JP5728185B2 - Method for producing fermented soybean meal using Bacillus subtilis - Google Patents

Description

  The present invention relates to a cheap and efficient method for producing fermented soybean meal that can improve or improve the quality of defatted soybean meal as a vegetable protein source, and a fermented soybean meal produced by this method.

  By determining diseases such as mad cow disease that cause fatal diseases in humans as a result of animal protein components added to the feed, animal proteins added to the feed worldwide are planted. The movement to replace with sex protein is spreading rapidly.

  The defatted soybean meal (hereinafter referred to as “soybean meal”) is the largest plant protein ingredient used in the feed market as a substitute for animal protein such as fish meal, meat and bone meal or plasma. is there. The soybean meal used as a vegetable protein feed source in Korea accounts for 60% of the total feed, and the amount reaches 2 million tons per year (2004, Korea Simple Feed Association).

  Typical general analytical values for soybean meal are 9.5% moisture, 49.4% crude protein, 22.1% invert sugar and 27.2% water-soluble nitrogen. In general, plant proteins have a relatively low protein content compared to animal proteins, and the composition of essential amino acids required for livestock is not as good as that of animal proteins, and some vitamins, minerals and UGF (Unknown Growth factor) is not high.

  In addition, soybean meal contains a variety of anti-nutritional factors (ANF), and thus has a problem that digestibility is inhibited when used as feed. Among them, a representative one is a trypsin inhibitor (hereinafter referred to as “TI”). In terrestrial animals, diet TI interferes with the proper enzymatic function of trypsin and chymotrypsin, resulting in a decrease in solubility of about 6% of the total protein present in soybean. In particular, since the effect of such anti-nutritive factors is significant in the case of young animals, the amount of soybean meal added to the feed for young animals is limited.

  The TI activity of raw soybean meal is about 39 mg / g. However, since defatted soybean meal destroys anti-nutritive factors by heat treatment in the production process, the TI activity of defatted soybean meal sold as a product is generally 4 mg / g. g or less. Therefore, while raising fattening pigs or mother pigs are less damaging, piglets are different from growing fattening pigs in that they limit the use of soybean meal as an anti-nutritional factor in soybean meal. Part must be used. For these reasons, processed soy products such as concentrated soy protein and refined soy protein are used as feeds, and specifications similar to those obtained when feeding mainly dairy feeds are obtained.

  Currently produced soy protein processed products such as concentrated soy protein (SPC), isolated soy protein (SPI) or hydrolyzed soy protein are produced by physicochemical treatment or enzymatic treatment and are mainly produced for food processing. It is expensive to use as feed.

  Therefore, in order to use soybean meal as a high-quality high-protein feed, it is required to develop a new processing method that can improve the quality of soybean protein and that is inexpensive, efficient, and capable of mass processing. Moreover, grain production has recently declined due to global climate changes such as global warming, and international prices for corn or soybean meal, the main raw material for feed, have risen, causing a major blow to the feed industry and livestock farmers. Because of the facts they are giving, the need is growing.

  In addition to the TI described above, soy oligosaccharides are known to induce diarrhea and abdominal pain, and soy polysaccharides inhibit the absorption of nutrient components. When soybean meal is mixed with feed, it often causes enteritis, but the cause has not been elucidated. It is estimated that it originates in the alcohol-soluble polysaccharide in soybean meal (refer nonpatent literature 1). In addition, soybean polysaccharides have been reported to inhibit the absorption of nutrient components in fish such as salmon and meat chicken chicks (see Non-Patent Document 2).

  Therefore, recently, as a method for producing a high-quality feed by removing such antinutritive factors, a product obtained by removing phytate using enzyme-treated soybean protein phytase (HP300 (Denmark)-www.hamletprotein. com) or fermented soybean protein (see Patent Document 1, Patent Document 2, Patent Document 3, and Non-Patent Document 3).

  Among these, fermented soy protein can remove many anti-nutritive factors during the fermentation process, and further decomposes protein or carbohydrate into a form that is easy to digest, so it is the highest digestive and high-quality feed. It can be said that it is a protein material.

  However, in the case of such a fermentation process, a long fermentation time is required in order to obtain an antinutritive factor removal effect of an appropriate level or more (in the case of Patent Document 1, 36 hours, in the case of Patent Document 2). 48 hours). Such a long fermentation time results in an overall cost increase by lowering the rotation rate of the koji making machine. As described above, the conventional technique has a problem in that a long fermentation time is required during the production process of the fermented soybean meal, resulting in an increase in overall cost.

  To date, there has been no report on research results of producing fermented soybean meal while dramatically reducing the fermentation time using Bacillus subtilis TP6, and in particular, Bacillus subtilis during solid fermentation of soybean meal. There have been no reports on proteases and functional polysaccharides produced by the plant and products that actively utilize the effectiveness of the microorganisms as probiotics.

  Therefore, as a result of diligent efforts to reduce the cost of the product by constructing a production system for fermented soybean meal for improving and improving soybean meal, which is a protein feed source, and reducing the fermentation time efficiently, By adopting Bacillus subtilis TP6 strain as a fermenting strain with characteristics suitable for solid fermentation of soybean meal, solid fermentation of soybean meal increases protein content, protein quality, and low molecular weight by hydrolysis of soybean protein , Inactivation of TI, increase in digestion absorption rate and feed efficiency due to decreased content of anti-nutritional factors such as non-digestible polysaccharides, as well as long fermentation time which is a problem of existing fermented soybean meal It was confirmed that a high-quality fermented soybean meal with an epoch-making improvement could be produced, and the present invention was completed.

