WO2011031020A2 - Method for preparing a fermented soybean meal using bacillus strains - Google Patents

Method for preparing a fermented soybean meal using bacillus strains Download PDF

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WO2011031020A2
WO2011031020A2 PCT/KR2010/005683 KR2010005683W WO2011031020A2 WO 2011031020 A2 WO2011031020 A2 WO 2011031020A2 KR 2010005683 W KR2010005683 W KR 2010005683W WO 2011031020 A2 WO2011031020 A2 WO 2011031020A2
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soybean meal
strain
bacillus
fermentation
fermented soybean
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PCT/KR2010/005683
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French (fr)
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WO2011031020A3 (en
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Sang Hyun Seo
Seong-Jun Cho
Young-Ho Hong
Je-Hoon Ryu
Ju Hui Kang
Hyun Chi
Seung-Won Park
Yu Ryang Pyun
Seck Cheol Cho
Moo Chang Kook
Hyun Ho Park
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Cj Cheiljedang Corporation
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Priority claimed from KR1020100028142A external-priority patent/KR101139027B1/en
Application filed by Cj Cheiljedang Corporation filed Critical Cj Cheiljedang Corporation
Publication of WO2011031020A2 publication Critical patent/WO2011031020A2/en
Publication of WO2011031020A3 publication Critical patent/WO2011031020A3/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms

Definitions

  • the present invention relates to an inexpensive and efficient method for producing a fermented soybean meal for the amelioration or improvement of the quality of defatted soybean meal as a plant protein source, and a fermented soybean meal produced by the same.
  • soybean meal A defatted soybean meal (hereinbelow, referred to as “soybean meal”) is the most common source of plant protein used in feed markets as a substitute for animal proteins such as fishmeal, meat bone powder or plasma. In Korea, soybean meal, used as a plant protein source, accounts for 60% of total meal supply, reaching 2 million tons annually (Korea Feed Ingredients Association, 2004).
  • Soybean meal is usually composed of 9.5% water, 49.4% crude protein, 22.1% invert sugar and 27.2% soluble nitrogen.
  • plant proteins have low protein content and are also low in the composition of essential amino acids required for livestock and in the content of some vitamins, minerals and UGF (Unknown Growth Factor), as compared to animal proteins.
  • soybean meal contains a variety of anti-nutritional factors (ANFs), which can problematically impair the rate of digestion when used as a feed.
  • ANFs anti-nutritional factors
  • TI trypsin inhibitor
  • dietary TI are known to interfere with the proper function of trypsin and chymotrypsin leading to a reduction in the availability of total protein present in soybean up to approximately 6%.
  • these anti-nutritional factors greatly affect young livestock, the use of soybean meal in the feed for young livestock is restricted.
  • TI activity of raw soybean meal is approximately 39 mg/g, but that of defatted soybean meal is generally less than 4 mg/g, because anti-nutritional factors are destroyed by heat treatment during commercial processing of the defatted soybean meal.
  • Growing-finishing pigs or sows are not greatly affected by anti-nutritional factors, but the use of soybean meal for weanling pigs is restricted due to anti-nutritional factors, unlike growing-finishing pigs. Therefore, use of alternative proteins should be considered distinguished by feeding steps. For this reason, processed soybean products such as soy protein concentrate or soy protein isolate are used as a feed to obtain growth performance similar to that of milk-based feeding.
  • soybean products such as soy protein concentrate (SPC), soy protein isolate (SPI) or hydrolyzed soy protein
  • SPC soy protein concentrate
  • SPI soy protein isolate
  • hydrolyzed soy protein are usually produced by physical/chemical treatment or enzymatic treatment for food processing, and are too expensive to feed livestock.
  • soybean meal as a high-quality protein feed
  • recent climate change such as global warming decreases grain yields
  • the international price of corn or soybean meal used as a feed source increases, inflicting severe damage on feed and livestock industries.
  • the need is rapidly growing.
  • Soybean meal is also known to contain soy oligosaccharides causing diarrhea and abdominal pain and soy polysaccharides inhibiting nutrient absorption, in addition to the above described TI.
  • An admixture of soybean meal with feed frequently induces enteritis, but the cause has not been clarified. It is suggested that the enteritis is attributed to alcohol-soluble polysaccharides in the soybean meal [Krogdahl, A. et al. Feeding atlantic Salmo salar L. soybean products. Aquac. Nutr. 6, 77-84, 200].
  • soy polysaccharides inhibit the absorption of nutrients in fish such as salmon and broiler chickens [Refstie, S. et al. Non-starch polysaccharides in soybean meals and effects on the absorption of nutrients in farmed Atlantic salmon and broiler chickens. Anim. Feed Sci. Technol. 79,331-345 1999].
  • the fermented soy proteins are said to be high-quality feed additives for easy digestion and best absorption, because a plurality of anti-nutritional factors can be removed and proteins or carbohydrates are degraded into digestible forms during the fermentation process.
  • Bacillus strain in particular, Bacillus subtilis TP6 is used to produce a fermented soybean meal while reducing the fermentation time. Also, there are no products produced by utilizing protease and functional polysaccharides, which are produced by Bacillus during solid fermentation of soybean meal, or by applying the efficacy of the probiotic microorganisms.
  • the present inventors have made many efforts to construct production systems of fermented soybean meals for the amelioration or improvement of the quality of soybean meals as a feed protein source and to reduce the fermentation time for reduction in overall costs.
  • Bacillus subtilis TP6 having properties suitable for solid fermentation of soybean meal, is adopted to perform solid fermentation of soybean meal, thereby increasing the content and quality of proteins, improving digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins, TI inactivation, or reduction in the content of anti-nutritional factors such as non-digestible polysaccharides, as well as remarkably improving the fermentation time that is an obstacle in the known production methods of fermented soybean meal.
  • Bacillus subtilis TP6 having properties suitable for solid fermentation of soybean meal, is adopted to perform solid fermentation of soybean meal, thereby increasing the content and quality of proteins, improving digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins, TI inactivation, or reduction in the content of anti-nutritional factors such as non-digestible polysacc
  • the present invention provides a method for producing a high-quality fermented soybean meal having ameliorated or improved properties by performing solid fermentation using Bacillus strain, in particular, Bacillus subtilis TP6 strain, which is effectively capable of removing anti-nutritional factors from soybean meal.
  • the present invention uses the Bacillus subtilis TP6 strain, which is capable of producing final products with the quality equivalent to or better than those produced by the method known in art while reducing the fermentation time up to 12 to 24 hrs.
  • the fermented soybean meal produced by the present invention is a high-quality protein source, in which powerful Bacillus-producing protease inactivates TI by hydrolysis and improves digestion and absorption rates by low-molecularization of soybean proteins into peptides.
  • Bacillus strain which is unusual in solid fermentation, is used as a fermentation strain.
  • the fermented soybean meal produced by the present invention becomes a high-quality protein source, in which powerful Bacillus-producing protease inactivates TI by hydrolysis and improves digestion and absorption rates by low-molecularization of soybean proteins into peptides.
  • the fermented soybean meal produced by the method of the present invention produces high-quality cellular proteins by active growth of a Bacillus strain, thereby increasing the absolute amount of protein and consuming carbohydrates in the soybean meal as an energy source required for the growth, leading to an increase in the relative protein content. Therefore, its value as a protein feed is more improved.
  • the fermented soybean meal produced by the present invention contains Bacillus strain having strong viability, and thus it has an advantage of helping intestinal regulation of the animals fed. Considering that the use of probiotics is positively recommended as an alternative to antibiotic-free feeds, the fermented soybean meal of the present invention will be more valuable in the future.
  • Bacillus strain with a capability of producing polyglutamic acid is used to perform the fermentation of soybean meal, thereby obtaining the additional effect of reducing the body fat of livestock.
  • Polyglutamic acid is a sticky mucilaginous substance found in Korean chungkukjang or Japanese natto, and is a functional ingredient having various functions. In particular, its effects on animal fat reduction have been recently revealed.
  • the fermented soybean meal of the present invention has high quality and various functions, thereby being widely applied to feeds as a high-protein source.
  • the fermentation time can be reduced up to 12 to 24 hrs, compared to the known method.
  • FIG. 1 is a schematic diagram showing a flow chart for producing the fermented soybean meal by using soybean meal as a raw material in accordance with one embodiment of the present invention
  • FIG. 2 is a graph showing changes in the activity of trypsin inhibitor, when each single culture of L.plantarum P23 and B.subtilis TP6 and co-culture of L.plantarum P23 and B.subtilis TP6 is performed;
  • FIG. 3 shows changes in the protein pattern, when each single culture of L.plantarum P23 and B.subtilis TP6 and co-culture of L.plantarum P23 and B.subtilis TP6 is performed;
  • FIG. 4 shows chromatography for sugar analysis of the heat-treated soybean meal inoculated no bacteria
  • FIG. 5 shows chromatography for sugar analysis of the soybean meal fermented by B. subtilis TP6
  • FIG. 6 shows chromatography for sugar analysis of the soybean meal fermented by B. subtilis TP6 and L.plantarum P23;
  • FIG. 7 shows the effects of B. subtilis TP6 expression on protein degradation and allergen removal (marker; (1): raw soybean meal; (2): 37°C 45%; (3): 37°C 50%; (4): 45°C 45%; (5): 45°C 50%).
  • the present invention relates to a method for producing a fermented soybean meal, comprising the steps of: (a) adding water to a soybean meal to perform heat-treatment; (b) cooling the heat-treated soybean meal, and then inoculating a Bacillus strain thereinto; and (c) acquiring a fermented soybean meal by solid fermentation of the Bacillus-inoculated soybean meal.
