1 ml. 5 . 30 soft 37 . 10
6 mg . 7 mg. 0.1 ml. 5 . 15 hard 36.05
7 mg . 7 mg. 0.1 ml. 5 . 00 hard 35.00
8 mg. 7 mg. 0.1 ml. 4.80 hard 33 . 60
9 mg. 7 mg. 0 .1 ml. 4.35 hard 30.45
10 mg. 7 mg. 0.1 ml. 3 . 20 hard 22.40

It was determined from the above tables and figure 7

that salts like potassium phosphate, calcium, chloride ,

manganous carbonate and ammonium sulfate, decreased the

clotting time and hence there was increased in the

vegetable proteinase activity, while salts like sodium

carbonate, sodium citrate prevented clotting.

Effect of pH

The effect of pH on the vegetable proteinase activity

was determined by changing the pH of milk on alkaline ssd

side with 1 N NaOH. On the acid side, the pH of the milk

was changed with the help of pure lactic acid .

 42

4 \

,- T- , ______.........- , --- ~

0 1 2 3 4 5 6 7 8 9 10
Amount of salts (mg)
Figure 7. Relationship between clotting time (minutes)
and different salts.(vegetable proteinase)
 43

It seems that the optimum pH of the vegetable prote-

inase is between 7-7.5.

Table 26 . Effect of pH on the plant proteinase activity

Amount pH Amount of Amount of Clotting Type K=ET

of milk enzyme rennet time of
(min.) curd

10 ml. 1.0 7 mg. 0.1 ml. none

10 ml. 3.0 7 mg. 0.1 ml. none
10 ml . 7 . 0 7 mg. 0.1 ml . 4 . 30 soft 29 . 10
10 ml. 7.5 7 mg. 0.1 ml. 5 . 30 soft 37.10
10 ml. 8.0 7 mg . 0.1 ml. 5 . 67 soft 39.69
10 ml . 8.5 7 mg . 0.1 ml. none
10 ml . 9.0 7 mg . 0.1 ml. none
10 ml . 9.5 7 mg . 0.1 ml . none
10 ml . 10 . 0 7 mg. 0 .1 ml . none
10 ml. 10.5 7 mg . 0.1 ml . none

Table 27 . Animal proteinase activity by A.O . A.C . method

Amount of Surrette Burette Difference Activity in

enzyme reading reading in cc . units / mg .
before after
incuba- incuba-
tion in tion in
cc . cc .

l mg. 0 .5 0.7 0.2 0.2

2 mg . 0.4 0.8 0.4 0.4
3 mg. 0.4 0 .9 0.5 0.5
4 mg . 0 .3 1.0 0 .7 0 .7
5 mg. 0 .6 1.2 0.6 0.6
6 mg. 0 .5 1. 3 0.8 0 .8
7 mg . 0.4 1 . 33 0.93 0 . 93
8 mg . 0.41 1.4 0.99 0.99
9 mg . 0.6 1.6 1.00 1.00
10 mg. 0 . 72 1.75 1.03 1.03
 44

Table 28 . Vegetable proteinase activity by A.O.A. C .

method

Amount of Burette Burette Difference Activity in

enzyme reading reading in cc . units / mg .
before after
incuba.:. incuba-
tion in tion in
cc. cc.

l mg. l l
0 1 .3 0 .3 0 .3
2 mg . 1 .1 1.4 0.3 0 .3
3 mg . 1 .2 1 .6 0.4 0 .4
4 mg . 1.34 1.74 0 .4 0 .4
5 mg. 1.5 2 . 01 0 . 51 0 . 51
6 mg. 1.8 2 . 34 0 . 54 0 . 54
7 mg . 1.9 2 . 50 0.60 0 . 60
8 mg. 2 .0 2 . 62 0.62 0 . 62
9 mg. 2 .1 2.82 0.73 0 . 73
10 mg. 2.2 2. 9 7 0 . 77 0. 77

Th e animal proteinas e was more active than the

v egetab l e proteinase.

Comp arisons of Bitterness of the Cu r d De v el o ped

by Animal and Vegetable Prote i nases

An experiment was run to determine the bitterness of

the curd by using different concentrations of animal and

vegetable proteinases .

Animal proteinase

Di fferent concentrations like 0 . 5 to 25 mg. of animal

proteinase were tried and tested for bitterness . It was

found that there was no bitterness in the lower concen-

trations . The curd was sweet . There was developed a

very slig ht b i tterness at the concentrat i on of 22 mg . of

 45

animal proteinase and the bitterness was incr eased in the

higher concentrations. When 27 mg. or above concentrations

of animal proteinase were used there was no clotting.

Vegetable proteinase
In the case of vegetable proteinase different concen-

trations like 0.5 mg . to 25 mg. of 0 . 5 percent, 1 percent ,

and 2 percent were used . It was found that bitterness

was developed at the lower concentrations of 3 mg . of 0.5

percent vegetable proteinase, where the clotting time was

very much delayed and the intensity of bitterness was

increased with the increase in concentration . The curd

was very much bitter at the concentration of 5 mg. of 2

percent vegetable proteinase concentration.

