KR102589538B1 - Supplementary feed composition for livestock using useful microorganisms and enzymes - Google Patents

Abstract

The present invention relates to a mixed strain of Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae MNRU0927, KACC 93265P) in a mixture of rice ethanol and soybean meal; About a supplementary feed composition for livestock, characterized in that it is a fermented product cultured for 24 to 48 hours and contains enzymes including alkaline protease, xylanase, amylase, and cellulase. As such, it can be used as a protein supplement for livestock and as a health-beneficial supplementary feed composition by substantially increasing degradable and non-degradable proteins in the feed composition and exhibiting antioxidant and antibacterial activities.

Description

{Supplementary feed composition for livestock using useful microorganisms and enzymes}

The present invention relates to a supplementary feed composition for livestock using useful microorganisms and enzymes.

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-Muslic 등(2011)에 의하면 평균 체중 18.0kg의 MIS 양의 경우, 해바라기 가루, 대두 가루 및 어분을 단백질 공급원을 급여하였을 경우, 평균 일일 증체량이 각각 0.169, 0.205 및 0.227kg으로, 어분과 같은 동물성 단백질이 어린 양의 성장에 효과적인 것으로 나타났다. 그러나 유럽연합 집행위원회가 동물성 단백질 사료를 법적으로 규제하면서 경제적이면서도 충분한 영양을 공급하는 사료에 대한 연구가 시급하게 대두되었다.As the population increases rapidly in modern society, livestock farming, which supplies animal protein, has developed along with it. However, the recent shortage of protein feed and rising costs are the biggest problems facing the livestock industry. Accordingly, research is being conducted on various protein sources to maintain optimal animal performance at low cost. Traditionally, feeds using meat meal or fish meal are more effective in increasing the average daily weight gain of livestock than grass or grain feeds. Ru

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-According to Muslic et al. (2011), for MIS sheep with an average weight of 18.0kg, when sunflower powder, soybean powder, and fishmeal were fed as protein sources, the average daily weight gain was 0.169, 0.205, and 0.227kg, respectively, which was 0.169kg, 0.205kg, and 0.227kg respectively, compared to animal-based sheep such as fishmeal. Protein has been shown to be effective in lamb growth. However, with the European Commission legally regulating animal protein feed, research on feed that is economical and provides sufficient nutrition has become urgent.

In particular, when feeding raw materials with insufficient content, livestock exposure to diseases increases due to reduced conception rates and reduced immunity. It is difficult to supply a sufficient amount of energy and protein due to the recent increase in raw material prices for high-capacity cattle, whose flow production has significantly improved, and the influx of low-priced and low-protein raw materials, so feed must be structured to enable efficient use of ingestible nutrients. .

Proteins consumed by ruminants, including cattle and dairy cows, can be divided into degradable proteins and non-degradable proteins. Degradable proteins refer to proteins that are broken down in the first stomach and destroyed by microorganisms and are later used for microbial protein synthesis. Some of the non-degradable proteins are destroyed in the first stomach after a sufficient period of time and are used by microorganisms, but they are mainly not destroyed and are absorbed and used while passing through the small intestine through the digestion process by digestive enzymes and pass to the fourth stomach without being destroyed. The combination of microbial proteins absorbed in the small intestine and non-degradable proteins is called metabolic protein. Since these metabolic proteins make up the cow's body and are used for milk production, supplying sufficient metabolic proteins is important to maintain high performance cows.

