Journal of Bioscience and Bioengineering

VOL. 134 No. 4, 301e306, 2022

www.elsevier.com/locate/jbiosc

Selection of pretreatment method and mannanase enzyme to improve the

functionality of palm kernel cake

Witida Sathitkowitchai,1 Francis Ayimbila,1 Sunee Nitisinprasert,1, 2 and Suttipun Keawsompong1, 2, *

Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand1 and Center for Advanced Studies for Agriculture and Food, Kasetsart
University Institute for Advanced Studies, Kasetsart University (CASAF, NRU-KU), Bangkok 10900, Thailand2

Received 19 November 2021; accepted 29 June 2022

Available online 13 August 2022
Palm kernel cake (PKC) is a by-product of palm kernel oil extraction with moderate nutritional value, containing
30e35% b-mannan, which is indigestible, slows growth, and reduces feed efficiency. PKC can be improved by mannanase
hydrolysis, but the effectiveness of mannanase is dependent on the microbial source. Thus, the effect of steam pre-
treatment and bacterial mannanases on PKC quality was investigated. PKC was pretreated by steaming and hydrolyzed in
the small intestine by various mannanases. The contents of reducing sugar, total sugar, and protein release were
measured. Steamed PKC had a significant increase in protein (16.95 ± 0.14 to 20.98 ± 0.13%) and a substantial decrease in
hemicellulose (29.52 ± 0.44 to 3.46 ± 0.88%) and lignin (8.94 ± 0.28 to 1.40 ± 0.22%). Mannanases from Escherichia coli-
KMAN-3 and E. coli-Man6.7 recorded the highest activities, followed by commercial mannanase, Bacillus circulans NT6.7
and B. amyloliquefaciens NT6.3 mannanases, orderly. B. circulans NT6.7 and B. amyloliquefaciens NT6.3 had multi-
activities that include glucanase (3.10 ± 0.04% and 2.47 ± 0.02%) and amylase (1.74 ± 0.03% and 1.38 ± 0.04%), respec-
tively. B. amyloliquefaciens NT6.3 mannanase hydrolyzed steamed PKC to release more reducing sugar, total sugar, and
protein than hydrolyzed raw PKC. In raw and steamed PKC, B. amyloliquefaciens NT6.3 mannanase produced the highest
reducing sugar release. As a result, steam pretreatment and mannanase hydrolysis, particularly from
B. amyloliquefaciens, can be used to increase the functioning of PKC and develop new feed ingredients for monogastric
animals at a reasonable cost.
Ó 2022, The Society for Biotechnology, Japan. All rights reserved.

[Keywords: Palm kernel cake; Monogastric animal; Mannanase; Steam pretreatment; Feed]

