CARBOHYDRASE ACTIVITY PROFILING OF Aspergillus niger AND Bacillus subtilis

DERIVED ENZYMES IN RICE BRAN AND COPRA MEAL SUBSTRATES

Hector Salazar Abes

Bachelor of Science in Agricultural Biotechnology

INTRODUCTION

Many countries, including the Philippines, have used soy bean meal as the staple and
primary feed ingredient when it comes to the supplementation of energy in the diet (Ng and
Chen, 2002). According to FAO, soy bean meals in the country are imported up to 99% from
other country.
However, one of the problems is that due to the fact that soy bean meals are 98%
imported. The enzymes also available and sold in the market are also limited to the enzymes
effective for these imported feed nutrients. Thus, many of our locally produced and potential
feed ingredients, like rice bran and copra meal, are not utilized since there are no specific
enzymes sold in the market to allow the utilization of said feed nutrients.
The solution to the problem is to produce an enzyme that would be specific in degrading
and making these locally available feed nutrients utilizable for our livestocks example: poultry
and swine. Feed enzyme usage has increased in the last decade. This technology is very
crucial to the animal feed industry as the feed enzyme usage has allowed the use of little feed
ingredient but with increased bioavailability. Studies have been conducted by Olude,
Alegbeleye, and Obasa, has shown that the copra meal can be used to partially substitute 2030% of the soy bean meal. Also another study which utilized the crude Aspergillus niger-derived
enzyme, termed coprase, on copra meal has been done and it has been concluded that with a
proper degradation the copra meal can be an alternative to soy bean meal as the staple feed
ingredient. In Nigeria, a pre-soaked copra meal has been used to supplement the soy bean
meal as feed ingredient for its fisheries diet.
Problem with copra meal use as feed ingredient is that despite the vast number of
researches regarding the improvement of copra meal for feed diet is that there are very few

researches conducted that deal with the specific enzyme inclusion in a diet fortified with copra
meal. Sundu (2009) has stated that the enzymes utilized in the researches to act on copra meal
is not entirely suited for copra meal. Thus, the copra meals potential as a supplementary feed
ingredient is not fully achieved. The latter has been associated by Sundu (2009), to the nonspecificity of enzymes commonly used and sold in the local markets.
Over the past years, there have been little researches or attempts to use enzymes for
the quality and bioavailability improvement of copra, specifically copra meal. Enzyme production
through the utilization of fungi, as microorganisms, has been commonly done to ferment a food.
Fermentation industry heavily relies their business on the enzyme produced by these fungi. The
fermentation allows food to be broken down to simpler units and this degradation allows,
otherwise unabsorbable complex feed ingredient, to be absorbed and utilized by the livestock.
In simpler terms, fermentation increases the absorbability of these complex feed ingredients via
enzyme action. (Sundu, 2006)
In the animal industry, enzyme inclusion in feed to increase the bioavailablity of feed
component for the livestock has grown popularity in the recent years. Lesser input of feed
component is now possible with the advent of feed enzymes as the feed can now be more
utilized by the livestock due to increased bioavailability. Submerged fermentation has been
industrially used to produce enzymes since the 1950s. (Farrell and Martin, 1993) Aspergillus
niger has also been industrially used for the production of enzymes and in fact it is the single
largest source of enzymes. (Subramaniyam, 2004)
The study aims to examine and characterize the specific enzymes, to be termed
coprase, produced by Aspergillus niger, one of the best organism for production of enzymes,
to degrade rice bran and copra.

REVIEW OF RELATED LITERATURE

Copra Meal
Traditionally, the major source of protein for animal feed is soybean up to the present.
However, soybean has also a relevance to human nutrition like usage in soy milk, baby formula,
and soypap. Due to the above usage, it has put limitation to the soy bean allocation. Also,
another detriment to the utilization of soy bean is the current decline trend of production of
soybean in tropic according to WHO. With the pressing concerns, it has been necessitated the
need to scout for alternative and cheaper animal feed ingredients which have little or no
relevance to human nutrition. (Olude et. Al.)
Copra is acquired by sun drying or artificially drying the coconut with the use of
machines. According to Swick 1999, the 2/3 (two-thirds) of worlds production of copra comes
from two major producers wherein Philippines is included. On the other hand, in terms of amino
acid balance and digestibility the copra protein quality is poor. Also, it is deficient from amino
acids such as histidine, threonine, and lysine but high in arginine. Fortifying the copra meal has
allowed it to keep up with its antagonistic properties. (Olude et. Al.)
Rice Bran
Rice bran is prone to rancidity, has a high phytate content, contains an enzyme inhibitor
(trypsin inhibitor), and is high in fiber (Gallinger et al., 2004). These characteristics have limited
the use of rice bran in poultry diets. A maximum of 10-20% is recommended in broiler diets,
depending on the geographical origin of the rice and the level of supplemental enzymes used
(Martin and Farrell, 1998a).
Recommended inclusion levels in broiler diets vary from 10 to 20% (Gallinger et al.,
2004 and Farrell, 1994, respectively). Gallinger et al. (2004) reported that inclusion of 20% rice