  Numerous patent references are referenced throughout the specification and their citations are displayed. The disclosures of the cited patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the level of the technical field to which the present invention belongs and the contents of the present invention.

Korean Patent Registration No. 10-0645284 Specification Korean Patent Registration No. 10-0459240 Specification Korean Patent Registration No. 10-0925173 Specification

Krogdahl, A. et al. Feeding atlantic Salmo salar L. soybean products. Aquac. Nutr. 6, 77-84, 200 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. 79,331-3451999 Livestock Research for Rural Development 20 (9) 2008

  An object of the present invention is a solid fermentation method using a Bacillus subtilis strain, particularly a TP6 strain, which has excellent antinutritive factor removal ability and proteolytic ability and can reduce the fermentation time to the maximum when producing fermented soybean meal. It is in providing the manufacturing method of fermented soybean meal by this.

  Another object of the present invention is to provide a high-quality fermented soybean meal fermented using the Bacillus subtilis.

  Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the drawings.

  As one aspect for achieving the above object, the present invention includes (a) adding water to soybean meal and heat-treating; and (b) cooling the heat-treated soybean meal and inoculating Bacillus subtilis. And (c) providing a fermented soybean meal by solid-culturing the fungus inoculated in the soybean meal.

  The present inventor can improve and improve the quality of soybean meal by fermentation, and at the same time, as a result of diligent efforts to construct a production system for reducing fermentation time, Bacillus subtilis is widely adopted as a fermentation strain. It was confirmed that fermented soybean meal with improved problems described above can be produced by solid fermentation of soybean meal.

  The method of the present invention for producing fermented soybean meal is described in detail as follows.

(A) Addition of moisture to soybean meal and heat treatment The soybean meal used in the method of the present invention is continuously the same from the same region so that the final product fermented soybean meal can maintain the same quality. It is preferable to use this type of soybean meal supplied, but the difference of fermented soybean meal as a product due to the difference in quality of soybean meal as a raw material is due to the change in the ingredients of the product due to the initial content of nutritional components on the raw material. Yes, the fermentation itself is not a problem. In particular, the difference in protein content depending on the type of soybean meal indicates a difference in the protein content of the final product. Preferably, the soybean meal used in the method of the present invention has a higher protein content and a lower trypsin inhibitor content, so that the quality of the fermented soybean meal increases.

  According to the method of the present invention, a step of adding water is necessary before heat-treating soybean meal as a raw material. That is, the raw soybean meal is subjected to a heat treatment for a certain period of time after the water content is adjusted by directly spraying and mixing water in an appropriate amount before solid fermentation. At this time, the heat treatment is to provide a chemical composition capable of actively growing the target microorganisms by killing various bacteria in the raw soybean meal and simultaneously destroying the soybean cell wall and denaturing the protein. .

  According to a preferred embodiment of the present invention, the moisture-added soybean meal of step (a) has a water content of 30 to 80% (v / w), preferably 30 to 70% (v / w). More preferably, it is 40 to 60% (v / w). The water content in the range of 30-80% (v / w) prevents the slowing of the fermentation rate due to low water content, improves many of the costly problems in the transfer of soybean meal and the drying process after fermentation, and in terms of thermal efficiency. preferable.

  Next, the soybean meal to which moisture has been added is heat-treated. For the heat treatment step, various methods known in the art can be used, but preferably heat treatment is performed using steam or superheated steam.

  In the method of the present invention, the heat treatment in the step (a) is steaming with steam at a temperature of 70 to 130 ° C. for 10 to 60 minutes, or heat treatment with superheated steam at 200 to 300 ° C. for a few seconds to several minutes, Preferably, it is steamed with steam at a temperature of 70 to 130 ° C. for 10 to 30 minutes, and more preferably steamed with steam at 80 to 121.1 ° C. for 10 to 30 minutes.

  When the heat treatment is performed by the method of the present invention, if the heat treatment temperature is low or the treatment time is short, there is a problem that the sterilizing effect of various bacteria is small and the subsequent fermentation process is not smoothly performed, and the heat treatment temperature is high. If the treatment time is long or the digestion rate due to denaturation of protein in soybean meal is reduced, the quality of the final product is lowered, so the heat treatment temperature or treatment time is set so that such a problem does not occur. It is preferable to adopt.

  In the method of the present invention, through the heat treatment process, the contaminating bacteria in soybean meal are almost killed, and there is an effect that a chemical environment in which solid fermentation as a subsequent process is smoothly performed is created, and TI that inhibits digestibility There is also an effect of slightly reducing.

  According to a preferred embodiment of the present invention, the method further includes a step of selecting and pre-culturing Bacillus subtilis excellent in protein resolution before or after the step (a).

In the selection of fermentation strains and pre-culture solid fermentation, molds such as Aspergillus oryzae that grow well with low moisture are mainly used because the moisture content of the solid substrate is generally low. However, when fermenting soybean meal with mold, the properties of the fermented soybean meal produced are often unsuitable for use as feed. There is Patent Document 1 in which soybean meal is produced using co-culture of mold and Bacillus subtilis, but it is not easy to grow the two at the same time (mold grows well with low moisture, and bacteria grows with moisture. However, there is a weak point that the product must be made by post-culturing in the form of post-addition. In this case, the overall process becomes complicated (seed culture / main culture must be performed for each strain, and the inoculation time and conditions must be different), and the water content and the like that meet each condition change. It is common to have to give Such a point eventually leads to an increase in cost due to the complexity of the process.

  This simplifies the process by using a single strain and having a quality equivalent to or better than a product that has previously been fermented for a long time through complex co-culture, etc. It is preferable to select a strain that can reduce the total cost by reducing the maximum cost.