Hemicelluloses, as cell wall polysaccharides, comprise one-third steamed PKC hydrolyzed by mannanase from various microbial
of the total components available in plants, accounting for 25e30% sources is limited.
of total wood dry weight (1). Mannan is a significant constituent of Mannanase is widely added in feedstuff to digest b-mannan in
hemicellulose in softwoods with widespread distribution in plant soybean meal and other plant proteins such as sesame meal and
tissues (2). Palm kernel cake (PKC) contains 35.2% mannan (3), cotton meal to release nutritional value. Mannanases are produced
which is a solid anti-nutritive factor for monogastric animals (4). by a variety of bacteria, fungi, actinomycetes, plants and animals
Mannan interferes with the digestibility, absorption, and utilization (8). Microbial enzymes are preferred for industrial applications
of nutrients, adversely affecting animal performance. The high because they can be easily and economically produced and have
mannan fraction makes bioconversion difficult, particularly in novel properties such as activity at a wide range of temperature and
enzymatic hydrolysis, to produce manno-oligosaccharides (2). As a pH. After proteases, cellulases and hemicellulases are the major
result, PKC must be subjected to pretreatments to break its struc- industrially important enzymes (9,10). The use of enzymes as feed
ture before bioconversion. additives is now well-established. For example, xylanases, man-
Compared to other pretreatment methods, steam explosion is a nanases and b-glucanases have been used throughout the past
much more environmentally friendly method that is widely used decade in cereal-based feed for monogastric animals which, con-
for many biomasses (5). During the steam explosion process, the trary to ruminants, are unable to fully degrade and utilize plant-
material is penetrated by high-pressure steam at the early stage based feeds containing high amounts of cellulose and hemicellu-
and disrupted by prompt decompression at the explosion stage. As lose (11). Our latest findings show that treating PKC with man-
a result, the pretreated material becomes porous and available for nanase from Bacillus amyloliquefaciens NT6.3 improves nutritional
subsequent enzymatic hydrolysis (6,7). However, even though content while also containing a significant amount of hemicellulose
steam explosion is effective for producing oligosaccharides from and acid detergent lignin (ADL), as well as less protein (12). Ng (13)
various materials, data comparing the nutritional content of demonstrated an alternative method to increase the nutrient and
protein contents of high fiber by-products such as PKC by extracting
and isolating the proteins by a combination of physical, chemical
and biological processes. Using steam explosion prior to hydrolysis
* Corresponding author at: Department of Biotechnology, Faculty of Agro-
Industry, Kasetsart University, Bangkok 10900, Thailand.
with engineered b-mannanase converted more than 80% of the
E-mail address: [email protected] (S. Keawsompong). mannan in PKC into manno-oligosaccharides (14). Effective

1389-1723/$ e see front matter Ó 2022, The Society for Biotechnology, Japan. All rights reserved.
https://doi.org/10.1016/j.jbiosc.2022.06.016
302 SATHITKOWITCHAI ET AL. J. BIOSCI. BIOENG.,

pretreatments increase gross material porosity, decrease crystal- cellulose (CMC) for cellulase activities. Activities of mannanase, xylanase, glucanase
linity of the cellulose fibrils, remove hemicellulose and reduce and cellulose were called non-starch polysaccharide hydrolase activities.

contained lignin (15). As a result, a combination of steam treatment In vitro hydrolysis of PKC samples One percent of raw PKC or steam-
pretreated PKC were hydrolyzed by mannanases from the six bacterial strains at
and enzymatic hydrolysis would be an effective method for 40  C for 2 h in phosphate buffer pH 6. Reactions were stopped by boiling the
increasing the nutritional content of PKC. In addition, recombinant samples for 15 min before centrifuging at 6000 rpm (2535 g). Supernatants
or modified b-mannanase has a better catalytic efficiency in acidic were tested for reducing sugar and total sugar by the dinitrosalicyclic acid method
and thermophilic conditions, making it more appropriate for (16) and phenol-sulfuric acid method (21), respectively. Protein release was
measured by the Bradford protein assay (22).
bioconversion of mannan-rich biomasses than original b-man-
Data analysis The statistical software package SPSS version 17 was used to
nanase (14). However, no research has been done to compare the
analyze the data. One-way analysis of variance (ANOVA) was used to determine
effects of mannanse from wild type and recombinant bacteria on statistical significance at P < 0.01 and P < 0.05. The nutrients released during
PKC functionality after steam pretreatment. In order to discover a mannanase hydrolysis of raw PKC and steam-pretreated PKC were compared
low-cost source of mannanse and boost the nutritional efficiency of between commercial enzyme and bacterial strains. The hydrolysis selectivity of
PKC for animal feed, we evaluated the effect of steam pretreatment B. amyloliquefaciens mannanase was also compared between raw PKC and steam-
pretreated PKC.
following hydrolysis by mannanase from wild-type and recombi-
nant bacteria.