Meanwhile, soybeans have become the most widely used vegetable protein source worldwide as livestock feed as the European Commission regulates animal protein feed. In particular, soybean meal is a product produced by extracting oil from soybeans, and currently accounts for more than 60% of the total protein feed used as animal feed, and is used in most livestock including chickens, pigs, cows, dairy cows, and companion animals. It is used as feed for. However, due to the anti-nutritional factors contained in soybean meal, there is a problem in that it cannot be decomposed efficiently in the intestines of livestock with short digestive organs such as pigs and chickens, and in the case of young livestock, digestion is poor and the digestion is difficult. Because it is harmful, its use in feed for young livestock is limited (Li, D.F., et al., 1990). Anti-nutritional factors in soybean meal include oligosaccharides such as raffinose and stachyose, which cause diarrhea and abdominal pain in livestock, trypsin inhibitor, which interferes with protein digestion, and hemagglutinin, a hemagglutinin. etc. Some of these are destroyed by heat treatment, but during this process, soy proteins may be denatured and essential amino acids such as lysine may be lost. Accordingly, various processing methods are being developed to remove anti-nutritional factors contained in soybean meal and to use soybean meal more efficiently as feed. In this regard, soy protein concentrates (SPC) are produced by ethanol extraction, or protein components are extracted under alkaline conditions, the protein is precipitated under acidic conditions, and then dried using a spray drying method to produce isolated soy protein. There is a known method of removing soy oligosaccharides by producing proteins (IPS), and recently, a processing method to decompose and reduce anti-nutritional factors using microorganisms has been developed (Korean Patent No. 10-2087673; Korean Patent No. No. 10-1139027; Korean Patent No. 10-1157618; Korean Patent No. 10-1214573). In particular, in Korean Patent No. 10-2087673, which is a prior patent of the present inventor, anti-nutritional factors are reduced in soybean meal by fermenting soybean meal by mixing it with Bacillus bacteria and kefir, and it contains beneficial organic acids such as lactic acid and physiologically necessary vitamins such as vitamins. An animal feed additive enriched with active substances is disclosed.

However, there were limitations in supplying sufficient metabolic proteins through soybean meal fermentation alone, such as for high-capacity cattle, and the metabolic proteins used to effectively reduce the anti-nutritional factors of soybean meal in the same weight of feed and substantially increase the body weight and flow rate of livestock. There was a need to develop supplementary feed that could increase the ratio.

Korean Patent No. 10-2087673, Animal feed additive containing fermented soybean meal fermented using Bacillus bacteria and kefir, registered on March 5, 2020. Korean Patent No. 10-1139027, Manufacturing method of fermented soybean meal using Bacillus bacteria, registered on April 16, 2012. Korean Patent No. 10-1157618, Method for producing soy protein and feed composition containing soy protein prepared therefrom, registered on June 12, 2012. Korean Patent No. 10-1214573, Fermented soybean meal obtained using Vicella coriensis and its manufacturing method, registered on December 14, 2012. Korean Patent No. 10-1600669, Feed additive for improving meat quality of poultry containing β-glucan and capers as active ingredients, feed composition and breeding method using the same, registered on February 29, 2016. Korean Patent No. 10-1771488, novel Bacillus coagulans NRR1207 strain, fermented ginseng and probiotic probiotic composition using the same, registered on August 21, 2017.

D. Rui-Muslic, M. P. Petrovic, M. M. Petrovic, Z. Bijelic, V. Pantelicand P. Periic, Effects of different protein sources of diet on yield and quality of lamb meat, African Journal of Biotechnology Vol. 10(70), pp. 15823-15829, 9 November, 2011. Li, D.F., et al., Transient hypersensitivity to soybean meal in the early-weaned pig, J. Anim. Sci., 68(6), 1790-1799, 1990.

D. Rui-Muslic, MP Petrovic, MM Petrovic, Z. Bijelic, V. Pantelicand P. Periic, Effects of different protein sources of diet on yield and quality of lamb meat, African Journal of Biotechnology Vol. 10(70), pp. 15823-15829, 9 November, 2011. Li, DF, et al., Transient hypersensitivity to soybean meal in the early-weaned pig, J. Anim. Sci., 68(6), 1790-1799, 1990.

The purpose of the present invention is to provide a supplementary feed composition for livestock using useful microorganisms and enzymes.