  • the present inventors have made an effort to construct production systems for soybean fermentation for the purpose of the amelioration or improvement of the quality of soybean meals as well as for reduction of the fermentation time. They found that a Bacillus strain is used as a fermentation strain to perform solid fermentation of soybean meals, thereby producing a fermented soybean meal devoid of the above mentioned problems.
  • the soybean meals used in the method of the present invention are supplied from the same source every time.
  • difference in the quality of the final fermented soybean meal is attributed to difference in the initial nutritional composition of the raw material, which merely affects final nutritional composition of the fermented soybean meal, but does not influence fermentation itself.
  • difference in protein content of soybean meal depending on its type influences protein content of the final product.
  • the quality of fermented soybean meal is improved.
  • a process of adding water is required prior to heat-treatment of the raw soybean meal. That is, before solid fermentation, a proper amount of water is directly sprayed onto the raw soybean meal, and is mixed to control the moisture content, followed by heat-treatment for a predetermined time.
  • the heat-treatment is performed for the purpose of killing a variety of germs in the raw soybean meal, destruction of the soybean cell wall, and denaturation of proteins, thereby providing chemical compositions for active growth of the desired microorganism.
  • the water-added soybean meal in the step (a) has a water content of 30 to 80% (v/w), more preferably 30 to 70% (v/w), and most preferably 40 to 60% (v/w).
  • the water content ranging from 30 to 80% (v/w) is preferred so that delay of fermentation due to low moisture can be prevented, the cost of transfer and drying process after fermentation can be reduced, and heating efficiency can be improved.
  • the water-added soybean meal is subjected to heat-treatment.
  • the heat-treatment process can be performed by various methods known in the art, but steam or superheated steam is preferably.
  • the heat-treatment of step (a) is performed using steam at a temperature ranging from 70 to 130°C for 10 to 60 min or superheated steam at a temperature ranging from 200 to 300°C for a short time of several seconds to minutes, more preferably steam at a temperature ranging from 70 to 130°C for 10 to 30 min, and most preferably steam at a temperature ranging from 80 to 121.1°C for 10 to 30 min.
  • the temperature of heat-treatment is low or the treatment time is short, there are problems that the sterilization effect on various germs is not sufficient or the subsequent fermentation process does not proceed smoothly. If the temperature of heat-treatment is high or the treatment time is long, protein denaturation occurs in the soybean meal to reduce the rate of digestion, resulting in deterioration of the quality of the final product. Therefore, it is preferable that the temperature and time of heat-treatment is adopted within acceptable ranges to avoid these problems.
  • the contaminants present in the soybean meal are almost completely removed, a chemical environment suitable for the subsequent solid fermentation is formed, and TI inhibiting the rate of digestion is slightly reduced.
  • a step of selecting and pre-culturing a bacillus strain with excellent protein degradation capacity is further included before or after step (a).
  • a fungus such as Aspergillus oryzae , which grows well under low moisture conditions, is generally used because of low moisture content of a solid substrate.
  • fungus when fungus is used to perform the fermentation of soybean meal, the produced, fermented soybean meal is in a state of not being suitable for feed use.
  • Production of soybean meals by co-culture of fungus with Bacillus is described in Korean Patent No. 10-0645284.
  • it is not practically easy to culture both of them at the same time fungus grows well at low moisture, but bacteria need more moisture to grow
  • post-culture by post-addition of any one of them should be performed to produce products.
  • Bacillus strain as a fermentation strain of soybean meal, which grows well under low oxygen conditions and has an excellent protein hydrolysis activity.
  • antibiotics to feed is prohibited worldwide.
  • probiotics having a variety of physiological functions such as health promotion by improvement of intestinal flora, inhibition of proliferation of harmful bacteria, and improvement in immunity, disease resistance or feed efficiency.
  • lactobacillus is used as a probiotic, but spore-forming bacteria are newly appreciated as probiotics, thereby increasing the interest in spore-forming bacteria as probiotics added to non-antibiotic feeds.
  • a representative strain of spore-forming probiotics is a Bacillus strain.
  • the Bacillus strain selected to be used in the present invention is Bacillus subtilis , Bacillus cereus , Bacillus megaterium or Bacillus clausii .
  • Bacillus subtilis is more preferred, and Bacillus subtilis TP6 strain (KFCC 11343P, Korean Patent No. 10-0753002) is most preferred.
  • Bacillus subtilis TP6 (hereinbelow, referred to as “TP6 strain”) strain grew well in soybean meal with a low water content of 40% or more under aerobic and anaerobic conditions, showed excellent hydrolysis activity of soybean proteins, and had the unique property of producing polyglutamic acid during the late fermentation period.
  • TP6 strain when used, the fermentation time was remarkably reduced, as compared to the known method.
  • the strain of the present invention may be further selected as a strain to be used for inoculation of Lactobacillus, or a mixed strain of Lactobacillus and Bacillus strain.
  • Plant Lactobacillus isolated from milk or cheese, grows using lactose, but plant Lactobacillus isolated from vegetables utilizes various sugars such as glucose, fructose, fructose or maltose and has high environmental adaptation to survive under harsh conditions such as low pH.
  • plant Lactobacillus shows higher survival rate in the stomach than animal Lactobacillus, and a number of strains reach the intestine.
  • plant Lactobacillus means Lactobacillus derived from plant sources.
  • Korean kimchi is said to be a representative source of plant Lactobacillus.
  • spices such as hot red pepper and garlic are added.
  • kimchi Lactobacillus is exposed to more harsh conditions than other Lactobacillus, it survives to show excellent ability of degradation and intake of nutrients and productivity of various physiologically active materials.
  • the present inventors have made many efforts to isolate Lactobacillus suitable for soybean meal fermentation from kimchi.
  • the selected Lactobacillus is preferably Lactobacillus sakei , Lactobacillus brevis , or Lactobacillus plantarum , more preferably Lactobacillus plantarum , and most preferably Lactobacillus plantarum P23 (KCCM 80048) which is isolated from kimchi by the present inventors.
  • the Lactobacillus plantarum P23 strain (hereinafter, referred to as “P23 strain”) was found to remarkably reduce non-starch polysaccharides, which are anti-nutritional factors present in soybean meal, and show excellent capability of producing organic acids and acid-resistance.
  • Media for seed culture of Lactobacillus isolated from kimchi may be any media known in the art, preferably MRS broth (deMan Rogosa Sharpe broth), APT (All Purpose with Tween) or BHI (Brain Heart Infusion), and more preferably MRS broth.
  • MRS broth deMan Rogosa Sharpe broth
  • APT All Purpose with Tween
  • BHI Brain Heart Infusion
  • MRS broth is too expensive for industrial use
  • the present inventors constructed an inexpensive media for seed culture of Lactobacillus, and CSL (Corn Steep Liquor) was used as carbon and nitrogen sources for seed culture of Lactobacillus.
  • modified medium (5 g/L soytone, 5 g/L beef extract and 20 g/L xylose, pH 7.0) is preferably used to culture at 37°C for 12 to 24 hrs.
  • This medium may also increase cost price upon large-scale production for industrial use, and thus it is preferable to culture Bacillus in an industrial medium based on CSL contrived by the present inventors.
  • Lactobacillus isolated from kimchi is inoculated into the industrial medium contrived by the present inventors, and cultured at 30°C for 8 to 24 hrs, thereby obtaining activity of the desired strain.
  • Each of two strains is cultured in the industrial medium for seed culture, until the final number of living cells in the seed culture medium becomes (1-5)x10 9 cfu/mL.
  • step (b) heat-treated soybean meal is cooled to a temperature at which solid fermentation can be performed, and then Bacillus is inoculated thereto.
  • the cooling of soybean meal is ordinarily performed after heat-treatment, in which the cooling process can be readily performed through a transfer process using a cooling conveyor in order to prevent overheating and cool uniformly by increasing cooling rate (FIG. 1).
  • the cooled soybean meal of step (b) in the method of the present invention has a temperature of 30 to 50°C, more preferably 35 to 45°C, and most preferably 37°C.
  • the pre-culture media of Bacillus strain is uniformly inoculated into the prepared soybean meal medium as it is or by dilution with sterilized water.
  • the number of microorganisms inoculated into the heat-treated soybean meal is an important factor that influences the solid fermentation of soybean meal.
  • the number of microorganisms immediately after inoculation into the heat-treated soybean meal is preferably 10 5 to 10 9 CFU/g. If the inoculation amount is less than 10 5 CFU/g, a small amount of seed fermentation broth is needed, but much time is required for the fermentation of soybean meal to increase incubation time and possibility of contamination. If the inoculation amount is more than 10 9 CFU/g, the fermentation time can be considerably reduced, but production of seed microorganism used for inoculation is problematic. In particular, since fermentation performance is greatly influenced by growth characteristics of microorganism and type of fermentor, it is preferable that the inoculation amount is properly determined depending on characteristics of the microorganism in the production step.
  • One of the characteristics of the present invention is to produce a fermented soybean meal by inoculating Bacillus into the soybean meal, in which unlike fungus or yeast, Bacillus is not usually used for solid fermentation (culture).
  • solid fermentation means that a defatted soybean meal obtained by removal of oil from soybean is used to culture microorganisms, and is distinguished from “liquid culture or liquid fermentation” using soybean meal extracts.
  • soybean meal is a solid substrate in a flake or particle state, and thus solid fermentation is an inexpensive and efficient method capable of improving the value of soybean meal as a feed using microorganisms.
  • a strain selected from Bacillus strains is used, and the strain type and inoculation method can be controlled depending on the properties of the final product of fermented soybean meal.
  • Bacillus subtilis TP6 strain is used to reduce the fermentation time of the present invention.