  In general, since bacteria do not grow well at lower moisture than mold, in order to achieve the object of the present invention by inoculating Bacillus subtilis into soybean meal, growth is vigorous even in soybean meal with insufficient moisture. It can be said that it is essential to use a strain that produces the target enzyme in large quantities.

  Moreover, since it is difficult to supply sufficient oxygen required for the growth of microorganisms in a large amount of solid-state fermentation apparatus used industrially, Bacillus subtilis that grows actively even under low oxygen conditions and has excellent proteolytic activity. Is preferred as a soybean meal fermenting strain. In addition, in order to further increase the future-oriented value of the fermented soybean meal of the present invention, it is more preferable to isolate a strain having a function as a spore-forming viable agent. Recently, when the addition of antibiotics to feed is banned worldwide, as an alternative to antibiotic-free feed, not only health promotion by improving gut microbiota, but also suppression of harmful bacteria growth, immunity improvement, disease The use of probiotics with various physiological functions such as increased resistance or increased feed efficiency is actively encouraged. Lactic acid bacteria have traditionally been the mainstream for probiotics, but recently, spore formation as probiotics added to antibiotic-free diets due to the newly recognized value of live spore-forming bacteria There is a growing interest in bacteria. A representative strain of a spore-forming viable agent is Bacillus subtilis.

  Much effort has been made to isolate Bacillus subtilis that satisfies the problems to be solved by the present invention from traditional Asian solid-phase soybean fermented foods such as Miso, Cheongukchan or Tempeh. As a result, we succeeded in isolating many excellent Bacillus subtilis.

  According to a preferred embodiment of the present invention, the Bacillus subtilis screened for use in the present invention is Bacillus subtilis, Bacillus cereus, Bacillus megaterium, or Bacillus megaterium. This includes Bacillus clausii.

  Bacillus subtilis is preferable, and Bacillus subtilis TP6 strain (KFCC11343P, Korean Patent No. 10-075302) is more preferable.

  In particular, Bacillus subtilis TP6 (hereinafter referred to as “TP6 strain”) is a low-moisture soybean meal having a moisture content of 40% or more and grows well not only in aerobic but also in non-aerobic conditions, and is effective in hydrolyzing soybean protein. In addition, it had a unique property of producing poly-γ-glutamic acid in the second half of the fermentation. Moreover, in the case of Bacillus subtilis TP6 strain, fermentation time was reduced epoch-making than the conventional method.

  Furthermore, the said strain of this invention can be selected as a strain used for inoculating lactic acid bacteria or a mixed strain of lactic acid bacteria and Bacillus subtilis.

  Animal lactic acid bacteria isolated from milk or cheese grow mainly using lactose, but plant lactic acid bacteria isolated from vegetables etc. can use various sugars such as glucose, fructose, sugar or maltose, Because it is highly adaptable to the environment, it can grow even in harsh environments such as low pH, so it has a higher survival rate in the stomach than animal lactic acid bacteria, and there are many bacteria that survive to reach the intestinal tract.

  Therefore, the term “plant lactic acid bacteria” in the present specification means lactic acid bacteria derived from plant raw materials.

  Korean kimchi is a representative treasure trove of plant lactic acid bacteria. When kimchi is pickled, spices such as chili powder or garlic are added, so the lactic acid bacteria in kimchi grow in a harsh environment than general lactic acid bacteria, and have the ability to decompose and ingest nutrients, as well as various physiologically active substances. It is known to be excellent in productivity.

  Therefore, when fermenting soybean meal using plant lactic acid bacteria isolated from kimchi instead of general lactic acid bacteria, it actively grows without the addition of special auxiliary materials, such as antimicrobial substances, functional peptides or organic acids during fermentation Can be expected to produce.

  In the present invention, as a result of efforts to select lactic acid bacteria suitable for fermentation of soybean meal from kimchi, according to a preferred embodiment of the present invention, the selected lactic acid bacteria are preferably Lactobacillus sakei, Lactobacillus brevis or Lactobacillus plantarum, more preferably Lactobacillus plantarum, most preferably Lactobacillus plantarum P23 (KCCM80048) separated from kimchi by the present inventors. .

  In the present invention, the Lactobacillus plantarum P23 strain (hereinafter referred to as “P23 strain”) significantly reduces non-starch polysaccharides, which are anti-nutritive factors in soybean meal, and has excellent organic acid production ability and acid resistance. showed that.

  Various culture media known in the art can be used as the culture medium for inoculating lactic acid bacteria isolated from kimchi, and preferably MRS broth (deMan Regosa Sharpe broth), APT (All Purpose with Tween) or BHI (Brain Heart). Infusion) medium, more preferably MRS broth medium. However, since the price of MRS broth is very high for industrial use, the present inventor has constructed a low-cost medium for lactic acid bacteria inoculum. The carbon source and nitrogen source of the medium for culturing the lactic acid bacteria inoculum were composed of CSL (Corn Steep Liquor).

  For inoculum culture of Bacillus subtilis, it is preferable to culture at 37 ° C. for 12 to 24 hours using a modified nutrient medium (5 g / L soyton, 5 g / L beef extract and 20 g / L xylose, pH 7.0). . Since the Bacillus subtilis medium can also be costly in mass production for industrialization, it is advantageous to grow it on an industrial medium based on CSL devised by the present inventors.

  When the lactic acid bacteria isolated from kimchi are inoculated into an industrial medium devised by the present inventor for lactic acid bacteria seed gold culture and cultured at 30 ° C. for 8 to 24 hours, the activity of the desired bacteria can be obtained.

By culturing the two strains in respective industrial inoculum culture media, the final viable cell count of the inoculum culture solution is in the range of (1-5) × 10 ⁹ cfu / mL.