RESULTS AND DISCUSSION

Nutritional and structural composition analysis Proximate

MATERIALS AND METHODS
and chemical analyses of raw PKC were determined and according
to the data, it contains crude fat (0.84%), protein (13.46%), carbo-
Materials PKC obtained from a palm oil factory in the south of Thailand was
sieved through a 1-mm aperture mesh. The remaining was ground using a Braun hydrate (56.05%), cellulose (36.73%), hemicellulose (29.52%) with
coffee grinder for 10 min and re-sieved. This was repeated twice and the material mannan content less than 29.52%. Neutral detergent fiber (NDF)
that did not pass through the mesh after the third grinding was discarded. analysis measured the fiber compositions; cellulose, hemicellulose,
Preparation of mannanase Four wild types of bacteria such as B. amyloli- and lignin, while neutral detergent soluble (NDS) determined
quefaciens NT6.3, Bacillus circulans NT6.7, Acinetobactor sp. ST1-1 and Klebsiella protein, starch, sugar, organic acids and pectin contents. The main
oxytoca CW2-3 and two recombinants as E. coli-KMAN-3 and E. coli-Man6.7 were
component was carbohydrate, with cellulose accounting for most
compared with a commercial mannanase. The bacteria were obtained
from 80  C stock culture (medium containing 20% (w/v) glycerol) and streaked of the fiber content. It was observed that PKC contained mainly
on medium agar for a single colony. One colony from each overnight culture was carbohydrates (65.8%) and a moderate amount of protein (16.5%)
inoculated into 5 mL of medium and grown overnight at 37  C, 250 rpm. (23). Typically, PKC consists of about 5e10% crude fat (4). The low-
Basic salt medium and producing enzyme medium for the four wild types and LB
fat content of PKC used could be due to the milling and solvent
broth for the two recombinants were prepared according to the modified procedure
(data not shown). A single colony of each selected bacteria from the overnight extraction processes used to extract palm oil. Alimon (24) found
cultures was inoculated into 5 mL of suitable media and condition. Cultures were little variation in the quality of expeller PKC and solvent extracted
transferred to 250 mL Erlenmeyer flasks containing 100 mL of the experimental PKC, though expeller PKC contained more oil (4e8%) than
conditions medium. solvent-extracted PKC (0.5e2%).
Enzyme harvest and determination of mannanase activity For enzyme A comparison of chemical composition between raw PKC as
harvest, supernatants of the culture medium were collected by centrifuging at 4  C,
 control and pretreated PKC showed different dry matter, carbohy-
8000 rpm for 20 min. All enzymes were kept at 20 C until required for use. En-
zymes were assayed for mannanase activity in a reaction mixture consisting of drate, protein and fiber percentages, as shown in Table 1. Steam
0.1 mL of 100 mM optimal pH containing 1% locust bean gum with 0.1 mL of su- pretreatment increased the level of protein (20.98  0.13%), cellu-
pernatant and incubated at 50  C for 60 min. The amount of reducing sugar released lose (67.68  2.66%) and NDS (26.92  0.73%), decreased hemi-
was determined by the dinitrosalicyclic acid method (16).
cellulose (3.46  0.88) and lignin (1.40  0.22) compared to raw
Steam pretreatment Steam pretreatment of PKC was performed following a PKC. Higher protein content suggests that the temperature and
modified method (17). PKC was pretreated by a steam explosion at 190  C for 10 min.
The hydrolysates were then dried at 60  C overnight to remove moisture content of
pressure during steam treatment broke down the structure of PKC
about 10% and stored at 20  C until required for use. and released bound proteins. Cross-linking of hemicellulose with
Chemical and structural composition analysis Crude lipid (18), moisture cellulose and lignin in plant cell walls promotes cell wall recalci-
(19), ash (18) crude fiber (18), crude protein (18) and nitrogen-free extracts trance (25). The reduction in hemicellulose and lignin indicates that
(digestible carbohydrates) of PKC and pretreated PKC were determined by steam treatment can reduce PKC’s recalcitrance. Pretreatment was
proximate analysis. In addition, NDF and ADF analysis was conducted following
required to convert the raw substrate to a suitable form via size
previous procedure (20).
reduction through grinding and physical, chemical, or enzymatic
Activities of non-starch polysaccharide hydrolases One hundred units of
mannanase enzymes produced from the six bacterial strains B. amyloliquefaciens NT hydrolysis to increase substrate availability and release nutrients
6.3, B. circulans NT 6.7, Acinetobactor sp. ST 1-1, K. oxytoca CW 2e3, E. coli-KMAN-3 and and sugar (26). The Carbohydrate content of the control was higher
E. coli-Man6.7 were performed at 40  C for 60 min in 0.1 M phosphate buffer pH 7.0 because pretreatment destroyed the carbohydrate structure of
containing different substrates to measure important enzyme activities in commer- hemicellulose, cellulose and sugar in PKC. Ng and Mohd Khan (27)
cial mannanase as b-mannanase, xylanase, b-1,3-(4)-glucancase, amylase and
cellulase activities. Substrates used in the experiment included locust bean gum for b-
described an extract of PKC with a protein content of 68.50%. The
mannanase activities, xylan from birch wood for xylanase activities, b-D-glucan for b- pretreatment sample had a crude fiber content compared to the
1,3-(4)-glucancase activity, potato starch for amylase activities and carboxymethyl control, but steamed PKC had a higher value. Steamed PKC may