  • the Bacillus strain In the case of producing the fermented soybean meal by inoculation of only Bacillus, the Bacillus strain actively grows to improve the quality of the fermented soybean meal, and at this time, the Bacillus strain first produces protease having powerful activity.
  • the Bacillus-producing protease is characterized in that it inactivates TI by hydrolysis, in which TI is a protein inhibiting the digestion of soybean meal, and improves digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins.
  • the Bacillus strain actively grows by utilizing carbohydrates among the components of soybean meal, the carbohydrates are converted into proteins constituting the cell.
  • the relative protein content of the soybean meal is increased during the fermentation, which is the most important factor among the evaluation items of the feed quality.
  • the Bacillus strains grown in the soybean meal form spores to survive at a high ratio, and thus they exist as viable cells even after drying the fermented soybean meal. Due to the spore formation of Bacillus, it is expected to show the effect of improving intestinal function as the above described spore-forming probiotics in livestock.
  • Lactobacillus may be inoculated alone or in a mixture with Bacillus.
  • plant Lactobacillus isolated from kimchi is only inoculated, it grows well by utilizing the soybean meal as a raw material to produce a large amount of organic acids.
  • plant Lactobacillus has an alpha galactosidase activity so as to exhibit a property of degrading galactooligosaccharides contained in the soybean meal, and hydrolyzes various polysaccharide anti-nutritional factors in the soybean meal so as to prevent enteritis and improve the rate of nutrient absorption.
  • the fermented soybean meal having all advantages of the two bacteria, they may be inoculated simultaneously or sequentially at time intervals.
  • growth conditions of two bacteria differ from each other, it is preferable that they are inoculated at a different ratio or sequentially, in terms of the quality of the final product.
  • simultaneous inoculation or inoculation at the same ratio fail to achieve the desired effects.
  • the soybean meal that is uniformly inoculated with the desired microorganism is fermented in a packed-bed fermentor in step (c).
  • the packed-bed fermentor is divided into batch, closed, and continuous stirred tank reactor.
  • the method of the present invention is not limited to any one of them, and it can be selected depending on the production scale.
  • the soybean meal is applied to the packed-bed fermentor in a thickness of 5 to 50 cm, and fermented at a temperature of 20 to 50°C for 12 to 72 hrs.
  • the method of the present invention further includes the step of (d) drying and pulverizing the fermented soybean meal at low temperature and moisture after step (c).
  • Water contained in the soybean meal is partially evaporated during the fermentation process, but the residual water content is considerably high at 20 to 50% (v/w) immediately after fermentation.
  • the preferred, final water content of the fermented soybean meal product is 10 to 12% (v/w), and thus a drying process is required.
  • the soybean meal When the soybean meal is fermented using fungus such as Aspergillus oryzae, it becomes hard and forms conglomerates by mycelium production, and it is very difficult to perform the drying and pulverization processes because of spores. Meanwhile, when the fermentation is performed using bacteria in the present invention, the fermented soybean meal is in good condition, but conglomerates are slightly formed. Therefore, after the drying process, the process of pulverizing the fermented soybean meal is required to form a uniform particle size.
  • fungus such as Aspergillus oryzae
  • the drying and pulverization processes may be performed by various methods known in the art. However, when the drying process is excessively performed at a high temperature, a number of live bacteria in the fermented soybean meal may be killed, and thus it should be performed with caution. Preferably, the drying process is performed at a low temperature without killing living bacteria. Most preferably, the drying process is performed with hot air at low temperature and humidity.
  • the fermented soybean meal may be pulverized to various sizes depending on the purpose of use, and preferably, by means of a hammer mill.
  • soybean meal When the soybean meal is fermented according to the above described method of the present invention, various anti-nutritional factors including TI in the soybean meal are reduced, the rate of digestion and absorption is improved by hydrolysis of proteins, and the protein content is also increased, thereby improving the absolute value as feed. Consequently, a high-quality fermented soybean meal can be obtained as an alternative to animal proteins.
  • the fermented soybean meal produced by the method of the present invention contains Bacillus strain having strong viability, and thus it has an advantage of helping intestinal regulation of the animals fed.
  • the present invention relates to a fermented soybean meal produced by the method of the present invention.
  • fermented soybean meal of the present invention is produced by the above mentioned method of the present invention, the common description will be omitted herein to avoid redundancies.
  • the Bacillus strain In the fermented soybean meal produced by the inoculation of Bacillus subtilis TP6 strain, the Bacillus strain actively grows to improve the quality of fermented soybean meal, and at this time, the Bacillus strain first produces protease having powerful activity.
  • the Bacillus-producing protease is characterized in that it inactivates TI by hydrolysis, in which TI is a protein inhibiting the digestion of soybean meal, and improves digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins.
  • TI is a protein inhibiting the digestion of soybean meal
  • improves digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins Further, while the Bacillus strain actively grows by utilizing carbohydrates among the components of soybean meal, the carbohydrates are converted into proteins constituting the cell. Thus, a relative protein content of the soybean meal is increased during the fermentation, which is the most important factor among the evaluation items of the feed quality.
  • the Bacillus strains grown in the soybean meal form spores to survive at a high ratio, and thus they exist as viable cells even after drying the fermented soybean meal. Due to the spore formation of Bacillus, it is expected to show an effect of improving intestinal function as the above described spore-forming probiotics in livestock.
  • the fermented soybean meal of the present invention contains vegetative cells or spores of microorganism selected from Bacillus strains, preferably, Bacillus subtilis TP6 strain.
  • the fermented soybean meal of the present invention contains poly- ⁇ -glutamic acid to reduce body fat accumulation in livestock.
  • the Bacillus strain includes Bacillus subtilis TP6 strain.
  • % used to indicate that a concentration of a specific material is (weight/weight)% for solid/solid, (weight/volume)% for solid/liquid, (volume/volume)% for liquid/liquid, unless otherwise specified.
  • Example 1 Growth of Bacillus and Lactobacillus depending on water contents of soybean meal
  • soybean meal suitable for culture of bacteria
  • water was added to the soybean meal at the water content of 30%, 40%, 50% and 60%, and 50 g of the water-added soybean meal was put in a beaker (250 mL). Then, the top portion of the beaker was covered with aluminium foil, and fastened with a rubber band. Heat-treatment was performed at 80°C for 30 min, followed by cooling.
  • the soybean meal fermentation was performed using the preferred Bacillus subtilis TP6 (KFCC 11343P) (hereinafter, referred to as “TP6” strain) and Lactobacillus plantrum P23 (KCCM 80048) (hereinafter, referred to as “P23” strain).
  • TP6 Bacillus subtilis TP6
  • KCCM 80048 Lactobacillus plantrum P23
  • a medium containing xylose 2%, soytone 0.5% and beef extract 0.5% was used for seed culture of the TP6 strain, and the P23 strain was pre-cultured using an MRSbroth (Difco) at a temperature of 37°C for 24 hrs.
  • MRSbroth MRSbroth
  • the bacterial growth became more active.
  • the final number of P23 and TP6 strains increased to 3.2 x 10 9 CFU/g after fermentation, and the pH value of the P23 strain was remarkably reduced by production of organic acids after fermentation.
  • the initial water content of soybean meal should be over 40% for industrial-scale production of bacteria.
  • heat-treatment of the water-added soybean meal is important in terms of preventing contamination and making the soybean meal to have a chemical environment suitable for fermentation.
  • the present inventors investigated heat-treatment conditions of soybean meal. To establish minimum heat-treatment conditions in which contamination can be prevented and the inoculated strain can grow actively during the fermentation period, the water content of soybean meal was adjusted to 60%, and then 50 g thereof was put into a 250 mL beaker and the top portion of the beaker is sealed with aluminium foil, followed by heat treatment at 60°C, 80°C, 105°C and 121.1°C autoclaves for 10-30 min, respectively.
  • Example 2 5% of each seed culture medium of TP6 and P23 strains was inoculated into each soybean meal that was heat-treated under different heat-treatment conditions, and incubated at 37°C for 72 hrs. The degree of contamination during fermentation was assessed and the number of live bacteria and pH in the fermented soybean meal were measured after cultivation.
  • the heat-treatment conditions affecting the soybean meal fermentation are shown in the following Table 2.
  • Example 3 Changes in quality and property of fermented soybean meal upon single culture of Lactobacillus and Bacillus
  • FIGs. 4, 5, and 6 The polysaccharide analysis results of the heat-treated soybean meal not inoculated, the soybean meal fermented with TP6 strain, and the soybean meal fermented with TP6 and P23 strains are shown in FIGs. 4, 5, and 6, respectively. Between FIG.4 and FIG. 5, there is little difference in sugar patterns by chromatography, indicating that the TP6 strain shows high enzymatic activity including protease, but hardly hydrolyzes soybean polysaccharides. However, comparing FIGs. 4 and 5 with FIG. 6 which shows the results of mixed fermentation of lactobacillus P23 isolated from Kimchi and the TP6 strain, it was found that polysaccharides, which are the anti-nutritional factors in soybean meal, were almost all hydrolyzed by the P23 strain.
  • Example 4 Mixed culture of lactobacillus and Bacillus in soybean meal
  • the water content of soybean meal was adjusted to 50%, and heat-treatment was performed at 110°C for 15 min.
  • the seed cultures of TP6 and P23 strains were inoculated into the treated soybean meal at different inoculation ratios or for different inoculation times, and cultured at 37°C for 48 hrs.
  • the “mixed culture 1” means that soybean meal was initially inoculated with 5% of TP6 strain and cultured for 20 hrs, and then 5% of P23 strain cultured in seed culture medium for 20 hrs was inoculated thereto, followed by additional fermentation for 28 hrs.