bran in broiler diets resulted in reduced growth performance. In addition, adding just 10% rice
bran reduced feed efficiency and tibia ash content. Others have recommended that rice bran not
be include in diets of broilers less than 21 days of age (Martin and Farrell, 1998b).
Aspergillus niger
According to Subramaniyon, in fungal enzyme production the genus Aspergillus have
been used as a model organism for more than a decade. In the industrial business point of view,
the Aspergillus has produced several crucial and important industrial enzyme for fermentation
and controlled degradation of food. Aspergillus niger, a species under the genus Aspergillus, is
in fact the largest single source of fungal enzymes in the industry of fermentation.
When fermented upon appropriate substrates, several fungi will produce a vast array of
enzymes for industrial use. Different subtrates spell different enzymes produced. In the enzyme
production with the use of fungi, solid state fermentation is preferred rather than submerged
fermentation. However, submerged is more advantageous to use if the production of the
enzymes would be in the industrial scale or mass produced. (Subramaniyon, 2004)
Feed Enzyme
In the recent decade, there has been a great improvement and progress in feed enzyme
technology. Commonly, origin of enzymes are from microbial source mostly from bacteria or
fungi. These enzymes are expected to have broad spectrum of effectivity: from the varying pH of
the digestive tract up to the elevated temperature during manufacturing. The said treatments are
required to be implemented in order to remove unwanted components such as Salmonella and
aflatoxins. Feed enzyme additions aims to target specific substances and sometimes the
targeted substrates are not clearly identified thus a cocktail of enzymes are applied. (Farrell and
Martin, 1993)

Feed enzymes are commonly used to degrade specific substrates and break chemical
bonds. Therefore, breaking down the substrate and most of the time causes an increase in the
bioavailability of a certain nutrient. According to Creswell (1993), upon the addition of enzyme
cocktails which targets a substrate has increased the standard metabolizable energy (ME) value
of the broiler diets by up to 6-10%.
Almost all enzymes, with the exception of ribozymes, are proteins. They consist of one
or more polypeptide chains with molecular weight that range from thousands to millions. They
are commonly known as the biological catalyst and without enzymes life would not be possible.
Enzymes govern almost all of the chemical reactions that occur in the body of all living things,
be it plant or animal. Enzymes are one of the most remarkable biomolecules known, as
reactions could permanently stop by its absence but proceed a thousand times faster in its
presence.
Feed Enzyme Importance
In the animal feed industry, application of enzymes has significantly increased in the last
decade due to the fact that the access of knowledge regarding enzyme and their properties has
risen with the advent of the internet. In turn, the enzymes application to feed as a feed
supplement has significantly increased. Enzyme supplementation to feed has increased feed
utilization since the livestock does not have to produce the certain enzyme to break down a feed
component as the enzyme to degrade the feed nutrient is included now to the diet. Therefore,
with the advent of enzyme supplementation the feed conversion ratio of animals has improved,
feed nutrients that were not previously possibly utilized by the monogastrics can now be utilized
with the addition of enzymes and countless more of applications. Feed enzymes have the
potential of further improving the nutritional range of feed components. (Farrell and Martin,
1993)

A study conducted by Sundu, applied crude coprase directly unto feeds that contained
copra. The crude coprase was produced by allowing the Aspergillus niger to ferment the
copra, the organisms only carbon source, to allow the Aspergillus niger to produce its own
coprase. Sundus experiment was conducted by feeding the coprase-treated feeds on
unsexed cobbs. The results yielded that upon the addition of 10% copra meal on the feed had
significant increments on the feed intake and improved the feed conversion ratio. However,
further increasing the concentration of copra meal above 30% has lessened the feed intake and
had detrimental effects to the feed conversion ratio of the cobbs. The reason stated by Sundu
for this is that the copra might contain anti-nutritional factors. The study has also provided that
spraying of coprase on the feed that contained copra will help increase the feed intake and
further improve the feed conversion ratio of chickens. (Sundu and Hatta, 2009)
Bradford Assay
Bradford protein assay is a relatively fast and simple method commonly used to
determine the total protein concentration of a sample. The Bradford assay is quick and uses
almost the same amount of protein as the Lowry assay. It works based on the proportional
binding of the Coomassie Brilliant Blue G-250 to protein. This assay is colorimetric. Thus as the
concentration of protein increase, the color intensity increase or the color becomes darker.
Samples subjected to Bradford assay is to be analyzed using spectrophotometer at 595nm.
(Robyt, 1987)