In order to solve the above problem, the present invention includes Bacillus coagulans NRR1207, KACC92114P and Saccharomyces cerevisiae in a mixture of rice ethanol and soybean meal. A supplementary feed composition for livestock containing a fermented product obtained by inoculating and culturing a mixed strain of (MNRU0927, KACC 93265P) is provided.

Strains used in the present invention Bacillus coagulans NRR1207, KACC92114P and yeast Saccharomyces cerevisiae cerevisiae MNRU0927, KACC 93265P) is a strain discovered by the present inventor in the process of studying the decomposition of anti-nutritional factors in soybean meal, and was deposited at the Korea Agricultural Microbial Resource Center.

In addition, the present invention provides the supplementary feed composition for livestock, adding an enzyme agent containing alkaline protease, xylanase, amylase, and cellulase to the rice ethanol and soybean meal mixture. Provided is a supplementary feed composition for livestock, characterized in that it is a cultured fermented product.

In the present invention, alkaline protease is a protease active in the neutral to alkaline pH range, has a serine center or is of the metallo type, and is mainly isolated from microorganisms and is widely used in the detergent, food, pharmaceutical and leather industries.

In the present invention, xylanase is an enzyme that decomposes the linear polysaccharide xylan into xylose.

In the present invention, amylase is an enzyme that hydrolyzes starch and catalyzes its decomposition into sugar.

In the present invention, cellulase is an enzyme that hydrolyzes cellulose.

In the present invention, co. India) and was used by Loomis, and each enzyme activity was maintained at ≥2500 units/g.

In the present invention, rice ethanol is a by-product produced in the process of making ethanol using rice, and refers to the residue remaining after filtering the alcohol. Rice Dried Distiller's Grains is high in energy (TDN 84%) and protein (CP 29.5%), is a by-pass protein source (about 60%), and can be used up to 25% of concentrated feed (Ensminger et al. , 1991).

In the present invention, soybean meal is a product produced after extracting oil from soybeans, and has a protein content of 50% (after removing the husk and extracting the oil), or about 44% if the husk is not removed, so it is highly palatable to livestock and contains methionine. ) is the limiting amino acid, and the protein content and amino acid composition are more uniform than other vegetable protein feeds.

The rice ethanol and soybean meal mixture of the present invention may be a mixture of rice ethanol and soybean meal in a weight ratio of 50 to 90:10 to 50. If the rice ethanol and soybean meal weight ratio is greater than the 90:10 weight ratio, the palatability of the feed for livestock is reduced and there is a problem of not containing sufficient protein, and the rice ethanol and soybean meal weight ratio is less than the rice ethanol and soybean meal weight ratio of 50:50. In this case, the ratio of degradable protein and non-degradable protein in the crude protein of the feed composition is lowered, which is not desirable. Preferably, the rice ethanol and soybean meal mixture may be a mixture of rice ethanol and soybean meal in a weight ratio of 50 to 60:40 to 50, and most preferably, the rice ethanol and soybean meal mixture may be a mixture of rice ethanol and soybean meal in a weight ratio of 50:50. will be.

The present invention is a supplementary feed composition for livestock, which contains Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae in the rice ethanol and soybean meal mixture. cerevisiae MNRU0927, KACC 93265P) mixed strain; Provided is a supplementary feed composition for livestock, characterized in that it is a fermented product containing alkaline phosphatase and an enzyme containing wheat and cultured for 24 to 48 hours.

The supplementary feed composition for livestock is Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae inoculated into a mixture of rice ethanol and soybean meal. The mixed strain of Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) 2×10 ⁷ ~2×10 ⁸ cell/g and Saccharomyces cerevisiae (MNRU0927, KACC 93265P) cerevisiae MNRU0927, KACC 93265P) can be cultured by inoculating 1×10 ⁶ ~1×10 ⁷ cell/g. If less than the number of strains is inoculated, sufficient fermentation may not occur, and if more than the number of strains is inoculated, it may not be economical.

The supplementary feed composition for livestock is characterized in that the ratio of degradable protein (DIP) and undegradable intake protein (UIP) in crude protein is 75% or more.