  • the “mixed culture 2 and 3” means that TP6 and P23 strains were initially inoculated at the different ratios of 1:0.5 and 1:1, followed by fermentation for 48 hrs.
  • the quality of fermented soybean meal has slight differences in the final number of bacteria, acidity, and flavor. Therefore, it is preferable to properly control the inoculation time and ratio of microorganisms selected from the group consisting of plant lactobacillus, Bacillus strains, and the mixture thereof, depending on the final product to be desired.
  • the water content of soybean meal was adjusted to 30%, and heat-treatment was performed at 121°C for 20 min. Sterilized water was uniformly sprayed to the sterilized soybean meal to adjust the water content to 60%.
  • TP6 and P23 strains cultured in each seed culture medium for 24 hrs were inoculated into the soybean meal to be 10%(v/w) of the soybean meal, and then fermentation was performed at 37°C for 48 hrs. After fermentation, the soybean meals were dried, and their physicochemical properties were examined, as shown in the following Table 4.
  • the fermented soybean meal fermented with only the TP6 strain of the present invention showed a highly increased protein content, as well as reduced content of the anti-nutritional factors including trypsin inhibitor, soybean oligosaccharides and polysaccharides, as compared to raw soybean meal, showing a highly improved value as feed.
  • the fermented soybean meal contained live bacteria of 2.0x10 9 CFU/g DM and polyglutamic acid, indicating that it has a much more improved value.
  • Example 6 Changes in TI content under different culture conditions
  • trypsin inhibitor which is one of anti-nutritional factors inhibiting digestion and absorption
  • the TI content was assessed by Korea Feed Ingredients Association in the AOAC method.
  • the experimental groups are as follows: “soybean” group: common soybean, “soybean meal” group: residue produced by removing soybean oil from soybean, “24 hr fermentation” group: 100 g of soybean meal was adjusted to have 45% water content, heat-treated (90°C for 15 min), and 5 ml of TP6 strain (2X10 9 cfu/ml) was inoculated into the heat-treated soybean meal, followed by fermentation at 37°C and constant humidity for 24 hrs. Changes in TI (trypsin inhibitor) content after fermentation were shown in the following Table 5.
  • Example 7 Changes in crude protein content depending on fermentation time upon single culture of TP6 strain
  • soybean meal 100 g was adjusted to have 45% water content, heat-treated (90°C for 15 min), and 5 ml of TP6 strain (2X10 9 cfu/ml) was inoculated into the heat-treated soybean meal, followed by fermentation at 37°C and constant humidity for 24 hrs. Changes in protein content during fermentation were shown in the following Table 6.
  • the protein content was obtained at a level equivalent to those of 36 hr fermentation of mixed strains (Korean Patent No. 10-0645284) and 48 hr fermentation of single strain (Korean Patent No. 10-0459240) while reducing the fermentation time up to 12 to 24 hrs, as compared to the prior art (the crude protein content is important in the soybean meal product as a protein source, because a total crude protein content is important in the composition).
  • the reduction in fermentation time reduces the fermentor batch cycle, resulting in an increase in the annual batch number. Consequently, consumers can be provided with high-quality fermented soybean meals at a low cost.
  • Example 8 Changes in crude protein content depending on fermentation time upon single culture of TP6 strain
  • Example 9 Effect of TP6 fermentation on protein degradation and allergen removal ( ⁇ -conglycinin, glycinin)
  • Fermentation experiments were performed under different conditions of temperature and moisture for 24 hrs.
  • the fermentation conditions are the same as in Examples 7 to 8, and water% was only changed during heat-treatment as follows: Lane 2; 37°C 45%, Lane 3; 37°C 50%, Lane 4; 45°C 45%, Lane 5; 45°C 50% in FIG. 7.
  • Fig. 7 it was found that the allergens in the raw soybean meal, ⁇ -conglycinin and glycinin were excellently removed at the water content of 45 to 50% and temperature of 37°C, indicating that high-molecular proteins are degraded into low-molecular proteins, and converted into a form suitable for digestion and absorption.

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Abstract

The present invention relates to a method for producing a fermented soybean meal, comprising the steps of: (a) adding water to a soybean meal to perform heat-treatment; (b) cooling the heat-treated soybean meal, and then inoculating a Bacillus strain thereinto; and (c) acquiring a fermented soybean meal by solid fermentation of the Bacillus-inoculated soybean meal, and to a fermented soybean meal produced by the method. The method of the present invention uses a Bacillus strain having excellent properties required for the production of fermented soybean meal, in particular, Bacillus subtilis TP6 strain as a fermentation strain. Therefore, the fermented soybean meal produced by the present invention is a high-quality plant protein source in that various anti-nutritional factors including trypsin inhibitors, soybean oligosaccharides and polysaccharides are almost all removed, its protein content is high, and digestion and absorption rates are also improved by low-molecularization of the proteins. In particular, the TP6 strain capable of removing anti-nutritional factors is used to improve the product quality equivalent to or better than those produced by the known fermented soybean meals while reducing the fermentation time, thereby remarkably increasing the annual production.

Description

METHOD FOR PREPARING A FERMENTED SOYBEAN MEAL USING BACILLUS STRAINS
The present invention relates to an inexpensive and efficient method for producing a fermented soybean meal for the amelioration or improvement of the quality of defatted soybean meal as a plant protein source, and a fermented soybean meal produced by the same.
As mad cow disease, fatal to humans, is proven to be caused by animal protein components in the cattle feed, there is an increasing worldwide trend of substituting plant proteins for animal proteins in the feed.
A defatted soybean meal (hereinbelow, referred to as “soybean meal”) is the most common source of plant protein used in feed markets as a substitute for animal proteins such as fishmeal, meat bone powder or plasma. In Korea, soybean meal, used as a plant protein source, accounts for 60% of total meal supply, reaching 2 million tons annually (Korea Feed Ingredients Association, 2004).
Soybean meal is usually composed of 9.5% water, 49.4% crude protein, 22.1% invert sugar and 27.2% soluble nitrogen. In general, plant proteins have low protein content and are also low in the composition of essential amino acids required for livestock and in the content of some vitamins, minerals and UGF (Unknown Growth Factor), as compared to animal proteins.
Moreover, soybean meal contains a variety of anti-nutritional factors (ANFs), which can problematically impair the rate of digestion when used as a feed. Among them, a trypsin inhibitor (hereinbelow, referred to as “TI”) is a representative ANF, and in terrestrial animals, dietary TI are known to interfere with the proper function of trypsin and chymotrypsin leading to a reduction in the availability of total protein present in soybean up to approximately 6%. In particular, since these anti-nutritional factors greatly affect young livestock, the use of soybean meal in the feed for young livestock is restricted.
TI activity of raw soybean meal is approximately 39 mg/g, but that of defatted soybean meal is generally less than 4 mg/g, because anti-nutritional factors are destroyed by heat treatment during commercial processing of the defatted soybean meal. Growing-finishing pigs or sows are not greatly affected by anti-nutritional factors, but the use of soybean meal for weanling pigs is restricted due to anti-nutritional factors, unlike growing-finishing pigs. Therefore, use of alternative proteins should be considered distinguished by feeding steps. For this reason, processed soybean products such as soy protein concentrate or soy protein isolate are used as a feed to obtain growth performance similar to that of milk-based feeding.
The current processed soybean products, such as soy protein concentrate (SPC), soy protein isolate (SPI) or hydrolyzed soy protein, are usually produced by physical/chemical treatment or enzymatic treatment for food processing, and are too expensive to feed livestock.
Therefore, to use soybean meal as a high-quality protein feed, there is a need to develop inexpensive and efficient, large-scale methods of processing soybean proteins for their quality improvement. Moreover, as recent climate change such as global warming decreases grain yields, the international price of corn or soybean meal used as a feed source increases, inflicting severe damage on feed and livestock industries. Thus, the need is rapidly growing.
Soybean meal is also known to contain soy oligosaccharides causing diarrhea and abdominal pain and soy polysaccharides inhibiting nutrient absorption, in addition to the above described TI. An admixture of soybean meal with feed frequently induces enteritis, but the cause has not been clarified. It is suggested that the enteritis is attributed to alcohol-soluble polysaccharides in the soybean meal [Krogdahl, A. et al. Feeding atlantic Salmo salar L. soybean products. Aquac. Nutr. 6, 77-84, 200]. In addition, there is a report that soy polysaccharides inhibit the absorption of nutrients in fish such as salmon and broiler chickens [Refstie, S. et al. Non-starch polysaccharides in soybean meals and effects on the absorption of nutrients in farmed Atlantic salmon and broiler chickens. Anim. Feed Sci. Technol. 79,331-345 1999].
Therefore, to produce high-quality feeds by removal of anti-nutritional factors, many studies have been made on a phytase-treated soy-protein product devoid of phytate (HP300 (Demark) - www.hamletprotein.com) or fermented soy proteins (Korean Patent Nos. 10-0645284, 10-0459240, and 10-0925173, Livestock Research for Rural Development 20(9) 2008).
Of these, the fermented soy proteins are said to be high-quality feed additives for easy digestion and best absorption, because a plurality of anti-nutritional factors can be removed and proteins or carbohydrates are degraded into digestible forms during the fermentation process.
During fermentation processing, however, a longer fermentation time is required to remove anti-nutritional factors beyond any predicted levels (36 hrs in Korean Patent No. 10-0645284, and 48 hrs in Korean Patent No. 10-0459240). Such a long fermentation time increases the fermentor batch cycle, resulting in an increase in overall costs. As such, the problems in the prior arts are to increase overall costs during the production process of fermented soybean meal due to long fermentation time.