TABLE 1. Chemical composition percentages of raw PKC and pretreated PKC.

Substrate Component (%)

DM CHO CP EE CF Ash Cel Hem Lig NDSa

Raw PKC 91.41  0.14 56.05  0.21 16.95  0.14 0.25  0.0 13.46  0.19 4.69  0.02 36.73  0.14 29.52  0.44 8.94  0.28 24.81  0.22
Steamed PKC 92.46  0.20 51.20  0.17 20.98  0.13 0.93  0.01 13.24  0.01 6.11  0.80 67.68  2.66 3.46  0.88 1.40  0.22 26.92  0.73

DM, dry matter; CHO, carbohydrate; CP, crude protein; EE, ether extract; CF, crude fiber; Cel, cellulose; Hem, hemicellulose; Lig, lignin.
a
NDS are protein, starch, sugar, organic acids and pectin.
VOL. 134, 2022 IMPROVING PKC BY PRETREATMENT AND MANNANASE 303

have had a slight rise in fiber content due to the considerable loss of sugar levels of enzymes from B. amyloliquefaciens NT6.3,
carbohydrate content. Steam pretreatment could permeate PKC cell B. circulans NT6.7, and the commercial enzyme were
walls and disrupt the structure of the fiber fraction to improve 20.36  4.2 mg/mL, 10.45  0.85 mg/mL and 11.91  3.67 mg/mL,
enzyme accessibility and digestibility of the complex fraction (14). respectively, whereas total sugar contents of B. amyloliquefaciens
NT6.3, B. circulans NT6.7, and commercial enzymes were
Non-starch polysaccharide hydrolase activities A recom-
200.60  7.78 mg/mL, 211.05  4.38 mg/mL and
binant or modified b-mannanase has been indicated to have a
206.61  5.12 mg/mL, orderly. Total sugar and reducing sugar
better catalytic efficiency in acidic and thermophilic conditions,
contents form the carbohydrate components (22); NDF and NDS
making it more appropriate for bioconversion of mannan-rich
in PKC. The results showed that these enzymes could break down
biomasses than original b-mannanase (14). The feed and animal
NDS and NDF to release lower molecular weight carbohydrates.
industries require a low-cost supply of enzymes to increase the
Previous work revealed that B. amyloliquefaciens NT6.3
nutritional content of PKC. Table 2 presents the results of enzyme
mannanase hydrolysis of PKC reduced NDF level from
activity for mannanase, xylanase, glucanase, cellulase and
75.19  0.22% to 66.31  0.22%, and increased NDS from 24.81 
amylase in the commercial enzyme and enzyme from six effective
0.22% to 33.69  0.22% (12). Additionally, enzyme hydrolysis
strains in mannan hydrolysis (four wild-type bacteria and two
increased the protein level in PKC. Protein release by the enzyme
recombinant bacteria). Mannanase, xylanase, glucanase and
from B. amyloliquefaciens NT6.3, B. circulans NT6.7, and
cellulase are non-starch polysaccharide hydrolases (28). The
commercial enzymes were 36.82  4.98%, 35.16  4.25% and
results showed the activities of mannanases of commercial
52.22  2.50%, respectively. When an appropriate enzyme
enzyme (6.73  0.084 U/mL), K. oxytoca CW2-3 (2.89  0.033 U/
degrades the fibrous component of PKC, proteins are released
mL) (29), Acinetobactor sp. ST1-1 (3.59  0.060 U/mL) (29), B.
along with sugars, manno-oligosaccharides, and other nutrients
amyloliquefaciens NT6.3 (0.46  0.009 U/mL) (30), B. circulans
(12). Thus, B. amyloliquefaciens NT6.3, B. circulans NT6.7, and
NT6.7 (0.65  0.017 U/mL) (30), E. coli-KMAN-3 (41.45  1.263 U/
commercial enzymes could digest the structure of raw PKC and
mL) (31) and E. coli-Man6.7 (50.40  0.390 U/mL) (32). Indicating
improve the nutrient content. Furthermore, the relative activity of
that mannanases from the two E. coli strains had the highest
non-starch polysaccharide hydrolase activities and hydrolyzed
activities, while both Bacillus strains had comparable mannanase