In the present invention, crude protein includes both non-protein nitrogen (NPN) such as urea, amino acids, and amines and true protein. Degradable proteins are composed of degradable proteins (DIP) and non-degradable proteins (UIP). Degradable proteins refer to proteins that are ingested by ruminants, broken down in the first stomach, and destroyed by microorganisms. Non-degradable protein refers to protein that is not mainly destroyed in the first stomach, but passes to the fourth stomach and is absorbed and used while passing through the small intestine through a digestion process using digestive enzymes. In order to increase the weight gain and flow rate of livestock, it is necessary to increase the ratio of degradable protein and non-degradable protein in feed protein, and the supplemental feed composition for livestock of the present invention increases the ratio of degradable protein and non-degradable protein in crude protein in fermented product to 75. It can be improved to more than %, so it can be used as an effective protein supplement for livestock.

The supplementary feed composition for livestock of the present invention is characterized by having antibacterial activity against Listeria monocytogenes 530. Listeria monocytogenes 530 is a Gram-positive intracellular parasitic bacterium with a rod shape and a low temperature that grows at 1 to 44°C and causes infectious food poisoning. Listeria, which was discovered in 1983 to be infectious to humans through food, has become one of the major public health diseases. The supplementary feed composition for livestock farming of the present invention is Listeria monocytogenes 530 ( Listeria monocytogenes 530), it can prevent food poisoning in fed livestock by having antibacterial activity.

The supplementary feed composition for livestock of the present invention is characterized by having antioxidant activity. Mixed strains of Bacillus coagulans NRR1207, KACC92114P and Saccharomyces cerevisiae MNRU0927, KACC 93265P in the rice ethanol and soybean meal mixture of the present invention; Fermented products cultured for 24 to 48 hours, including enzymes including alkaline phosphatase and (wheat), exhibit higher antioxidant activity compared to non-fermented products, thereby preventing inflammation caused by reactive oxygen species in the bodies of livestock fed. It may have beneficial effects on health.

The present invention relates to a mixed strain of Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae MNRU0927, KACC 93265P) in a mixture of rice ethanol and soybean meal; About a supplementary feed composition for livestock, characterized in that it is a fermented product cultured for 24 to 48 hours and contains enzymes including alkaline protease, xylanase, amylase, and cellulase. As such, it can be used as a protein supplement for livestock and as a health-beneficial supplementary feed composition by substantially increasing degradable and non-degradable proteins in the feed composition and exhibiting antioxidant and antibacterial activities.