Until now, there have been no reports that Bacillus strain, in particular, Bacillus subtilis TP6 is used to produce a fermented soybean meal while reducing the fermentation time. Also, there are no products produced by utilizing protease and functional polysaccharides, which are produced by Bacillus during solid fermentation of soybean meal, or by applying the efficacy of the probiotic microorganisms.
Accordingly, the present inventors have made many efforts to construct production systems of fermented soybean meals for the amelioration or improvement of the quality of soybean meals as a feed protein source and to reduce the fermentation time for reduction in overall costs. the present inventors found that Bacillus subtilis TP6, having properties suitable for solid fermentation of soybean meal, is adopted to perform solid fermentation of soybean meal, thereby increasing the content and quality of proteins, improving digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins, TI inactivation, or reduction in the content of anti-nutritional factors such as non-digestible polysaccharides, as well as remarkably improving the fermentation time that is an obstacle in the known production methods of fermented soybean meal. Finally, they prepared high-quality fermented soybean meals, leading to the present invention.
Throughout this application, various publications are cited. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
It is an object of the present invention to provide a method for producing a fermented soybean meal by solid fermentation using Bacillus strain, in particular, TP6 strain, which is effectively capable of removing anti-nutritional factors and has superior protein digestibility to minimize fermentation time upon the production of fermented soybean meal.
It is another object of the present invention to provide a high-quality fermented soybean meal which is fermented by the Bacillus strain.
Other objects and advantages of the present invention will become apparent upon reading the following detailed description and appended claims, and upon reference to the accompanying drawings.
The characteristics and advantages of the present invention are summarized as follows:
(a) The present invention provides a method for producing a high-quality fermented soybean meal having ameliorated or improved properties by performing solid fermentation using Bacillus strain, in particular, Bacillus subtilis TP6 strain, which is effectively capable of removing anti-nutritional factors from soybean meal.
(b) In particular, the present invention uses the Bacillus subtilis TP6 strain, which is capable of producing final products with the quality equivalent to or better than those produced by the method known in art while reducing the fermentation time up to 12 to 24 hrs. The fermented soybean meal produced by the present invention is a high-quality protein source, in which powerful Bacillus-producing protease inactivates TI by hydrolysis and improves digestion and absorption rates by low-molecularization of soybean proteins into peptides.
(c) The reduction in fermentation time ruduces the fermentor batch cycle to remarkably increase the annual production, resulting in a reduction in overall costs. Consequently, consumers can be provided with high-quality fermented soybean meals at a low cost.
(d) In the method of the present invention, Bacillus strain, which is unusual in solid fermentation, is used as a fermentation strain. Thereby, the fermented soybean meal produced by the present invention becomes a high-quality protein source, in which powerful Bacillus-producing protease inactivates TI by hydrolysis and improves digestion and absorption rates by low-molecularization of soybean proteins into peptides.
(e) Moreover, the fermented soybean meal produced by the method of the present invention produces high-quality cellular proteins by active growth of a Bacillus strain, thereby increasing the absolute amount of protein and consuming carbohydrates in the soybean meal as an energy source required for the growth, leading to an increase in the relative protein content. Therefore, its value as a protein feed is more improved.
(f) Even during distribution, the fermented soybean meal produced by the present invention contains Bacillus strain having strong viability, and thus it has an advantage of helping intestinal regulation of the animals fed. Considering that the use of probiotics is positively recommended as an alternative to antibiotic-free feeds, the fermented soybean meal of the present invention will be more valuable in the future.
(g) Moreover, in the present invention, Bacillus strain with a capability of producing polyglutamic acid is used to perform the fermentation of soybean meal, thereby obtaining the additional effect of reducing the body fat of livestock.
When livestock are fed with high-energy feed in order to promote their growth, body fat accumulation is increased. Recently, as consumers’ concerns on health increase, they wish to ingest reduced fat. Therefore, needed are breeding methods or feed development capable of reducing the body fat of livestock. Polyglutamic acid is a sticky mucilaginous substance found in Korean chungkukjang or Japanese natto, and is a functional ingredient having various functions. In particular, its effects on animal fat reduction have been recently revealed.
(h) As described above, the fermented soybean meal of the present invention has high quality and various functions, thereby being widely applied to feeds as a high-protein source. In addition, the fermentation time can be reduced up to 12 to 24 hrs, compared to the known method.
FIG. 1 is a schematic diagram showing a flow chart for producing the fermented soybean meal by using soybean meal as a raw material in accordance with one embodiment of the present invention;
FIG. 2 is a graph showing changes in the activity of trypsin inhibitor, when each single culture of L.plantarum P23 and B.subtilis TP6 and co-culture of L.plantarum P23 and B.subtilis TP6 is performed;
FIG. 3 shows changes in the protein pattern, when each single culture of L.plantarum P23 and B.subtilis TP6 and co-culture of L.plantarum P23 and B.subtilis TP6 is performed;
FIG. 4 shows chromatography for sugar analysis of the heat-treated soybean meal inoculated no bacteria;
FIG. 5 shows chromatography for sugar analysis of the soybean meal fermented by B. subtilis TP6;
FIG. 6 shows chromatography for sugar analysis of the soybean meal fermented by B. subtilis TP6 and L.plantarum P23; and
FIG. 7 shows the effects of B. subtilis TP6 expression on protein degradation and allergen removal (marker; (1): raw soybean meal; (2): 37℃ 45%; (3): 37℃ 50%; (4): 45℃ 45%; (5): 45℃ 50%).
In accordance with one aspect to achieve the above objects, the present invention relates to a method for producing a fermented soybean meal, comprising the steps of: (a) adding water to a soybean meal to perform heat-treatment; (b) cooling the heat-treated soybean meal, and then inoculating a Bacillus strain thereinto; and (c) acquiring a fermented soybean meal by solid fermentation of the Bacillus-inoculated soybean meal.
The present inventors have made an effort to construct production systems for soybean fermentation for the purpose of the amelioration or improvement of the quality of soybean meals as well as for reduction of the fermentation time. They found that a Bacillus strain is used as a fermentation strain to perform solid fermentation of soybean meals, thereby producing a fermented soybean meal devoid of the above mentioned problems.
The method for producing a fermented soybean meal of the present invention will be described in detail with respect to the following steps:
(a) Addition of water to soybean meal and heat-treatment
To maintain the quality of fermented soybean meal as a final product, it is preferable that the soybean meals used in the method of the present invention are supplied from the same source every time. However, difference in the quality of the final fermented soybean meal is attributed to difference in the initial nutritional composition of the raw material, which merely affects final nutritional composition of the fermented soybean meal, but does not influence fermentation itself. In particular, difference in protein content of soybean meal depending on its type influences protein content of the final product. Preferably, as the soybean meal used in the method of the present invention shows a higher protein content and lower trypsin inhibitor content, the quality of fermented soybean meal is improved.
According to the method of the present invention, a process of adding water is required prior to heat-treatment of the raw soybean meal. That is, before solid fermentation, a proper amount of water is directly sprayed onto the raw soybean meal, and is mixed to control the moisture content, followed by heat-treatment for a predetermined time. In this regard, the heat-treatment is performed for the purpose of killing a variety of germs in the raw soybean meal, destruction of the soybean cell wall, and denaturation of proteins, thereby providing chemical compositions for active growth of the desired microorganism.
In accordance with the preferred embodiment of the present invention, the water-added soybean meal in the step (a) has a water content of 30 to 80% (v/w), more preferably 30 to 70% (v/w), and most preferably 40 to 60% (v/w). The water content ranging from 30 to 80% (v/w) is preferred so that delay of fermentation due to low moisture can be prevented, the cost of transfer and drying process after fermentation can be reduced, and heating efficiency can be improved.
Subsequently, the water-added soybean meal is subjected to heat-treatment. The heat-treatment process can be performed by various methods known in the art, but steam or superheated steam is preferably.
In the method of the present invention, the heat-treatment of step (a) is performed using steam at a temperature ranging from 70 to 130℃ for 10 to 60 min or superheated steam at a temperature ranging from 200 to 300℃ for a short time of several seconds to minutes, more preferably steam at a temperature ranging from 70 to 130℃ for 10 to 30 min, and most preferably steam at a temperature ranging from 80 to 121.1℃ for 10 to 30 min.
In the performance of heat-treatment according to the method of the present invention, if the temperature of heat-treatment is low or the treatment time is short, there are problems that the sterilization effect on various germs is not sufficient or the subsequent fermentation process does not proceed smoothly. If the temperature of heat-treatment is high or the treatment time is long, protein denaturation occurs in the soybean meal to reduce the rate of digestion, resulting in deterioration of the quality of the final product. Therefore, it is preferable that the temperature and time of heat-treatment is adopted within acceptable ranges to avoid these problems.
Through the heat-treatment in the method of the present invention, the contaminants present in the soybean meal are almost completely removed, a chemical environment suitable for the subsequent solid fermentation is formed, and TI inhibiting the rate of digestion is slightly reduced.
In the preferred embodiment of the present invention, a step of selecting and pre-culturing a bacillus strain with excellent protein degradation capacity is further included before or after step (a).
Selection and pre-culture of fermentation strain
In the solid fermentation, a fungus such as Aspergillus oryzae, which grows well under low moisture conditions, is generally used because of low moisture content of a solid substrate. However, when fungus is used to perform the fermentation of soybean meal, the produced, fermented soybean meal is in a state of not being suitable for feed use. Production of soybean meals by co-culture of fungus with Bacillus is described in Korean Patent No. 10-0645284. However, there are disadvantages in that it is not practically easy to culture both of them at the same time (fungus grows well at low moisture, but bacteria need more moisture to grow), and post-culture by post-addition of any one of them should be performed to produce products. In this regard, the entire process becomes complicated (seed culture/main culture of each strain should be performed, and inoculation time and conditions differ depending on each strain), and water content should be also changed depending on each condition. Consequently, complication of the process causes a substantial increase in cost.