(B) Cooling of the heat-treated soybean meal and inoculation with microorganisms Next, according to the method of the present invention, the soybean meal treated in the step (b) is cooled to a temperature capable of solid fermentation, and then Bacillus subtilis. Inoculate.

  In the present invention, the soybean meal may be cooled naturally after cooking, but in order to prevent overheating by increasing the cooling rate and to cool uniformly, the transfer process using a conveyor type cooler is performed. After that, it can be easily performed (FIG. 1).

  According to a preferred embodiment of the present invention, in the method of the present invention, the temperature of the cooled soybean meal in the step (b) is 30-50 ° C, preferably 35-45 ° C, more preferably 37. ° C.

  After cooling the heat-treated soybean meal, according to a preferred embodiment of the present invention, the pre-culture solution obtained by culturing Bacillus subtilis on the produced soybean meal medium is diluted as it is or appropriately with sterilized water as much as possible. It is preferable to inoculate uniformly.

The amount of microorganisms inoculated into the heat-treated soybean meal is an important factor that affects the solid fermentation of soybean meal. The amount of microorganisms inoculated into the steamed soybean meal is preferably 10 ⁵ to 10 ⁹ CFU / g immediately after the inoculation. When the inoculation amount is less than 10 ⁵ CFU / g, the required amount of the inoculum fermentation solution is small, but it takes a lot of time to ferment soybean meal, and the culture time for product production becomes longer, and various bacteria may be contaminated. There is a drawback of high nature. On the other hand, when the inoculation amount exceeds 10 ⁹ cfu / g, the fermentation time can be considerably shortened, but there is a drawback that it is a burden on the production of inoculum for inoculation. In particular, since the fermentation results greatly depend on the growth characteristics of the strain used and the type of fermentation apparatus, it is preferable to appropriately select the inoculation amount in consideration of the characteristics of the strain at the production stage.

(C) Obtaining fermented soybean meal by solid culture of the fungus inoculated in the soybean meal One of the greatest features of the present invention is not mold and yeast but hay that is not generalized in solid fermentation (culture) The purpose is to produce fermented soybean meal by inoculating the fungus into soybean meal and culturing.

  As used herein, the term “solid fermentation (culture)” means culturing microorganisms using defatted soybean meal left after separation of fat (soybean oil) from soybean, and an extract of soybean meal Is a method that is distinguished from “liquid culture or liquid fermentation”.

  Since certain microorganisms sometimes grow well in solid fermentation processes and produce large amounts of extracellular enzymes and various other metabolites, solid fermentation has long been adopted mainly in the Asian region for the production of traditional foods or alcoholic beverages. It was. Since soybean meal is a flake or particulate solid substrate, the cheapest and most efficient fermentation method that can significantly improve the feed value of soybean meal using microorganisms is the solid fermentation method.

  In the method of the present invention, a strain selected from Bacillus subtilis is used to use the solid fermentation method, but the type of strain and the inoculation method can be adjusted according to the final product characteristics of the fermented soybean meal. Preferably, the Bacillus subtilis TP6 strain is used, thereby reducing the fermentation time of the present invention.

  Fermented soybean meal produced by inoculating only Bacillus subtilis acts to increase the quality of the fermented soybean meal while Bacillus subtilis actively grows, but preferentially produces an active and powerful protease. The protease produced by Bacillus subtilis hydrolyzes and inactivates protein TI, which inhibits the digestion of soybean meal, and hydrolyzes soy protein as a macromolecule to lower its molecular weight. It shows the characteristics of increasing rate.

  Bacillus subtilis grows actively using carbohydrates in the components of soybean meal and is converted to the proteins that make up the cells. Soybean meal has a relatively high protein content during fermentation. Show the effect of This has an important meaning of increasing the content of the most important protein among the items for evaluating the quality of the feed.

  Bacillus subtilis grown in soybean meal has a characteristic that it exists as a living microorganism even after the fermented soybean meal is dried by surviving at a high ratio while forming spores during drying. The spores of Bacillus subtilis are expected to improve the intestinal function of livestock with the spore-forming probiotics described above.

  The method of the present invention can inoculate lactic acid bacteria alone or mixed bacteria of lactic acid bacteria and Bacillus subtilis. At this time, when only plant lactic acid bacteria isolated from kimchi are inoculated, a large amount of organic acid is produced while growing vigorously using soybean meal as a raw material, and it is contained in soybean meal to show the activity of α-galactosidase. In addition to showing the property of degrading galactooligosaccharides, various polysaccharide anti-nutritional factors in soybean meal can also be hydrolyzed to prevent enteritis and the like, and the absorption rate of nutrient components is significantly improved.

  In order to produce a fermented soybean meal including all the advantages of the two fungi, the two fungi can be inoculated simultaneously or sequentially with a time difference, and the growth conditions of the two fungi are not the same. It is preferable from the standpoint of improving the quality of the finished product that the inoculation is different or the inoculation is performed sequentially. However, even if they are inoculated at the same time or at the same ratio, they only differ in their degree and do not mean that the desired effect cannot be achieved.

  According to a preferred embodiment of the present invention, the soybean meal that has been uniformly inoculated with the target microorganism in the step (c) is fermented in a packed-bed fermentor. There are various types of packed bed fermenters such as a batch aeration culture apparatus, a closed culture apparatus, and a continuous aeration culture apparatus. In any of these apparatuses, the method of the present invention is not limited, and an appropriate apparatus is selected and used according to the production scale.