It was found that bitterness in the case of anima l

proteinase developed only at the higher concentrations ,

while in the case of vegetable proteinase the bitterness

was developed at the lower concentrations and the intensity

of bitterness was increased with the increase of concen-

trations . The bitterness might be due to peptone formation

causing the breakdown of casein.

Nitrogen Determinations

Total nitrogen can be determined by Kjeldahl method

while amino nitrogen can be determined by Vd n Skyke

method. The free carboxyl groups can be determined by

Formal titration method . During the study formal titration

method was used to determine the free carboxyl groups in

 46

the proteolytic material .

Met bod

The milk was first heated to 370 C. In the case of

animal proteinase enzyme, 5 mg. of enzyme solution and

0.3 ml. of rennet were added while in the case of

vegetable proteinase, 7 mg. of enzyme and 0 . 1 ml of

rennet were added. The clotting took place in a few

minutes. The tubes were then centrifuged to separate

casein from the proteolytic material at 18,000 r.p.m. for

15 minutes. The supernatent material was poured into the

separate tubes and was analysed for the nitrogen content.

Formal titration method

One ml. of the sample was taken in a 50 ml. Erlenmeyer

flask and 2 drops of phenolptalein indicator was added

and titrated to a faint pink color with 0.25 N. NAOH .

The burette reading was recorded. Then 10 ml . of neutral

formalin (40 percent HCHO) was added and titrated again

to a definite pink end point . The difference in the

burette reading is the formal value.

Table 29 . Analysis of nitrogen determination

Amount of Amount of Nitrogen by Average

enzyme rennet formal Nit. / ml.
titration
method

1. Animal I II
proteinase 5 mg. 0.3 ml. 36.6 36 . 2 34.6

2. Vegetable
proteinase 7 mg. 0.1 ml. 27.85 27 . 80 27 . 82
 47

Reactions During Formal Titration

No. Ot-1
CH.fCOO
I
to P P Nt-1 t
3

pH 3·3
"'neu r"Y"o.li z.ed

L \l,iCOO- CH"'-- coo-

[_ XC"S 'S

I
HCHO
I No.Orl CH.t- coo
NH + 1'\ Ht- I
3 t-

/~
N No..

C H_z OH C li.{DH
/~
CH.,_(lH C~OH
 su~mARY

During the study three methods were studied, i.e .,

Fuld Gross Method (A .O.A. C.), Curd Tension Method, and

the Clotting Method . The C l otting Method was selected

and then modified for practical purposes for determining

the proteinase activity .

The modified clotting method for determining animal

proteinase activity is as follows: Weigh accurately 0.7

grams of an unactive proteinase and dissolve in 100 cc.

of distilled water. Then add 0.3 grams of active enzyme

to activate an unactive enzyme. Stir well and centrifuge

the enzyme solutions at 1,000 rpm for 15 minutes. The

sediment is removed and only the supernatent clear

solution is used for the test .

9 cc. of rennet extract is dissolved in 91 cc. of

cold water. 10 cc . of milk is heated to 370 C. before

using the enzyme. Then add 0.5 ml . (5 mg.) of enzyme

solution and 0.3 ml. of rennet solution, and invert the

tube 4 times to mix it properly and allow it to stand.

Note the time of clotting with the help of a stopwatch.

The clotting can be seen by slightly bending the tube.

The modified clotting method for determining the

plant proteinase activity is as follows: Weigh accurately

1 gram of the active plant proteinase and dissolve in 100

 49

cc of di s tilled water. The solution is then centrifuged

at 1 , 000 rpm for 15 minutes. The sediment is removed and

the clear supernatent solution is taken for the test.

Nine cc. of rennet extract is dissolved in 91 cc. of

cold distilled water. Ten cc. of milk is heated to 40° C .

before using the enzyme. Then 0 . 7 ml. (7 mg . ) of enzyme

solution is added, followed by 0 . 1 ml . of rennet solution .

Then invert the tube 4 times to mix the enzyme properly .

Note the time of clotting with the help of a stopwatch .

 CONCLUSION

l. The curd formed by the animal proteinase tasted

sweeter at the lower concentrations and slight bitterness

developed at the higher concentrations . The curd formed

by the plant proteinase tasted bitter at all concentrations.

The intensity of bitterness was increased as the concen-

trations of both the proteinases were increased . The

developed bitterness might be due to the peptone formation

from the breakdown of casein by the proteolytic enzymes .

2. It was concluded from the experiments that the

activity of the animal proteinase is more than that of

the plant proteinase.

3. The modified clotting method gave fairly accurate

results . The replicate values of the clotting time agreed

with each other. It is also a rapid and practical method

as compared to other methods.

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