PKC could imply that multiple enzyme activities could hydrolyze
activities but were lower than commercial mannanase.
some complex structures. The sequence of enzyme activity from
Importantly, enzymes other than mannanase including xylanase,
commercial enzyme was mannanase, amylase, glucanase,
glucanase, cellulase and amylase of B. amyloliquefaciens NT6.3 and
xylanase, and cellulase, based on relative activity calculated from
B. circulans NT6.7 exhibited significant enzyme activities.
non-starch polysaccharide hydrolase activities. The enzymes of
Furthermore, amylase activities (U/mL) in B. amyloliquefaciens
B. amyloliquefaciens NT6.3 were ranked in order of relative
NT6.3 (1.38  0.042) and B. circulans NT6.7 (1.74  0.031)
activity as mannanase, glucanase, amylase, xylanase, and
enzymes were comparable to commercial enzyme (1.30  0.018),
cellulase, while B. circulans NT6.7 enzymes were glucanase,
but higher than amylase from the other bacteria. Again,
mannanase, amylase, xylanase and cellulase. Notably, the
B. amyloliquefaciens NT6.3 (2.47  0.158) and B. circulans NT6.7
activities of commercial and B. amyloliquefaciens NT6.3
(3.10  0.037) had significantly higher glucanase activities (U/mL)
mannanases, amylases, and glucanases were generally similar.
than commercial enzyme (0.23  0.055). Jørgensen et al. (33)
The primary activity sequences of enzymes in B. circulans NT6.7
reported that incorporating mannanase, mannosidase and
were glucanase, then mannanase, but for commercial enzyme
glucanase enzymes could release glucose and mannose. When
and B. amyloliquefaciens NT6.3, the sequences of NSP hydrolase
non-starch polysaccharide enzymes (primarily xylanase, b-
activity were mananase, then glucanase. It has been shown that
glucanase, cellulose disaccharidase, pectinase, protease, and
the cell wall of PKC was a complex structure consisting of 58%
mannanase) are added, growth performance and feed nutrient
mannan, 12% cellulose and 4% xylan (4,32), so the enzyme activity
digestibility improve (34). This suggests that enzymes from
sequences for all tested enzymes could be mannanase, glucanase,
B. amyloliquefaciens NT6.3 and B. circulans NT6.7 with various
and amylase because the main structure was mannan.
activities may be more suited and cost-effective for hydrolyzing
Commercial enzymes, B. amyloliquefaciens NT6.3 and B. circulans
PKC and releasing nutrients.
NT6.7 enzymes were found to be suitable for hydrolyzing raw
In vitro enzymatic hydrolysis of raw PKC The selection of PKC to yield additional nutrients. However, it contains significant
hydrolysis enzyme in simulated chicken intestine conditions was amounts of hemicellulose (mannan and xylan), cellulose and
first tested on raw or untreated PKC. All enzymes digested raw PKC lignin, as well as bound proteins, which may impede its efficient
and released scant reducing sugar, total sugar, and protein, but use. Thus, it required efficient pretreatment.
commercial enzyme, B. amyloliquefaciens NT6.3, and B. circulans The results confirmed that B. amyloliquefaciens NT6.3 man-
NT6.7, produced more nutrients (Fig. 1). The reducing sugar and nanase could be suitable for digesting raw PKC because of higher
total sugar released by B. amyloliquefaciens NT6.3, B. circulans nutrient released during hydrolysis than other enzymes. Therefore,
NT6.7, and the commercial enzymes after hydrolysis were the B. amyloliquefaciens NT6.3 enzyme was selected to hydrolyze
significantly higher than raw PKC before hydrolysis. Reducing raw PKC to improve nutrients.