  The soybean cake is placed in a packed bed fermenter with a thickness of 5 to 50 cm by the method of the present invention and fermented at a culture temperature of 20 to 50 ° C. for 12 to 72 hours. As much as possible, the thickness of the packed bed of soybean meal is preferably as high as possible, and it is preferable to ferment at a culture temperature of 30 to 45 ° C. for 12 to 48 hours. More preferably, it is fermented at 37 ° C. for 24 hours.

(D) Drying and grinding of the obtained fermented soybean meal

  According to a preferred embodiment of the present invention, the method of the present invention further includes, after the step (c), (d) drying and crushing the fermented soybean meal at low temperature and low humidity.

  Although the water in soybean meal partially evaporates during the fermentation process, the residual water content immediately after the end of fermentation is considerably high at 20 to 50% (v / w). However, since the final moisture content of the fermented soybean meal product is preferably 10 to 12% (v / w), a drying step is required.

  In addition, when soybean meal is fermented with fungi such as Aspergillus oryzae, the soybean cake is hardened due to the formation of mycelia, spores are scattered and drying and grinding are very difficult. On the other hand, when fermented by bacteria in the present invention, the state of fermented soybean meal is very good, but it forms a partially weak and hardened lump. It is necessary to grind uniformly.

  Drying and grinding can be carried out in various ways known in the art, but care should be taken as most of the viable bacteria in the fermented soybean meal are killed, especially when dried at excessively high temperatures. Preferably, it must be dried at a low temperature at which viable bacteria do not die, more preferably dried with hot air at low temperature and low humidity. The pulverization process can be pulverized into various sizes according to the purpose of using the fermented soybean meal, and it is preferable to use a hammer mill as a pulverization method.

  By fermenting soybean meal by the method of the present invention described above, various anti-nutritive factors such as TI contained in soybean meal are reduced, digestion and absorption rate is improved by protein hydrolysis, and protein content is increased. By doing so, since the absolute value as a feed is improved, fermented soybean meal is obtained as a high-quality protein feed source that can replace animal protein.

  In addition, since fermented soybean meal produced by the method of the present invention contains Bacillus subtilis having a high viability even during distribution, it has an advantage of having a function that is useful for the intestinal action of animals that have ingested feed. have.

  In another aspect, the present invention relates to a fermented soybean meal produced by the method of the present invention described above.

  Since the fermented soybean meal of the present invention is produced by the method of the present invention described above, the description common to both of them is omitted in order to avoid excessive complexity of the specification due to repeated description. To do.

  Fermented soybean meal produced by inoculating the Bacillus subtilis TP6 strain acts to increase the quality of the fermented soybean meal while Bacillus subtilis is actively growing, but preferentially produces a protease with strong activity. The protease produced by Bacillus subtilis hydrolyzes and inactivates protein TI, which inhibits digestion of soybean meal, and hydrolyzes high molecular weight soy protein to lower its molecular weight. Indicates an increasing feature. In addition, Bacillus subtilis grows actively using carbohydrates in the components of soybean meal and is converted to the protein that composes the cells. Soybean meal has the effect of relatively increasing the protein content during fermentation. Indicates. This has an important meaning of increasing the content of the most important protein among the items for evaluating the quality of feed. The Bacillus subtilis grown in soybean meal has a characteristic that it exists as a living microorganism even after the fermented soybean meal is dried, by surviving at a high ratio while forming spores during drying. The Bacillus subtilis spores are expected to improve the intestinal function of livestock as the above-mentioned spore-forming probiotics.

  According to a preferred embodiment of the present invention, the fermented soybean meal of the present invention contains vegetative cells or spores of microorganisms selected from Bacillus subtilis. Bacillus subtilis TP6 strain is preferable.

  According to another preferred embodiment of the present invention, the fermented soybean meal of the present invention contains poly-γ-glutamic acid that reduces the accumulation of body fat in livestock.

  According to another preferred embodiment of the present invention, the Bacillus subtilis comprises a Bacillus subtilis TP6 strain.

The features and advantages of the present invention can be summarized as follows:
(A) The present invention has improved or improved characteristics as a protein feed source by inoculating soybean meal with Bacillus subtilis excellent in ability to remove soybean antinutritional factors, in particular, Bacillus subtilis TP6 strain and solid culture. A method for producing high quality fermented soybean meal is provided.
(B) In particular, the present invention reduces the solid fermentation time by 12 to 24 hours compared to existing patents or papers, but can ensure a quality equal to or higher than that of existing strains (Bacillus). Subtilis TP6) strain was used. Compared with conventional soybean meal, fermented soybean meal produced according to the present invention is almost inactivated by hydrolysis of TI by a powerful protease produced by Bacillus subtilis, and soy protein is reduced to peptides. It is an excellent high quality protein source with improved absorption and feed efficiency.
(C) Such a decrease in fermentation time increases the annual rate of production by increasing the rotation rate of the iron making machine. This has the advantage of reducing the cost of the product and providing consumers with high quality fermented soybean meal at a low price.
(D) In the method of the present invention, Bacillus subtilis produced by the present invention is produced by Bacillus subtilis produced by the present invention by using Bacillus subtilis not generally used for solid fermentation as a fermenting strain. By powerful protease, TI is hydrolyzed and almost inactivated, soy protein is reduced to peptide, and it is an excellent high quality protein source with improved absorption rate and feed efficiency.
(E) Moreover, since the fermented soybean meal produced by the method of the present invention produces bacterial proteins, which are high-quality proteins, by vigorous growth of Bacillus subtilis, the absolute amount of the protein increases and at the same time Consumption of the carbohydrates contained in the soybean meal as an energy source necessary for the growth of microorganisms further increases the value as a protein feed, such as a relative increase in protein content.
(F) The fermented soybean meal produced according to the present invention contains a large amount of Bacillus subtilis that is highly viable even during distribution, and therefore has a function useful for the intestinal action of animals that have consumed the fermented soybean meal. Has characteristics. Considering that probiotics are recommended as an alternative to prohibiting the use of antibiotics, the future-oriented value of this fermented soybean meal is even greater.
(G) In the present invention, by fermenting soybean meal using Bacillus subtilis having the ability to produce poly-γ-glutamic acid, an effect capable of additionally reducing the accumulation of body fat in livestock is also obtained. be able to.
Ingestion of high energy feed to promote livestock growth will increase livestock body fat. Recently, however, consumers want to reduce the intake of animal fat as much as possible as health concerns increase, so there is a wide demand for the development of breeding methods or feeds that can reduce the body fat of livestock. . Poly-γ-glutamic acid is a main ingredient of sticky mucilage of Korean natto sauce or Japanese natto and is a functional ingredient with various functions. It has been found that it reduces the accumulation of body fat.
(H) As described above, the fermented soybean meal of the present invention can be widely applied to feed as a high protein feed source having excellent quality and various functions. Moreover, fermentation time can be reduced 12 to 24 hours compared with the existing time.