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

This modified gel electrophoresis involves the use of a detergent and denaturing agent,
sodium dodecyl sulfate, while running the electrophoresis. The SDS-PAGE is a high resolution
method that has been preferred by almost all chemists. There are two major advantages with
the use of SDS-PAGE over the native one. First, is that it aggregates and insoluble particles are
solubilized and converted to single polypeptides. Second, is that the mobility is directly related
to the polypeptide size. Thus, an immediate indication of molecular weight for each component
is provided. (Scopes, 1998)

Figure 1. Action of dodecyl sulfate in denaturing proteins

Separation of proteins has been possible with the exploitation of its charge and
molecular weight. SDS-PAGE is a separation technique used to separate protein. SDS-PAGE
uses reagents like SDS which adheres to the backbone of proteins, puts a negative charge on
the protein, and denatures it. Also, SDS-PAGE uses -mercaptoethanol which breaks the
disulfide bonds between proteins thus further denaturing the protein molecule. In the SDSPAGE, proteins are separated based on their molecular weight alone and not influenced by their
net charge. Another difference between native PAGE and SDS-PAGE is that by using native
PAGE the determination of the sample concentration while with the use of SDS-PAGE
determines the count of subunits or monomers the sample protein has. (Scopes, 1998)

MATERIALS AND METHODS

Sample collection
Copra and rice bran samples will be obtained from copra farms and rice milling
industries, respectively. The samples will then be transferred immediately and subjected to
grinding. The grinding, to pass through 2mm siever, is to be done in order to produce the copra
meal and rice bran meal which will be the sole carbon source of the Aspergillus niger and
Bacillus subtilis in its fermentation broth later.
As for the microorganism to be used, three (3) test tube slants of Aspergillus niger _____
and Bacillus subtilis will be acquired from the Philippine National Collection of Microorganisms,
BIOTECH. It will be transferred in a chilled condition using an ice box and be quickly stored in a
refrigerator at 4oC. The Aspergillus niger and Bacillus subtilis shall be mass cultured by
inoculating it in a potato dextrose agar (PDA) plates at 30 oc and shall be stored at 4oc prior to its
inoculation to the fermentation broth reported by Maldonado and Strasser de Saad (1998).
Enzyme Extraction
Aspergillus niger and Bacillus subtilis derived enzymes will be extracted via the method
reported by Maldonado and Strasser de Saad (1998). This protocol for extraction of protein
would allow the specific enzyme for the degradation of copra and rice bran to be rapidly
extracted from the solution.
The fermentation broth reported by Maldonado and Strasser de Saad (1998), will be
used to house the nutrients and preferred substrate of the Aspergillus niger and Bacillus
subtilis. Component of the said fermentation broth is provided in Table 1. All of the components
will be mixed in a 250-ml Erlenmeyer flask. After all of the components are mixed, initial pH for

the fermentation broth for Aspergillus niger shall be adjusted to 4.5 with the use of 1M HCl.
Literature has provided that at pH 4.5 the Aspergillus niger will be able to have its normal
metabolism. In relation with the pH, the buffer to be used for Aspergillus niger shall be H3BO3,
with a buffering range of 3.5-5.5. As for Bacillus subtilis, the optimum pH is provided by
Vijayalakshmi (2012) at pH 7.0 therefore the fermentation broth for Bacillus subtilis shall be
adjuSted to the neutral pH of 7.0. The buffer to be used for its fermentation broth shall be
phosphate buffer with the buffering range of 6.2-8.2. A fifteen (15) minute sterilization at 120 oC
will be applied to the Erlenmeyer flask containing the fermentation broth. Then with the aid of
hemocytometer, inoculation of 2x106 per ml count of Aspergillus niger and Bacillus subtilis shall
be conducted.
KH2PO4
4 g/L
[NA2HPO4 FeSO4 7H20 ]
0.2 g/L
CaCl2
0.01 g/L
MnSO4 7H2O
70 g/L
(NH4)2 SO4
2 g/L
H3BO3
10 g/L
Rice Bran / Copra Meal
15 g/L
Table 1. Fermentation broth components
Fermentation shall be carried out using the rotary shaker with the settings of 300 rpm at
30oC. Aliquots of 10mL will be intermittently taken at 12-hour intervals during the 72-hour
fermentation period. The samples will be filtered through Whatmann No. 595 filter paper,
centrifuged at 20000 rpm for 20 minutes and will be transferred to a test tube. Then the samples
will be stored at -20oC for further assays.