TABLE 2. Non-starch polysaccharide hydrolase from various source.

Enzyme Enzyme activity (U/mL)

Mannanase Xylanase Glucanase Cellulase Amylase

Commercial mannanase 6.73  0.084 0.09  0.012 0.23  0.055 0.07  0.047 1.30  0.018
Crude mannanase from E. coli-KMAN-3 41.45  1.263 0.08  0.035 0.13  0.381 0.36  0.039 0.21  0.102
Crude mannanase from E. coli-Man6.7 50.40  0.390 0.68  0.046 0.58  0.089 0.22  0.172 0.49  0.051
Crude mannanase from B. circulans NT6.7 2.89  0.033 0.04  0.009 3.10  0.037 0.01  0.006 1.74  0.031
Crude mannanase from B. amyloliquefaciens NT6.3 3.59  0.060 0.05  0.004 2.47  0.158 0.04  0.006 1.38  0.042
Crude mannanase from K. oxytoca CW2-3 0.46  0.009 0.01  0.003 0.01  0.008 0.01  0.007 0.07  0.011
Crude mannanase from Acinetobactor sp. ST1-1 0.65  0.017 0.01  0.003 0.003  0.002 0.02  0.006 0.01  0.001
304 SATHITKOWITCHAI ET AL. J. BIOSCI. BIOENG.,

FIG. 1. Carbohydrate and protein released in steamed PKC hydrolysate obtained from hydrolysis: raw PKC in phosphate buffer pH 6 without enzyme hydrolysis (control) and raw PKC
hydrolyzed by various enzymes. Commercial mannanase enzyme (commercial), Klebsiella oxytoca CW2-3 mannanase (CW2-3), Acinetobactor sp. ST1-1 mannanase (ST1-1), Bacillus
amyloliquefaciens NT6.3 mannanase (NT6.3), Bacillus circulans NT6.7 mannanase (NT6.7), E. coli-KMAN-3 mannanase (Kman3) and E. coli-Man6.7 mannanase (Man6.7). Different or
similar numbers, uppercase letters and lowercase letters denote significant (P < 0.01) or no significant (P > 0.01) variations in reducing sugars, total sugars and proteins,
respectively.

FIG. 2. Carbohydrate and protein released in steamed PKC hydrolysate obtained from hydrolysis: steamed PKC in phosphate buffer pH 6 without enzyme hydrolysis (control) and
steamed PKC hydrolyzed by different enzymes. Commercial mannanase enzyme (commercial), Klebsiella oxytoca CW2-3 mannanase (CW2-3), Acinetobactor sp. ST1-1 mannanase
(ST1-1), Bacillus amyloliquefaciens NT6.3 mannanase (NT6.3), Bacillus circulans NT6.7 mannanase (NT6.7), E. coli-KMAN-3 mannanase (Kman3) and E. coli-Man6.7 mannanase
(Man6.7). Different or similar numbers, uppercase letters and lowercase letters denote significant (P < 0.01) or no significant P > 0.01) variations in reducing sugars, total sugars and
proteins, respectively.