It is the schematic which shows one Example of this invention which manufactures fermented soybean meal using soybean meal as a raw material. L. L. plantarum P23 culture alone, B. subtilis TP6 single culture and L. subtilis. Plantarum P23 and B.I. It is a graph which shows the activity change of a trypsin inhibitor in the case of mixed culture with B. subtilis TP6. L. L. plantarum P23 culture alone, B. subtilis TP6 single culture and L. subtilis. Plantarum P23 and B.I. It is a figure which shows the change of the protein pattern in the case of mixed culture with B. subtilis TP6. It is a graph which shows the sugar analysis chromatogram of the steamed soybean meal which is not inoculated with a fungus. B. It is a graph which shows the saccharide | sugar analysis chromatogram of the fermented soybean meal fermented with B. subtilis TP6 strain. B. B. subtilis TP6 and L. It is a graph which shows the sugar analysis chromatogram of the soybean meal fermented material fermented by the plantarum (L. plantarum) P23 strain. B. The proteolytic effect and allergen-inducing substance removal effect by subtilis (B. subtilis) TP6 expression are shown. M: Marker, (1): Raw soybean meal, (2): 37 ° C 45%, (3): 37 ° C 50%, (4): 45 ° C 45%, (5): 45 ° C 50%.

  Hereinafter, the present invention will be described in more detail with reference to examples. These examples are merely illustrative of the present invention. It will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

  Throughout this specification, “%” used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid and (weight / volume)% for solid / liquid unless otherwise stated. , Liquid / liquid is (volume / volume)%.

Example 1: To determine the moisture content of soybean meal suitable for cultivation of Bacillus subtilis and lactic acid bacteria growing bacteria according to the moisture content of soybean meal, the moisture content of soybean meal is 30%, 40%, 50% and 60%. Add 50 g of the soy bean cake to a beaker (250 mL), cover the top of the beaker with aluminum foil and tie it with a rubber string, boil at 80 ° C for 30 minutes, and then release it. Chilled.
Here, for soybean meal fermentation, Bacillus subtilis TP6 (KFCC11343P) (hereinafter referred to as “TP6” strain) and Lactobacillus plantarum (Lactobacillus plantrum) selected as the most preferred strains according to the embodiment of the present invention. ) P23 (KCCM80048) (hereinafter referred to as “P23” strain) was used. For the seed culture of the TP6 strain, a medium containing 2% xylose, 0.5% soyton and 0.5% beef extract was used, and the P23 strain was used with MRS broth (Difco) at a temperature of 37 ° C. for 24 hours. Cultured.
When the temperature of the steamed soybean meal is cooled to about 30 ° C., 5% inoculation of the various fungi pre-cultured with the above-mentioned various bacteria culture mediums on a clean bench into each steamed soybean meal with different moisture content, contamination and moisture In order to prevent evaporation, the beaker was sealed with aluminum foil and fermented at 37 ° C. for 72 hours using an incubator, and then the number of bacteria and the pH value of each fermented soybean meal were measured. The results are summarized in Table 1 below.

As can be seen from Table 1, considering that the initial number of bacteria after inoculation is about 1 × 10 ⁷ CFU / g, P23 and TP6 bacteria hardly grow after fermentation when the water content of soybean meal is 30%. I can confirm that. When the water content of soybean meal is 40%, (4.5 to 7.0) × 10 ⁸ CFU / g, when the moisture content of soybean meal is 50%, (1.3 to 2.5) × Each grew to a level of 10 ⁹ CFU / g.
As the water content increased, the growth of the bacteria became more vigorous. When the water content was 60%, the final number of P23 and TP6 bacteria increased to a level of 3.2 × 10 ⁹ CFU / g after fermentation. In the case of the P23 strain, the pH decreased significantly after fermentation due to the production of organic acids.
Therefore, the result of this experiment was able to confirm the fact that the initial moisture content of soybean meal must be 40% or more in order to grow the bacteria to the industrial level.