Figure 1 is a graph showing the number of viable cells in soybean meal fermented with each strain. (LF; L. fermentum DSHA, LS; L. salivarus Nam27, BC2; B. coagulans NRR1207, BC4; B. coagulans S254, SC; Saccharomyces cerevisiae MNRU0927, SB; Saccharomyces boulardi )
Figure 2 shows the results of HPLC analysis of hydrolyzed carbohydrates in fermented soybean meal. (Control (non-fermented soybean meal), LF 24 h; L. fermentum 24 h, LF 48 h; L. fermentum 48 h, LS 24 h; L. salivatus 24 h, LS 48 h; L. salivarus 48 h, BC2 24 h; B. coagulans NRR1207 24 h, BC2 48 h; B. coagulans NRR1207 48 h, BC4 24; B. coagulans S254 24 h, BC4 48 h; B. coagulans S254 48 h, SB 24 h; S. boulardi 24 h, SB 48 h; S. boulardi 48 h, SC 24 h; S. cerevisiae MNRU0927 24 h, SC 48 h; S. cerevisiae MNRU0927 48 h)
Figure 3 shows TLC results of hydrolyzed carbohydrates of fermented soybean meal. (F; Fructose, S; Sucrose, G; Glucose, M; Melibiose, R; Raffinose, St; Stachyose, Co; Control (non-fermented soybean meal), LF 24 h; L. fermentum 24 h, LF 48 h; L. fermentum 48 h, LS 24 h; L. salivatus 24 h, LS 48 h; L. salivarus 48 h, BC2 24 h; B. coagulans NRR1207 24 h, BC2 48 h; B. coagulans NRR1207 48 h, BC4 24 ; B. coagulans S254 24 h, BC4 48 h; B. coagulans S254 48 h, SB 24 h; S. boulardi 24 h, SB 48 h; S. boulardi 48 h, SC 24 h; S. cerevisiae MNRU0927 24 h, SC 48 h; S. cerevisiae MNRU0927 48 h)
Figure 4 is a graph showing the pH change of fermented soybean meal by the strain and enzyme composition of the present invention.
Figure 5 shows lactic acid bacteria ( B. coagulans NRR1207) and yeast ( Saccharomyces ) in fermented soybean meal using the strain and enzyme composition of the present invention. This is a graph showing the change in viable cell count of cerevisiae MNRU0927).
Figure 6 shows the protein SDS-PAGE results of soybean meal fermented with the strain and enzyme composition of the present invention. (STD: standard marker, Cont: soybean meal, fermented soybean meal: 4 hours, 8 hours, 12 hours, 24 hours, 48 hours)
Figure 7 shows the results of carbohydrate HPLC analysis of soybean meal fermented using the strain and enzyme composition of the present invention. (Fermentation time: 0 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours)
Figure 8 shows the results of carbohydrate TLC analysis of soybean meal fermented with the strain and enzyme composition of the present invention. (F; Fructose, S; Sucrose, G; Glucose, M; Melibiose, R; Raffinose, St; Stachyose, fermentation time: 0 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours)
Figure 9 shows the results of HPLC analysis of organic acids of fermented soybean meal using the strain and enzyme composition of the present invention. (Fermentation time: 0 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours)
Figure 10 shows the results showing the pH change according to the composition ratio of rice ethanol and soybean meal by the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, soybean meal; SBM, A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+SBM 10%)
Figure 11 shows the results showing changes in the number of viable bacteria in mixed fermented rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, soybean meal; SBM, A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+SBM 10%)
Figure 12 shows the SDS-PAGE results of mixed fermentation of rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (M; molecular weight marker, rice ginseng; RDDG, soybean meal; SBM, Lane 1; RDDG 50%+SBM 50% (0 h), Lane 2; RDDG 50%+SBM 50% (24 h), Lane 3; RDDG 50 %+SBM 50% (48 h), Lane 4; RDDG 60%+SBM 40% (0 h), Lane 5; RDDG 60%+SBM 40% (24 h), Lane 6; RDDG 60%+SBM 40% (48 h), Lane 7; RDDG 70%+SBM 30% (0 h), Lane 8; RDDG 70%+SBM 30% (24 h), Lane 9; RDDG 70%+SBM 30% (48 h), Lane 10; RDDG 80%+SBM 20% (0 h), Lane 11; RDDG 80%+SBM 20% (24 h), Lane 12; RDDG 80%+SBM 20% (48 h), Lane 13; RDDG 90 %+SBM 10% (0 h), Lane 14; RDDG 90%+SBM 10% (24 h), Lane 15; RDDG 90%+SBM 10% (48 h), Rice DDG; RDDG, Soybean meal; SBM)
Figure 13 shows the results of HPLC analysis of carbohydrates in mixed fermentation of rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, soybean meal; SBM A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E : RDDG 90%+SBM 10%, fermentation time; A0-0 h, A24-24 h, A48-48 h, B0-0 h, B24-24 h, B48-48 h, C0-0 h, C24-24 h, C48-48 h, D0-0 h, D24-24 h, D48-48 h, E0-0 h, E24-24 h, E48-48 h)
Figure 14 is a graph showing changes in carbohydrate components of mixed fermented rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice; Rice ginseng meal; Stalk; Soybean meal)
Figure 15 shows the results of TLC analysis of mixed fermentation of rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, Soybean meal; SBM, G: Glucose, F: Fructose, Suc: Sucrose, M: Melibiose, R: Raffinose, Sta: Stachyose, A: RDDG 50%+SBM 50%, B: RDDG 60%+ SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+SBM 10% Fermentation time: A0-0 h, A24-24 h, A48-48 h , B0-0 h, B24-24 h, B48-48 h, C0-0 h, C24-24 h, C48-48 h, D0-0 h, D24-24 h, D48-48 h, E0-0 h , E24-24 h, E48-48 h)
Figure 16 shows the results of HPLC analysis of organic acids in the mixed fermentation of rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, soybean meal; SBM, A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+SBM 10%, fermentation time; A0-0 h, A24-24 h, A48-48 h, B0-0 h, B24-24 h, B48-48 h, C0-0 h, C24- 24 h, C48-48 h, D0-0 h, D24-24 h, D48-48 h, E0-0 h, E24-24 h, E48-48 h)
Figure 17 is a photograph showing the antibacterial activity of mixed fermentation of rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal: RDDG, soybean meal: SBM, control group: Cont, A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+ SBM 20%, E: RDDG 90%+SBM 10%, fermentation time: A0-0 h, A24-24 h, A48-48 h, B0-0 h, B24-24 h, B48-48 h, C0-0 h, C24-24 h, C48-48 h, D0-0 h, D24-24 h, D48-48 h, E0-0 h, E24-24 h, E48-48 h)
Figure 18 is a graph showing the change in radical scavenging activity (antioxidant activity) of mixed fermented rice ethanol and soybean meal according to the composition ratio of rice ethanol and soybean meal using the strain and enzyme composition of the present invention. (Rice ethanol meal; RDDG, soybean meal; SBM, A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+SBM 10%)