Therefore, to simplify the process, it is preferable to select a strain capable of producing final products with the quality equivalent to or better than those produced by the known complicated co-culture for a long fermentation time, and minimizing fermentation time to reduce overall costs.
Compared to fungus, bacteria do not grow well under low moisture conditions. Thus, to achieve the objects of the present invention by inoculation of Bacillus into soybean meal, it is essential to use a strain which grows well in soybean meal with a low content of water and produces a large amount of the desired enzyme during growth.
In industrial-scale solid fermentation equipment, it is difficult to supply oxygen being sufficient to culture microorganisms. Therefore, it is preferable to use a Bacillus strain as a fermentation strain of soybean meal, which grows well under low oxygen conditions and has an excellent protein hydrolysis activity. In addition, to further improve the value of the fermented soybean meal of the present invention, it is more preferable to isolate strains which function as spore-forming probiotics. Recently, the addition of antibiotics to feed is prohibited worldwide. As an alternative to antibiotic-free feeds, it is positively recommended to use probiotics having a variety of physiological functions such as health promotion by improvement of intestinal flora, inhibition of proliferation of harmful bacteria, and improvement in immunity, disease resistance or feed efficiency. Traditionally, lactobacillus is used as a probiotic, but spore-forming bacteria are newly appreciated as probiotics, thereby increasing the interest in spore-forming bacteria as probiotics added to non-antibiotic feeds. A representative strain of spore-forming probiotics is a Bacillus strain.
There are many efforts to isolate Bacillus strain satisfying the objects of the present invention from Asian traditional solid-fermented soybean foods such as soybean paste, chungkukjang or Tempeh, leading to isolation of a large number of Bacillus strains.
In accordance with the preferred embodiment of the present invention, the Bacillus strain selected to be used in the present invention is Bacillus subtilis, Bacillus cereus, Bacillus megaterium or Bacillus clausii.
Bacillus subtilis is more preferred, and Bacillus subtilis TP6 strain (KFCC 11343P, Korean Patent No. 10-0753002) is most preferred.
In particular, the Bacillus subtilis TP6 (hereinbelow, referred to as “TP6 strain”) strain grew well in soybean meal with a low water content of 40% or more under aerobic and anaerobic conditions, showed excellent hydrolysis activity of soybean proteins, and had the unique property of producing polyglutamic acid during the late fermentation period. In addition, when the Bacillus subtilis TP6 strain was used, the fermentation time was remarkably reduced, as compared to the known method.
The strain of the present invention may be further selected as a strain to be used for inoculation of Lactobacillus, or a mixed strain of Lactobacillus and Bacillus strain.
Animal Lactobacillus, isolated from milk or cheese, grows using lactose, but plant Lactobacillus isolated from vegetables utilizes various sugars such as glucose, fructose, fructose or maltose and has high environmental adaptation to survive under harsh conditions such as low pH. Thus, plant Lactobacillus shows higher survival rate in the stomach than animal Lactobacillus, and a number of strains reach the intestine.
Accordingly, the term “plant Lactobacillus”, as used herein, means Lactobacillus derived from plant sources.
Korean kimchi is said to be a representative source of plant Lactobacillus. When making kimchi, spices such as hot red pepper and garlic are added. At this time, since kimchi Lactobacillus is exposed to more harsh conditions than other Lactobacillus, it survives to show excellent ability of degradation and intake of nutrients and productivity of various physiologically active materials.
Therefore, when plant Lactobacillus isolated from kimchi is used to perform fermentation of soybean meal, it is expected that the Lactobacillus actively grows without addition of subsidiary materials and produces antimicrobial materials, functional peptides, or organic acids during fermentation.
In the present invention, the present inventors have made many efforts to isolate Lactobacillus suitable for soybean meal fermentation from kimchi. In accordance with the preferred embodiment of the present invention, the selected Lactobacillus is preferably Lactobacillus sakei, Lactobacillus brevis, or Lactobacillus plantarum, more preferably Lactobacillus plantarum, and most preferably Lactobacillus plantarum P23 (KCCM 80048) which is isolated from kimchi by the present inventors.
In the present invention, the Lactobacillus plantarum P23 strain (hereinafter, referred to as “P23 strain”) was found to remarkably reduce non-starch polysaccharides, which are anti-nutritional factors present in soybean meal, and show excellent capability of producing organic acids and acid-resistance.
Media for seed culture of Lactobacillus isolated from kimchi may be any media known in the art, preferably MRS broth (deMan Rogosa Sharpe broth), APT (All Purpose with Tween) or BHI (Brain Heart Infusion), and more preferably MRS broth. However, since MRS broth is too expensive for industrial use, the present inventors constructed an inexpensive media for seed culture of Lactobacillus, and CSL (Corn Steep Liquor) was used as carbon and nitrogen sources for seed culture of Lactobacillus.
In addition, for seed culture of Bacillus, modified medium (5 g/L soytone, 5 g/L beef extract and 20 g/L xylose, pH 7.0) is preferably used to culture at 37℃ for 12 to 24 hrs. This medium may also increase cost price upon large-scale production for industrial use, and thus it is preferable to culture Bacillus in an industrial medium based on CSL contrived by the present inventors.
For seed culture of Lactobacillus, Lactobacillus isolated from kimchi is inoculated into the industrial medium contrived by the present inventors, and cultured at 30℃ for 8 to 24 hrs, thereby obtaining activity of the desired strain.
Each of two strains is cultured in the industrial medium for seed culture, until the final number of living cells in the seed culture medium becomes (1-5)x109 cfu/mL.
(b) Cooling of the heat-treated soybean meal and inoculation of microorganism
According to the method of the present invention, in step (b), heat-treated soybean meal is cooled to a temperature at which solid fermentation can be performed, and then Bacillus is inoculated thereto.
In the present invention, the cooling of soybean meal is ordinarily performed after heat-treatment, in which the cooling process can be readily performed through a transfer process using a cooling conveyor in order to prevent overheating and cool uniformly by increasing cooling rate (FIG. 1).
In accordance with the preferred embodiment of the present invention, the cooled soybean meal of step (b) in the method of the present invention has a temperature of 30 to 50℃, more preferably 35 to 45℃, and most preferably 37℃.
After cooling the heat-treated soybean meal, in accordance with the preferred embodiment of the present invention, it is preferable that the pre-culture media of Bacillus strain is uniformly inoculated into the prepared soybean meal medium as it is or by dilution with sterilized water.
The number of microorganisms inoculated into the heat-treated soybean meal is an important factor that influences the solid fermentation of soybean meal. The number of microorganisms immediately after inoculation into the heat-treated soybean meal is preferably 105 to 109 CFU/g. If the inoculation amount is less than 105 CFU/g, a small amount of seed fermentation broth is needed, but much time is required for the fermentation of soybean meal to increase incubation time and possibility of contamination. If the inoculation amount is more than 109 CFU/g, the fermentation time can be considerably reduced, but production of seed microorganism used for inoculation is problematic. In particular, since fermentation performance is greatly influenced by growth characteristics of microorganism and type of fermentor, it is preferable that the inoculation amount is properly determined depending on characteristics of the microorganism in the production step.
(c) Acquisition of fermented soybean meal by solid fermentation of the Bacillus-inoculated soybean meal
One of the characteristics of the present invention is to produce a fermented soybean meal by inoculating Bacillus into the soybean meal, in which unlike fungus or yeast, Bacillus is not usually used for solid fermentation (culture).
As used herein, the term “solid fermentation (culture)” means that a defatted soybean meal obtained by removal of oil from soybean is used to culture microorganisms, and is distinguished from “liquid culture or liquid fermentation” using soybean meal extracts.
Since some microorganisms grow well during solid fermentation and produce a large amount of extracellular enzymes and other metabolites, solid fermentation has been used for the production of traditional foods or alcoholic beverages in Asia. The soybean meal is a solid substrate in a flake or particle state, and thus solid fermentation is an inexpensive and efficient method capable of improving the value of soybean meal as a feed using microorganisms.
To employ the solid fermentation in the method of the present invention, a strain selected from Bacillus strains is used, and the strain type and inoculation method can be controlled depending on the properties of the final product of fermented soybean meal. Preferably, the Bacillus subtilis TP6 strain is used to reduce the fermentation time of the present invention.
In the case of producing the fermented soybean meal by inoculation of only Bacillus, the Bacillus strain actively grows to improve the quality of the fermented soybean meal, and at this time, the Bacillus strain first produces protease having powerful activity. The Bacillus-producing protease is characterized in that it inactivates TI by hydrolysis, in which TI is a protein inhibiting the digestion of soybean meal, and improves digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins.
In addition, while the Bacillus strain actively grows by utilizing carbohydrates among the components of soybean meal, the carbohydrates are converted into proteins constituting the cell. Thus, the relative protein content of the soybean meal is increased during the fermentation, which is the most important factor among the evaluation items of the feed quality.
During a drying process, the Bacillus strains grown in the soybean meal form spores to survive at a high ratio, and thus they exist as viable cells even after drying the fermented soybean meal. Due to the spore formation of Bacillus, it is expected to show the effect of improving intestinal function as the above described spore-forming probiotics in livestock.