Example 2: Heat treatment conditions of soybean meal The heat treatment of soybean meal that has been hydrolyzed in the fermentation of soybean meal not only prevents contamination of bacteria, but is also an important factor that makes the chemical environment of soybean meal suitable for fermentation. The inventor examined the heat treatment conditions of soybean meal. In order to establish the minimum heat treatment conditions so that the inoculated strains can grow vigorously without causing contamination during the fermentation period, the water content of soybean meal is adjusted to 60% and then placed in a 250 mL beaker. 50 g of each was added, and the upper part of the beaker was sealed with aluminum foil, heat-treated in an autoclave at 60 ° C., 80 ° C., 105 ° C. and 121.1 ° C. for 10 to 30 minutes, respectively, and then allowed to cool.
Each cooked soybean meal with different cooking conditions was inoculated with 5% seed culture solution of TP6 and P23 strains precultured in the same manner as in Example 1 and cultured at 37 ° C. for 72 hours. The presence or absence of miscellaneous bacteria during the fermentation process and the number of viable bacteria and the pH in the fermented soybean meal after the cultivation were measured. The heat treatment conditions affecting the fermentation of soybean meal are shown in Table 2 below.

As can be confirmed from Table 2, under the condition of 60 ° C., when sterilization was performed for 30 minutes, the desired fermentation could not be performed due to contamination after 24 hours of fermentation. However, when the heat treatment was performed at 80 ° C. or higher for 10 minutes or longer, no contamination occurred during the fermentation for 72 hours, and the bacteria grew to 1 × 10 ⁹ CFU / g or more.

Example 3: Change in quality characteristics of fermented soybean meal when lactic acid bacteria and Bacillus subtilis were cultured respectively 70% and 100% of the original soybean meal weight was uniformly sprayed on soybean meal and cooked at 0 ° C for 30 minutes. Soybean meal is inoculated with 5% each of P23 and TP6 strain seed cultures precultured in the same manner as in Example 1, or 5% seed cultures of the two strains are simultaneously inoculated and cultured while performing fermentation. The content of trypsin inhibitor (TI) and the degree of proteolysis by time were analyzed by SDS-PAGE and shown in FIGS. 2 and 3, respectively.
As can be seen from FIG. 2, when the TP6 strain was cultured alone, the change in the TI activity was measured. As a result, when the water content was 70%, the TI activity significantly decreased after 24 hours of culture. Thereafter, it gradually decreased and was completely inactivated after 72 hours of culture. When the amount of water was 100%, the inactivation rate was further increased, and TI was almost completely inactivated after 24 hours of fermentation. There was little change after that. On the other hand, when the P23 strain was cultured alone, the residual amount of TI was high even after 72 hours of fermentation. When the TP6 strain and the P23 strain were mixed and cultured, the same tendency was observed as when the TP6 strain was cultured alone.
Moreover, when the change of the protein pattern by fermentation shown in FIG. 3 is considered, in the case of TP6 single culture | cultivation, it turns out that the high molecular peptide was hydrolyzed 48 hours after fermentation, and the band almost disappeared. However, when the P23 strain was cultured alone, there was almost no change in the protein pattern compared to the original. On the other hand, when the TP6 strain and the P23 strain were mixed and cultured, it was almost the same as when the TP6 strain was cultured alone.
On the other hand, the polysaccharide analysis result of the steamed soybean meal not inoculated with the fungus is FIG. 4, the polysaccharide analysis result of the fermented soybean meal fermented with the TP6 strain is the fermented soybean meal fermented with the TP6 and P23 strains. The polysaccharide analysis results of the product are shown in FIG. Comparing FIG. 4 and FIG. 5, it can be seen that the sugar patterns of the chromatogram are almost the same. This indicates that the TP strain has high activity of various enzymes including protease, but hardly hydrolyzes soybean polysaccharide. However, comparing FIG. 4, FIG. 5, and FIG. 6 in which lactic acid bacteria P23 isolated from kimchi and TP6 strain were mixed and fermented, polysaccharides, which are anti-nutritive factors in soybean meal, were almost hydrolyzed by P23 strain. You can see that it has disappeared.

Example 4: Mixed culture water content in soybean meal of lactic acid bacteria and Bacillus subtilis was adjusted to 50%, and the inoculation ratio or inoculation time of TP6 and P23 strains cultured in the inoculum culture medium on soybean meal treated at 110 ° C. for 15 minutes. Different cultures were carried out at 37 ° C. for 48 hours. Specifically, “Mixed Culture 1” is an inoculation of 5% of P23 strain cultured for 20 hours in a seed culture medium in a fermented soybean meal initially inoculated with 5% of TP6 strain and cultured for 20 hours, followed by further fermentation for 28 hours. “Mixed cultures 2 and 3” were initially inoculated with different inoculation ratios of TP6 and P23 strains of 1: 0.5 and 1: 1, respectively, and fermented for 48 hours.

As can be seen from Table 3, when the TP6 strain was cultured alone, when the P23 strain was additionally inoculated, or when the TP6 strain and the P23 strain were mixed and cultured simultaneously at different inoculation ratios, There is not much difference. In view of these results, fermented soybean meal expected in the present invention can be obtained even if fermentation methods such as single and mixed culture are different.
However, since the quality of the fermented soybean meal has slight differences in the final bacterial count, acidity, flavor and the like, it is preferable that the plant lactic acid bacteria, Bacillus subtilis, and the plant lactic acid bacteria and the hay bean according to the specifications of the target product. It is necessary to appropriately adjust the inoculation time and the inoculation ratio of microorganisms selected from the group consisting of mixed bacteria with bacteria.

Example 5: Quality characteristics of fermented soybean meal After the moisture content was adjusted to 30%, the moisture content was adjusted to 60% by spraying soybean milk uniformly on soybean meal for 20 minutes at 121 ° C. After inoculating soybean koji with adjusted water content with TP6 and P23 strains cultured in each seed medium for 24 hours to 10% (v / w) of soybean koji, fermentation was carried out at 37 ° C. for 48 hours. Table 4 below shows the results of examining physicochemical properties after drying fermented soybean meal.