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms, and the content introduced here is provided to sufficiently convey the spirit of the present invention.

< Example 1. Selection of useful microorganisms>

1.1 Measurement of enzyme activity

Useful microorganisms include Lactobacillus fermentum DSHA, which the present inventor isolated as a strain with activity in decomposing anti-nutritional factors and proteins of soybean meal in order to select strains with excellent ability to produce acid as a metabolite and to produce enzymes that decompose stachyose and raffinose, which are anti-nutritional factors of soybean meal; Lactobacillus salivarus Nam27, Bacillus coagulans NRR1207, Bacillus coagulans S254, Saccharomyces cerevisiae The enzyme activity of MNRU0927 and Saccharomyces boulardi was measured.

rieux, Mercy I’Etoile, France)을 사용하여 조사하였다. 배양 상자를 준비하고 약 5 mL의 멸균 증류수를 트레이에 부어 수분을 유지시켰다. 그 후 스트립을 트레이 위에 올려놓고 각 균주를 액체배지 (0.85% NaCl, Ref 20070, BioM

rieux, France)에 현탁한 다음, 표준 탁도 농도를 McFarland No 5.0-6.0 standard (BioM

rieux, France)를 맞추었다. API ZYM 19 탈수 발색 효소 기질(dehydrated chromogenic enzyme substrates)에 50 μl씩 분주 및 현탁한 다음 37℃에서 4시간 동안 배양하였다(version 4.0 of API LAB plus; BioM

rieux, France). 이후, ZYM A와 ZYM B시약을 각각의 커플에 한 방울씩 떨어뜨린 후 ZYM A가 표면의 활성을 증가시켜 ZYM B의 용해를 돕게 하고 5분간 후에 판독하였다. 큐플 위의 10 cm 위치에 강력한 빛(1,000 W bulb)을 10초간 쪼이도록 하고 반응 결과를 읽고 색의 변화 정도에 따라 0-5까지의 값으로 표시하였다. 0은 음성반응, 5는 최대 강도의 반응이고 1, 2, 3, 4는 중간 반응 값이며 3 이상이면 양성으로 판정하고 그 결과를 하기 표 1에 나타내었다.The enzyme activity of the strain was tested using the API ZYM enzyme system (version 4.0 of API LAB plus, BioM