Enzyme Assay
The method for dinitrosalicylic acid assay (DNSA) provided by Miller (1957) will be the
protocol to be followed for the enzyme assay. 3ml aliquot of the DNSA reagent is to be added to

3ml aliquot of the samples in test tubes. The prepared mixtures shall be heated in a boiling
water bath for 5 minutes and then cooled to ambient temperature by allowing it to be subjected
to running water. After the samples have cooled, the color densities will be measured in the
spectrophotometer at 575nm.
Partial Characterization
The samples with the highest absorbance per sample will be subjected to ammonium
sulfate precipitation to further purify the solution. A _____g of ammonium sulfate ((NH 4)2SO4)
shall be added to the test tube containing the samples and then the solutions will be stand at
cold water bath with occasional stirring for 30 minutes. After standing, 1mL aliquot of the
solution will be transferred to 1.5mL eppendorf tubes. The eppendorf tubes will be spun at
20000rpm for 20 minutes. The supernatant shall be discarded. Precipitate formed is to be
diluted with distilled H2O or buffer.
Bradford Assay
Preparation of the Bradford reagent shall be done first. It will be done by mixing 1 part
Bradford: 4 parts distilled H2O then filtered through a Whatmann 540 paper. If the agent will not
be immediately used, store at -20oC.
Addition of 10-20 L of the sample to an mL of the prepared reagent will be done then
mixed. Test tube will be the container used in the mixture of the protein extract and the reagent.
Absorbance is to be read at 595nm wavelength. The preparation of standard curve will be done
via a serial dilution series (0.1-1.0 mg/ml) of a known protein sample concentration.
SDS-PAGE
The stacking and separating gels will be prepared prior to the experiment in a small
beaker and the gel components are presented in tables 2, and 3. The final concentration of

components is variable since there are still no available studies regarding the molecular weight
of the coprase to be studied.

Acrylamide Percentage
H2O
Acylamide/Bis-Acrylamide
(30%/0.8% w/v)
1.5M Tris (pH=8.8)
10% (w/v) SDS
10% (w/v) ammonium
persulfate (AP)
TEMED

For a 10mL separating gel

6%
8%
10%
5.2mL
4.6mL
3.8mL

12%
3.2mL

15%
2.2mL

2mL

2.6mL

3.4mL

4mL

5mL

2.6mL
0.1mL

2.6mL
0.1mL

2.6mL
0.1mL

2.6mL
0.1mL

2.6mL
0.1mL

100L

100L

100L

100L

100L

10L

10L

10L

10L

10L

Table 2. Separating gel concentration of components

For a 5mL stacking gel

H2O
0.5M Tris-HCl, pH 6.8
10% (w/v) SDS
Acylamide/Bis-Acrylamide (30%/0.8% w/v)
10% (w/v) ammonium persulfate (AP)
TEMED

2.975mL
1.25mL
0.05mL
0.67mL
0.05mL
0.005mL

Table 3. Components of the stacking gel

Table 4 presents the ideal acrylamide concentration in relation with the molecular weight
range of the protein. The concentrations 7% and 15% is to be tested first and optimization is
done accordingly.
Acrylamide %
7%
10%
12%
15%

Molecular Weight Range

50 kDa 500 kDa
20 kDa 300 kDa
10 kDa 200 kDa
3 kDa 100 kDa

Table 4. Ideal acrylamide concentration in relation with the molecular weight range

Casting of the frames by clamping two glass plates in the frames will be done first. After
that, the separating gel will be loaded by pipetting the appropriate amount of the gel into the gap

of the glass plates. Water will also be loaded after the gel is filled in order to allow the top of the
separating gel to become horizontal. Wait for the gelation for about 20-30 minutes.
After it has gelated, discard the water loaded until the separating gel is the only one left
in the casting frames. Pipette in the stacking gel until it overflows. Create wells by inserting the
comb and wait for it to gelate for about 20-30 minutes again. While waiting, prepare the sample
proteins to be loaded by mixing the samples with the sample buffer. When the gel has already
achieved complete gelation, take out the comb and put the glass plates in the cell buffer dam.
Load the running buffer inside the inner chamber until the buffer has reached the required level
in the outer chamber.
The samples will be loaded in the wells along with the molecular ladder and also with the
protein marker. After the samples have been loaded, cover the top and connect the anodes.
Stop the electrophoresis when the protein marker has reached the bottommost part of the gel.