In the method of the present invention, Lactobacillus may be inoculated alone or in a mixture with Bacillus. When plant Lactobacillus isolated from kimchi is only inoculated, it grows well by utilizing the soybean meal as a raw material to produce a large amount of organic acids. In addition, plant Lactobacillus has an alpha galactosidase activity so as to exhibit a property of degrading galactooligosaccharides contained in the soybean meal, and hydrolyzes various polysaccharide anti-nutritional factors in the soybean meal so as to prevent enteritis and improve the rate of nutrient absorption.
To produce the fermented soybean meal having all advantages of the two bacteria, they may be inoculated simultaneously or sequentially at time intervals. In addition, since growth conditions of two bacteria differ from each other, it is preferable that they are inoculated at a different ratio or sequentially, in terms of the quality of the final product. However, it does not mean that simultaneous inoculation or inoculation at the same ratio fail to achieve the desired effects.
In accordance with the preferred embodiment of the present invention, the soybean meal that is uniformly inoculated with the desired microorganism is fermented in a packed-bed fermentor in step (c). The packed-bed fermentor is divided into batch, closed, and continuous stirred tank reactor. The method of the present invention is not limited to any one of them, and it can be selected depending on the production scale.
In the present invention, the soybean meal is applied to the packed-bed fermentor in a thickness of 5 to 50 cm, and fermented at a temperature of 20 to 50℃ for 12 to 72 hrs.
(d) Drying and pulverization of the acquired fermented soybean meal
In accordance with the preferred embodiment of the present invention, the method of the present invention further includes the step of (d) drying and pulverizing the fermented soybean meal at low temperature and moisture after step (c).
Water contained in the soybean meal is partially evaporated during the fermentation process, but the residual water content is considerably high at 20 to 50% (v/w) immediately after fermentation. However, the preferred, final water content of the fermented soybean meal product is 10 to 12% (v/w), and thus a drying process is required.
When the soybean meal is fermented using fungus such as Aspergillus oryzae, it becomes hard and forms conglomerates by mycelium production, and it is very difficult to perform the drying and pulverization processes because of spores. Meanwhile, when the fermentation is performed using bacteria in the present invention, the fermented soybean meal is in good condition, but conglomerates are slightly formed. Therefore, after the drying process, the process of pulverizing the fermented soybean meal is required to form a uniform particle size.
The drying and pulverization processes may be performed by various methods known in the art. However, when the drying process is excessively performed at a high temperature, a number of live bacteria in the fermented soybean meal may be killed, and thus it should be performed with caution. Preferably, the drying process is performed at a low temperature without killing living bacteria. Most preferably, the drying process is performed with hot air at low temperature and humidity. The fermented soybean meal may be pulverized to various sizes depending on the purpose of use, and preferably, by means of a hammer mill.
When the soybean meal is fermented according to the above described method of the present invention, various anti-nutritional factors including TI in the soybean meal are reduced, the rate of digestion and absorption is improved by hydrolysis of proteins, and the protein content is also increased, thereby improving the absolute value as feed. Consequently, a high-quality fermented soybean meal can be obtained as an alternative to animal proteins.
Even during distribution, the fermented soybean meal produced by the method of the present invention contains Bacillus strain having strong viability, and thus it has an advantage of helping intestinal regulation of the animals fed.
In another aspect, the present invention relates to a fermented soybean meal produced by the method of the present invention.
Since the fermented soybean meal of the present invention is produced by the above mentioned method of the present invention, the common description will be omitted herein to avoid redundancies.
In the fermented soybean meal produced by the inoculation of Bacillus subtilis TP6 strain, the Bacillus strain actively grows to improve the quality of fermented soybean meal, and at this time, the Bacillus strain first produces protease having powerful activity. The Bacillus-producing protease is characterized in that it inactivates TI by hydrolysis, in which TI is a protein inhibiting the digestion of soybean meal, and improves digestion and absorption rates by low-molecularization due to hydrolysis of soybean proteins. Further, while the Bacillus strain actively grows by utilizing carbohydrates among the components of soybean meal, the carbohydrates are converted into proteins constituting the cell. Thus, a relative protein content of the soybean meal is increased during the fermentation, which is the most important factor among the evaluation items of the feed quality. Furthermore, during a drying process, the Bacillus strains grown in the soybean meal form spores to survive at a high ratio, and thus they exist as viable cells even after drying the fermented soybean meal. Due to the spore formation of Bacillus, it is expected to show an effect of improving intestinal function as the above described spore-forming probiotics in livestock.
In accordance with one preferred embodiment of the present invention, the fermented soybean meal of the present invention contains vegetative cells or spores of microorganism selected from Bacillus strains, preferably, Bacillus subtilis TP6 strain.
In accordance with another preferred embodiment of the present invention, the fermented soybean meal of the present invention contains poly-ν-glutamic acid to reduce body fat accumulation in livestock.
In accordance with still another preferred embodiment of the present invention, the Bacillus strain includes Bacillus subtilis TP6 strain.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these Examples are for illustrative purposes only, and the invention is not intended to be limited in scope thereby.
Example
Throughout this application, “%” used to indicate that a concentration of a specific material is (weight/weight)% for solid/solid, (weight/volume)% for solid/liquid, (volume/volume)% for liquid/liquid, unless otherwise specified.
Example 1: Growth of Bacillus and Lactobacillus depending on water contents of soybean meal
To determine the water content of soybean meal suitable for culture of bacteria, water was added to the soybean meal at the water content of 30%, 40%, 50% and 60%, and 50 g of the water-added soybean meal was put in a beaker (250 mL). Then, the top portion of the beaker was covered with aluminium foil, and fastened with a rubber band. Heat-treatment was performed at 80℃ for 30 min, followed by cooling.
In accordance with one embodiment of the present invention, the soybean meal fermentation was performed using the preferred Bacillus subtilis TP6 (KFCC 11343P) (hereinafter, referred to as “TP6” strain) and Lactobacillus plantrum P23 (KCCM 80048) (hereinafter, referred to as “P23” strain). A medium containing xylose 2%, soytone 0.5% and beef extract 0.5% was used for seed culture of the TP6 strain, and the P23 strain was pre-cultured using an MRSbroth (Difco) at a temperature of 37℃ for 24 hrs.
When the heat-treated soybean meal was cooled to approximately 30℃, 5% of each strain pre-cultured in the seed culture medium was inoculated into the heat-treated soybean meals having different water contents in a clean bench. To prevent contamination and water evaporation, the beaker was sealed tightly with aluminium foil, and fermented in an incubator at 37℃ for 72 hrs. Subsequently, the number of bacteria in each fermented soybean meal and pH value were determined. The results are summarized in the following Table 1.
Table 1
Figure PCTKR2010005683-appb-T000001
As shown in Table 1, considering that the number of bacteria was approximately 1x107 CFU/g early after inoculation, it can be seen that P23 and TP6 strains hardly grow at a 30% water content of soybean meal. When the water content of soybean meal were 40% and 50%, the strains grew to the levels of (4.5-7.0)x108 CFU/g and (1.3-2.5)x109 CFU/g, respectively.
As the water content increased, the bacterial growth became more active. At 60% water content, the final number of P23 and TP6 strains increased to 3.2 x 109 CFU/g after fermentation, and the pH value of the P23 strain was remarkably reduced by production of organic acids after fermentation.
In accordance with the result of the present experiment, the initial water content of soybean meal should be over 40% for industrial-scale production of bacteria.
Example 2: Heat-treatment conditions of soybean meal
In the fermentation of soybean meal, heat-treatment of the water-added soybean meal is important in terms of preventing contamination and making the soybean meal to have a chemical environment suitable for fermentation. The present inventors investigated heat-treatment conditions of soybean meal. To establish minimum heat-treatment conditions in which contamination can be prevented and the inoculated strain can grow actively during the fermentation period, the water content of soybean meal was adjusted to 60%, and then 50 g thereof was put into a 250 mL beaker and the top portion of the beaker is sealed with aluminium foil, followed by heat treatment at 60℃, 80℃, 105℃ and 121.1℃ autoclaves for 10-30 min, respectively.
In the same manner as in Example 1, 5% of each seed culture medium of TP6 and P23 strains was inoculated into each soybean meal that was heat-treated under different heat-treatment conditions, and incubated at 37℃ for 72 hrs. The degree of contamination during fermentation was assessed and the number of live bacteria and pH in the fermented soybean meal were measured after cultivation. The heat-treatment conditions affecting the soybean meal fermentation are shown in the following Table 2.
Table 2
Figure PCTKR2010005683-appb-T000002
As shown in Table 2, when sterilization was performed at 60℃ for 30 min, contamination occurred after 24 hr fermentation, and thus the desired fermentation could not be performed. However, when heat-treatment was performed at 80℃ over 10 min, contamination did not occur and the number of bacteria was 1x109 CFU/g or more, even though the fermentation was performed for 72 hr.
Example 3: Changes in quality and property of fermented soybean meal upon single culture of Lactobacillus and Bacillus
70% and 100% water relative to weight of the raw soybean meal were uniformly sprayed on the soybean meal, and heat-treatment was performed at 0℃ for 30 min. In the same manner as in Example 1, 5% of each or both of the pre-cultured TP6 and P23 strains were inoculated into the heat-treated soybean meals, and fermentation was performed. The content of trypsin inhibitor (TI) and protein degradation during fermentation were analyzed by SDS-PAGE at each culture time, as shown in FIGs. 2 and 3.
As shown in FIG. 2, when the TP6 strain was cultured alone, TI activity was remarkably reduced at an added water of 70% after 24 hrs, and thereafter, it was gently reduced, and completely inactivated after 72 hrs. At an added water of 100%, the inactivation speed increased more rapidly, and TI was completely inactivated 24 hr after fermentation. Thereafter, no changes were observed. Meanwhile, when the P23 strain was cultured alone, TI remained even after 72 hrs. Upon the mixed culture of TP6 and P23 strains, the results were almost similar to those of a single culture of TP6 strain.
