Global demand for seafood is growing rapidly and more than 40% of the
demand is met by aquaculture. Conventional aquaculture diets used fishmeal as the
protein source. The limited production of fishmeal cannot meet increase of
aquaculture production. Therefore, people try to partially or totally replace fishmeal
with less-expensive protein sources in fish feeds, such as poultry by-product meal,
feather meal blood meal, and meat and bone meal. However, these feeds are deficient
in one or some of the essential amino acids, especially lysine, isoleucine and
methionine. And animal protein sources are increasingly less or un-acceptable due to
health concerns. People transferred to sustainable, economic and safe protein sources,
the plant proteins, especially soybean source. Soybean industry has been very popular
in many countries in these 20 years. The world soybean production has increased by
106% from 4.5 billion bushels in 1996 to 9.3 billion bushels in 2010.
Soybean protein is becoming the best choice of sustainable, economic and safe
protein sources. Defatted soybean flour contains about 53% proteins and 32%
carbohydrates. In order to get rid of the un-digestible and anti-nutritional factors and
enrich protein content, the soybean flour needs processing before consumption. The
soy proteins can be concentrated by hydrolyzing the carbohydrates through enzymatic
separation process. The soy proteins produced by the enzymatic separation process
has much higher protein contents and therefore will make better aquaculture diet
formulations. Further, with the hydrolysis of carbohydrates, the possible indigestion
problem for young animals/fish can be avoided. The hydrolyzed soluble
carbohydrates can be used as carbon sources for microorganisms to produce biofuel
and other value-added chemicals. The objective of this project is to produce effective
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enzymes for soybean hydrolysis by fungal fermentation. The producer screening,
culturing conditions, inducers, enzyme assay and stabilities were investigated and
optimized to maximize the yields and productivities of the enzymes.
T. reesei is an efficient producer of carbohydrate-degrading enzymes cellulase,
xylanase, and polygalacturonase. Therefore, the study of enzyme productions of T.
reesei fermentations was carried out to induce more effective enzyme production with
T. reesei. The effects of inclusion of defatted soybean flour and a variety of soluble
and solid carbon sources were evaluated. The fermentation pH effect was also
investigated. The enzyme of T. reesei brought 68% total sugar and 40% reducing
sugar conversion. Based on the low sugar achievement especially reducing sugar, the
strain screening was investigated to select the optimum strain for enzyme production
to hydrolyze the soy carbodydrates. The production of various carbohydrase activities
under soybean hull induction of different strains was studied and two optimum strains
322 and 341 were selected as the most promising strains for producing the enzyme
mixtures more effective for enzymatic upgrading of soybean flour. The selection was
based on both measurements of their enzyme productivities and soy carbohydrate
hydrolysis efficiencies. To achieve high and efficient enzyme production, the
optimization of culture medium and condition were studied of these two optimum
strains. Different carbon, nitrogen sources, carbon to nitrogen ratio, and other medium
compositions were studied to find the optimum. Different inducers were studied to
find the best inducer. Different fermentation controls like pH, temperature, dissolved
oxygen were investigated to find the optimum. With our developed enzyme
production of strain 341, 78% total sugar and 76% reducing sugar conversion were
obtained, which were much higher than the previous work with T. reesei. Accurate,
consistent, and efficient assays for enzyme activities were developed. The stabilities
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and storage time effect were also studied. Besides the soy meal hydrolysis, our
enzyme mixtures were also applied to soy hull hydrolysis to develop the high
carbohydrate syrup with enzyme hydrolysis. With the presence of protease in our
enzyme mixtures, the soy protein analysis was evaluated after hydrolysis to find out
the protease effect on protein degradation.
Overall, this research developed high efficiency production of carbohydrases,
which can separate the soy protein from soy carbohydrate of soy meal. The 70% SPC
and 90% SPI were obtained with 1ml enzyme broth/g soy meal and 25% solid loading,
which were much higher than original 53% protein in soy meal. Besides the high
protein products, high content soluble carbohydrates were achieved with 76%
reducing sugar conversion, which increase the value. In future work, in order to
further increase the enzyme productivities, fed-batch will be suggested. The inducer
soy hull as the carbon source will be gradually added into the fermentor to
continuously stimulate enzyme production. The soy flour will be suggested to
investigate as inducer for enzyme production. Better pH and dissolved oxygen
synthetic control study will be suggested. The future study on soy protein degradation
after hydrolysis will be recommended. The storage investigation of other enzymes
besides a-galactosidase and pectinase will be recommended in future study.