References:
Creswell, D.C. (1993). Proc. Finfeeds & Pharmochem Feed Enzyme seminar. Feb. 8, 1993.
Sydney Marriot Hotel
FAO

2001 Food

and

Agriculture

Organization

of

the

United

Nations.

FAO

STAT.

Database. http://apps.fao.org/default.htm
Farrell, D.J. (1991). I. Animal Physiology Animal Nutrition. 65: 146-153
Farrell, D.J. and Green, A.G. (1991). Proc. Australian Poultry Science Symposium. 1991, pp.
60-63 University of Sydney, N.S.W.

Farrell, D.J. and Martin, E. 1993. Feed enzymes in poultry nutrition: Recent findings. Recent
advances in Animal Nutrition in Austalia. The University of England, Armidale, Australia,
pp: 266-276
Farrell, D.J., Takhar, B.S., Varr, A.R., and Pell, A.S. (1991). In: Recent Advances in Animal
Nutrition in Austalia
Gallinger CI, Suarez DM, Irazusta A (2004) Effects of rice bran inclusion on performance and
bone mineralization in broiler chicks. Journal of Poultry Research 13, 183190.
Holker, U., Hofer, M. and Lenz, J. (2004) Biotechnological advantages of laboratory-scale
solidstate fermentation with fungi. Applied Microbiology and Biotechnology, 64: 175-186.
International Institute for Tropical Agriculture 1990 Soybean for Good Health: How to grow and
use Soybean in Nigeria, Ibadan. 25pp.
Jobling M 1994 Fish Bioenergetics, Effect of processing by fermentation on nutrient, In: R S
Hami and E Kamas (editors.).Nutritional Evaluation of food processing. Avi Publishing
Co. Inc. Westport, Connecticut. 324pp
Maldonado, M.C., and Strasser de Saad, A.M. (1998). Production of pectinesterase and
polygalacturonase by Aspergillus niger in submerged and solid state fermentations.
Journal of Industrial Microbiology & Biotechnology. pp 34-38
Miller, G.L. (1957). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar.
Analytical Chemistry. pp 426-428
Nampoothiri, K.M., Tomes, G.J., Roopesh, K., Szakacs, G., Nagy, V., Soccol, C.R. and Pandey,
A. (2004) Thermostable phytase production by Thermoascus aurantiacus in submerged
fermentation. Applied Biochemistry and Biotechnology 118(1-3): 205-214.
Ng W K and Chen M L 2002 Replacement of soybean meal with palm kernel meal in practical
diets of hybrid Asian-African catfish, Clarias macrocephalus x Clarias gariepinus. Journal
of Applied Aquaculture 12:67-76

Olude, O.O., Alegbeleye, W.O.A., and Obasa, S.O., The use of soaker copra meal as a partial
substitute for soybean meal in the diet of Nile Tilapia (Oreochromis nilotius) fingerlings.
Department of Aquaculture and Fisheries Management, University of Agriculture, Nigeria
Robyt, J.F. and White, B.J. (1987). Biochemical Techniques: Theory and Practice. California:
Brooks/Cole Publishing Company.
Scopes, R. K., Protein Purification, 3rd edition. Bundoora, Victoria: Springer Verlag New York
Inc, 1998
Subramaniyam, R. and Vimala, R. (2004). Solid State and Submerged Fermentation for the
Production of Bioactive Substances: A Comparative Study. Internation Journal of
Science and Nature, pp 480-486
Sundu, B., and Hatta, U. (2009). Coprase Produced by Aspergillus niger and Trichoderma spp.
Improved Broiler Performance Fed Copra Meal Based Diets. The 1 st International
Seminar on Animal Industry, pp 176-179
Sundu, B., Kumar, A. and Dingle, J. (2006). Response of broiler chicks fed increasing levels of
copra meal and enzymes. International Journal of Poultry Science, 5: 13-18.
Vijayalakshmi, K. Sushma, S. Abha, and P. Chander. (2012). Isolation and Characterization of
Bacillus subtilis KC3 for Amylolytic Activity. International Journal of Bioscience,
Biochemistry, and Bioinformatics. Vol. 2., No. 5. : 336-341
Warren, B.E., and Farrell, D.J. (1990). Animal Feed Science Technology. 27: 247-257