As can be seen from Table 4, the single fermented soybean meal of the TP6 strain of the present invention has a significantly increased protein content as compared with the original soybean meal, and also has trypsin inhibitor, soybean oligosaccharide and It can be said that this is a product in which polysaccharides are reduced and feed value is increased by several levels. Moreover, the fermented soybean meal contains 2.0 × 10 ⁹ CFU / g DM of live bacteria and poly-γ-glutamic acid, and it can be said that the value is even higher.

Example 6: Change in TI content depending on culture conditions In order to measure the change in the content of trypsin inhibitor, which is one of the antinutritional factors that prevent digestion and absorption, the content of trypsin inhibitor in fermented soybean meal was measured. The measurement of TI content was carried out by requesting the Korea Simple Feed Association. The AOAC method was used as the method. Considering the following experimental group, "soybean" group: general soybean, "soybean meal" group: the portion left by taking soybean oil from soybean, "fermentation 24 hours" group: 100g soybean meal is adjusted to 45% moisture The steamed soybean meal was inoculated with 5 mL of 2 × 10 ⁹ cfu / mL of steamed soybean meal (90 ° C. for 15 minutes) and fermented at 37 ° C. for 24 hours under constant humidity conditions. The change in the content of TI (trypsin inhibitor) after fermentation is as shown in Table 5 below.

  In 24 hours of fermentation, the content of trypsin inhibitor is 0.32 mg / g, which is a 50% level of the TI content of 0.6 mg / g as a result of 36 hours of fermentation described in Patent Document 1, and is 24 hours. Only the fermentation showed the same or better anti-nutritive factor reduction effect than the previous experiment.

Example 7: Change in crude protein content according to soybean meal fermentation time during single culture of TP6 strain (fermentation temperature is 37 ° C.)
100 g of soybean cake was steamed to a moisture content of 45% (90 ° C. for 15 minutes), 5 mL of 2 × 10 ⁹ cfu / mL of TP6 strain was inoculated into the cooked soybean cake, and constant humidity was maintained at 37 ° C. for 24 hours. Fermented under the following conditions. Changes in protein content during fermentation are shown in Table 6 below.

  As a result of 36 hours fermentation using a mixed strain of an existing patent (Patent Document 1) at a level of fermentation time of 24 hours, the result of fermentation for 48 hours using a single strain of an existing patent (Patent Document 2) Compared to existing patents or papers, the fermentation time could be reduced by 12 to 24 hours compared to existing patents or papers (because the total crude protein content is important in the blend ratio, For processed soybean meal that plays a role, the content of crude protein is important.) Such a decrease in fermentation time increases the rotation rate of the koji making machine. This leads to an increase in the number of media that can be produced annually, and the superiority of the present invention can be confirmed from the point that it is possible to supply consumers with a good quality fermented soybean meal at a lower price.

Example 8: Change in crude protein content depending on soybean meal fermentation time during single culture of TP6 strain (fermentation time is 24 hours)
100 g of soybean cake was steamed to a moisture content of 45% (90 ° C. for 15 minutes), 5 mL of 2 × 10 ⁹ cfu / mL of TP6 strain was inoculated into the cooked soybean cake, and constant humidity was maintained at 37 ° C. for 24 hours. Fermented under the following conditions. Changes in protein content during fermentation are shown in Table 7 below.

  As a result of performing fermentation for 24 hours using the selected TP6 strain, the ratio of raffinose and stachyose, which are anti-nutritive factors, was significantly reduced. Moreover, the result of the level substantially equivalent to the 48-hour fermentation result of the existing patent (the said patent document 2) was able to be obtained by 24-hour fermentation.

Example 9: Proteolytic effect by TP6 fermentation and removal effect of allergens (β-conglycinin, glycinin) Fermentation time was all the same as 24 hours, and fermentation experiments were carried out by changing temperature conditions and water conditions. The fermentation conditions were the same as in Examples 7 and 8, and only the moisture percentage during cooking was changed. The respective moisture percentages are 37 ° C. 45% in the case of 2 rains in FIG. 7 and 37 ° C. 50% in the case of 3 rains, 45% 45% in the case of 4 rains and 45% in the case of 5 rains. The temperature was 50%. From the results shown in FIG. 7, it can be confirmed that β-conglycinin and glycinin, which are allergens in raw soybean meal, were best removed at a moisture content of 45 to 50% under 37 ° C. culture conditions. Thus, it can be seen that the high molecular protein was decomposed into a low molecular peptide form and changed into a form suitable for digestion and absorption.

Claims (6)

(A) a step of heat-treating soybean meal with moisture added so as to have a moisture content of 40 to 80% (v / w) at a temperature of 80 to 130 ° C. for 10 to 30 minutes ;
(B) after cooling the heat-treated soybean meal, inoculating Bacillus subtilis TP6 (KCCM11438P) strain;
(C) A method for producing fermented soybean meal, comprising solid-culturing the bacteria inoculated in the soybean meal for 12 to 24 hours to obtain fermented soybean meal.   The method according to claim 1, wherein the temperature of the cooled soybean meal in step (b) is 30-50 ° C.   The method according to claim 1, wherein the solid culture in the step (c) is performed at a temperature of 20 to 50C.   The method according to claim 1, wherein the manufacturing method further comprises, after the step (c), (d) drying and pulverizing the fermented soybean meal.   A fermented soybean meal produced by the method according to claim 1, wherein the fermented soybean meal contains vegetative cells or spores of a Bacillus subtilis TP6 (KCCM11438P) strain. Bean candy. The fermented soybean meal according to claim 5 , wherein the fermented soybean meal contains poly-γ-glutamic acid.