In vitro enzymatic hydrolysis of steam-pretreated PKC Ng hydrolysis of steam-pretreated PKC improved reducing sugar,
(13) demonstrated an alternative way to increase nutrition and total sugar and protein levels compared with the control (raw
protein content of high fibre by-products such as PKC by PKC) (Fig. 2) and enzymatic hydrolysis of raw PKC. In general,
extracting and isolating the proteins using a combination of protein contents were more than 5-folds higher than the
physical, chemical and biological processes. The bioconversion of enzymatic hydrolysis of raw PKC and more than 20-folds higher
PKC to manno-oligosaccharides was efficient when PKC was than the control (raw PKC). This effect was most noticeable
processed by steam explosion in combination with an engineered during enzyme hydrolysis in B. amyloliquefaciens NT6.3 and
b-mannanase (mRmMan5A) hydrolysis (14). As seen in Table 1, B. circulans NT6.7. Again, hydrolysis of B. circulans NT6.7 and
steam treatment increased protein and cellulose content while commercial enzymes increased total sugar contents significantly
decreasing hemicellulose and lignin concentration, allowing PKC more than the control (steam-pretreated PKC), and more than
to be more easily accessed by enzymes. As a result, enzymatic twice as much as hydrolysis of raw PKC. The digestion of raw PKC
VOL. 134, 2022 IMPROVING PKC BY PRETREATMENT AND MANNANASE 305

FIG. 3. Carbohydrate and protein released during hydrolysis of substrates by B. amyloliquefaciens NT6.3 mananases; raw PKC (control), enzyme hydrolyzed raw PKC (PKC) and
pretreated raw PKC using steam explosion (control-steamed) and enzyme hydrolyzed steamed PKC (steamed PKC). Different or common numbers, uppercase letters and lowercase
letters denote significant (P < 0.01) or no significant (P > 0.01) variations in reducing sugars, total sugars and proteins, respectively.

(Fig. 1) and steamed PKC (Fig. 2) by B. amyloliquefaciens NT6.3 protein. PKC that had been steamed was more susceptible to
enzymes improved reducing sugar content significantly more mannanase hydrolysis. B. amyloliquefaciens NT6.3 enzymes
than the control. Furthermore, when compared to their enhanced the reducing sugar content of raw and steamed PKC
respective controls (unhydrolyzed), B. amyloliquefaciens NT6.3 considerably more than commercial enzymes and the control.
mannanase significantly (P < 0.01) digested steam-pretreated Mannanase from B. amyloliquefaciens NT6.3 increased nutrient
PKC, releasing higher reducing sugar and protein contents than release and may provide a low-cost way to improve raw and
raw PKC, as shown in Fig. 3. Clearly, steam pretreatment steamed PKC for use in animals, particularly monogastric animals.
improved the efficiency of the various enzymes in digesting PKC,
particularly with the enzyme of B. amyloliquefaciens NT6.3. Galbe ACKNOWLEDGMENTS
and Zacchi (35) reported that hemicellulose enzymes catalyze
hydrolysis, and without pretreatment, conversion of native This research was financially supported by the Research and
hemicellulose to sugar is extremely slow since hemicellulose is Researchers for Industries (RRI) PhD program (PHD56I0043), the
well protected by lignin. Using an engineered b-mannanase to Thailand Research Fund (TRF), Betagro Science Center Co., Ltd., and
hydrolyze steam explosion pretreated PKC; more than 80% of the Betagro Public Co., Ltd. We also appreciate the technical assistance
mannan in PKC was converted into manno-oligosaccharides (22). provided by the Biotechnology Laboratory of the Department of
Hence, pretreatment with steam could have permeated PKC cell Biotechnology. The authors declare no conflicts of interest.
walls, disrupted the structure of the fibrous fraction, and
improved fibre digestibility and enzyme accessibility in PKC. References
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