As shown in FIG. 3, changes in protein patterns were examined during fermentation. When TP6 strain was cultured alone, high-molecular peptides were hydrolyzed 48 hr after fermentation, and their bands almost disappeared. However, when P23 strain was cultured alone, there were no changes in the protein pattern, compared to raw matter. Meanwhile, the mixed culture of TP6 and P23 strains showed almost similar results to the single culture of TP6 strain.
The polysaccharide analysis results of the heat-treated soybean meal not inoculated, the soybean meal fermented with TP6 strain, and the soybean meal fermented with TP6 and P23 strains are shown in FIGs. 4, 5, and 6, respectively. Between FIG.4 and FIG. 5, there is little difference in sugar patterns by chromatography, indicating that the TP6 strain shows high enzymatic activity including protease, but hardly hydrolyzes soybean polysaccharides. However, comparing FIGs. 4 and 5 with FIG. 6 which shows the results of mixed fermentation of lactobacillus P23 isolated from Kimchi and the TP6 strain, it was found that polysaccharides, which are the anti-nutritional factors in soybean meal, were almost all hydrolyzed by the P23 strain.
Example 4: Mixed culture of lactobacillus and Bacillus in soybean meal
The water content of soybean meal was adjusted to 50%, and heat-treatment was performed at 110℃ for 15 min. The seed cultures of TP6 and P23 strains were inoculated into the treated soybean meal at different inoculation ratios or for different inoculation times, and cultured at 37℃ for 48 hrs. Specifically, the “mixed culture 1” means that soybean meal was initially inoculated with 5% of TP6 strain and cultured for 20 hrs, and then 5% of P23 strain cultured in seed culture medium for 20 hrs was inoculated thereto, followed by additional fermentation for 28 hrs. The “ mixed culture 2 and 3” means that TP6 and P23 strains were initially inoculated at the different ratios of 1:0.5 and 1:1, followed by fermentation for 48 hrs.
Table 3
Figure PCTKR2010005683-appb-T000003
As shown in Table 3, there were no differences in the final number of bacteria and TI value between single culture of TP6 strain, additional inoculation of P23 strain, and mixed culture at the different ratios, suggesting that fermented soybean meal desired in the present invention can be obtained even though the fermentation methods of such single and mixed cultures are changed.
However, the quality of fermented soybean meal has slight differences in the final number of bacteria, acidity, and flavor. Therefore, it is preferable to properly control the inoculation time and ratio of microorganisms selected from the group consisting of plant lactobacillus, Bacillus strains, and the mixture thereof, depending on the final product to be desired.
Example 5: Quality characteristics of fermented soybean meal
The water content of soybean meal was adjusted to 30%, and heat-treatment was performed at 121℃ for 20 min. Sterilized water was uniformly sprayed to the sterilized soybean meal to adjust the water content to 60%. TP6 and P23 strains cultured in each seed culture medium for 24 hrs were inoculated into the soybean meal to be 10%(v/w) of the soybean meal, and then fermentation was performed at 37℃ for 48 hrs. After fermentation, the soybean meals were dried, and their physicochemical properties were examined, as shown in the following Table 4.
Table 4
Figure PCTKR2010005683-appb-T000004
As shown in Table 4, the fermented soybean meal fermented with only the TP6 strain of the present invention showed a highly increased protein content, as well as reduced content of the anti-nutritional factors including trypsin inhibitor, soybean oligosaccharides and polysaccharides, as compared to raw soybean meal, showing a highly improved value as feed. Moreover, the fermented soybean meal contained live bacteria of 2.0x109 CFU/g DM and polyglutamic acid, indicating that it has a much more improved value.
Example 6: Changes in TI content under different culture conditions
To assess changes in the content of trypsin inhibitor, which is one of anti-nutritional factors inhibiting digestion and absorption, the content of trypsin inhibitor in the fermented soybean meal was measured. The TI content was assessed by Korea Feed Ingredients Association in the AOAC method. The experimental groups are as follows: “soybean” group: common soybean, “soybean meal” group: residue produced by removing soybean oil from soybean, “24 hr fermentation” group: 100 g of soybean meal was adjusted to have 45% water content, heat-treated (90℃ for 15 min), and 5 ml of TP6 strain (2X109 cfu/ml) was inoculated into the heat-treated soybean meal, followed by fermentation at 37℃ and constant humidity for 24 hrs. Changes in TI (trypsin inhibitor) content after fermentation were shown in the following Table 5.
Table 5
TI(mg/g)
Soybean 5-20
Soybean meal 4-8
24 hr fermentation 0.32
24 hr after fermentation, the content of trypsin inhibitor was 0.32 mg/g reaching 50% level of 0.6 mg/g, which is a TI content measured after 37 hr fermentation, described in Korean Patent No. 10-645284. This result indicates that the effect of reducing anti-nutritional factors can be obtained at a level equivalent to or better than prior experimental results, even after 24 hr fermentation.
Example 7: Changes in crude protein content depending on fermentation time upon single culture of TP6 strain
(Fermentation was performed at 37℃)
100 g of soybean meal was adjusted to have 45% water content, heat-treated (90℃ for 15 min), and 5 ml of TP6 strain (2X109 cfu/ml) was inoculated into the heat-treated soybean meal, followed by fermentation at 37℃ and constant humidity for 24 hrs. Changes in protein content during fermentation were shown in the following Table 6.
Table 6
Crude protein % (correction to 10% moisture)
Raw soybean meal 49.11261
Heat-treated 49.73485
0 hr 49.83154
18 hrs 56.55319
21 hrs 56.23199
24 hrs 57.85988
After 24 hr fermentation, the protein content was obtained at a level equivalent to those of 36 hr fermentation of mixed strains (Korean Patent No. 10-0645284) and 48 hr fermentation of single strain (Korean Patent No. 10-0459240) while reducing the fermentation time up to 12 to 24 hrs, as compared to the prior art (the crude protein content is important in the soybean meal product as a protein source, because a total crude protein content is important in the composition). The reduction in fermentation time reduces the fermentor batch cycle, resulting in an increase in the annual batch number. Consequently, consumers can be provided with high-quality fermented soybean meals at a low cost.
Example 8: Changes in crude protein content depending on fermentation time upon single culture of TP6 strain
(Fermentation time: 24 hrs)
100 g of soybean meal was adjusted to have 45% water content, heat-treated (90℃ for 15 min), and 5 ml of TP6 strain (2X109 cfu/ml) was inoculated into the heat-treated soybean meal, followed by fermentation at 37℃ and constant humidity for 24 hrs. Changes in protein content during fermentation were shown in the following Table 7.
Table 7
Raffinose (%) Stachyose (%)
Raw soybean meal 1.35 4.71
Heat-treated 0.50 5.42
0 hr 0.25 1.11
18 hrs 0.03 0.75
21 hrs 0.01 0.04
24 hrs ND 0.03
When the selected TP6 strain was used to perform the fermentation for 24 hrs, the ratio of anti-nutritional factors, raffinose and stachyose were remarkably reduced, and the result was at a level equivalent to that of 48 hr fermentation (Korean Patent No. 10-049240).
Example 9: Effect of TP6 fermentation on protein degradation and allergen removal (β-conglycinin, glycinin)
Fermentation experiments were performed under different conditions of temperature and moisture for 24 hrs. The fermentation conditions are the same as in Examples 7 to 8, and water% was only changed during heat-treatment as follows: Lane 2; 37℃ 45%, Lane 3; 37℃ 50%, Lane 4; 45℃ 45%, Lane 5; 45℃ 50% in FIG. 7. As shown in Fig. 7, it was found that the allergens in the raw soybean meal, β-conglycinin and glycinin were excellently removed at the water content of 45 to 50% and temperature of 37℃, indicating that high-molecular proteins are degraded into low-molecular proteins, and converted into a form suitable for digestion and absorption.

Claims (12)

  1. A method for producing a fermented soybean meal, comprising the steps of:
    (a) adding water to a soybean meal to perform heat-treatment;
    (b) cooling the heat-treated soybean meal, and then inoculating a Bacillus strain thereinto; and
    (c) acquiring a fermented soybean meal by solid fermentation of the Bacillus-inoculated soybean meal.
  2. The method according to claim 1, wherein the water-added soybean meal of step (a) has a water content of 30 to 80% (v/w).
  3. The method according to claim 1, wherein the heat-treatment of soybean meal of step (a) is performed at a temperature of 70 to 130℃ for 10 to 30 min.
  4. The method according to claim 1, wherein the cooled soybean meal of step (b) is at a temperature of 30 to 50℃.
  5. The method according to claim 1, wherein the Bacillus strain of step (b) is selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Bacillus clausii.
  6. The method according to claim 5, wherein the Bacillus strain is Bacillus subtilis TP6 strain.
  7. The method according to claim 1, wherein the solid fermentation of step (c) is performed at a temperature of 20 to 50℃ for 12 to 72 hrs.
  8. The method according to claim 1, wherein the method further includes the step of (d) drying and pulverizing the fermented soybean meal after step (c).
  9. A fermented soybean meal produced by the method according to any one of claims 1 to 8.
  10. The fermented soybean meal according to claim 9, wherein the fermented soybean meal contains vegetative cells or spores of Bacillus strain.
  11. The fermented soybean meal according to claim 10, wherein the fermented soybean meal contains poly-ν-glutamic acid.
  12. The fermented soybean meal according to claim 10, wherein the Bacillus strain is Bacillus subtilis TP6 strain.
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