rieux, Mercy I'Etoile, France). A culture box was prepared, and approximately 5 mL of sterile distilled water was poured into the tray to maintain moisture. Afterwards, the strip was placed on a tray and each strain was cultured in liquid medium (0.85% NaCl, Ref 20070, BioM

rieux, France), and then the standard turbidity concentration was measured using McFarland No 5.0-6.0 standard (BioM

rieux, France). 50 μl each was dispensed and suspended in API ZYM 19 dehydrated chromogenic enzyme substrates and incubated at 37°C for 4 hours (version 4.0 of API LAB plus; BioM

rieux, France). Afterwards, one drop of ZYM A and ZYM B reagent was added to each couple, and ZYM A increased surface activity to help dissolve ZYM B, and the results were read after 5 minutes. A strong light (1,000 W bulb) was irradiated for 10 seconds at a position 10 cm above the cupule, and the reaction results were read and expressed as a value from 0 to 5 depending on the degree of color change. 0 is a negative response, 5 is the maximum intensity response, 1, 2, 3, and 4 are intermediate response values, and if it is 3 or more, it is judged positive. The results are shown in Table 1 below.

No Enzymes L. fermentum
DSHA L. salivarus
Nam27 B. coagulans
1207 B. coagulans
S254 S. cerevisiae MNRU0927 S. boulardi One Control 0 0 0 0 0 0 2 Alkaline phosphatase 0 3 3 3 2 2 3 Esterase (c4) 2 5 3 4 2 2 4 Esterase lipase (C8) 2 4 2 3 One 2 5 Lipase (c14) 0 3 0 0 0 0 6 Leucinearylamidase 5 5 4 2 5 5 7 Valinearylamidase 2 5 2 One One 2 8 Cystinearylamidase 2 3 One One 0 One 9 Trypsin 0 0 0 0 0 0 10 α-chymotrypsin 0 3 0 0 0 0 11 Acid phosphatase One 5 4 5 5 5 12 Naphtol-AS-BO-phosphohydrolase 2 5 4 5 4 5 13 α-galactosidase 3 5 3 3 0 0 14 β-galactosidase One 5 5 5 0 One 15 β-glucuronidase 0 0 0 2 One 0 16 α-glucosidase 2 5 5 5 0 0 17 β-glucosidase 0 2 One 4 0 0 18 N-acetyl-β-glucosamidase 0 0 3 2 0 0 19 α-mannosidase 0 0 0 0 0 0 20 α-fucosidase 0 0 0 0 0 0 * Quantity of hydrolyzed substrate, 0: 0 nanomoles, 1: 5 nanomoles, 2: 10 nanomoles, 3: 20 nanomoles, 4: 30 nanomoles, 5: ≥40 nanomoles

As shown in Table 1, all four types of bacteria and two types of yeast used in the experiment showed esterase and esterase lipase enzyme activities involved in fat decomposition. In addition, proteolytic enzymes such as leucine arylamidase, valine arylamidase, and cystine arylamidase showed activity of 5 to 40 ≤ nmol, and trypsin showed no activity. . α-galactosidase activity, which can decompose raffinose and stachyose, which are anti-nutritional factors in soybean meal, was observed in all four types of bacteria, and β-galactosidase, which is involved in sugar decomposition. Active also L. fermentum It was found to be high in three types of bacteria excluding DSHA. B. coagulans 1207 and B. coagulans S254 exhibited N-acetyl-β-glucosamidase activity that targets and hydrolyzes oligosaccharides including chitin and S. cerevisiae MNRU0927 showed beta-glucuronidase activity, a glycosidase family enzyme that catalyzes the decomposition of complex carbohydrates.

1.2. Sugar availability survey

Lactobacillus sp . and Bacillus sp .

rieux, Mercy I’Etoile, France)을 사용하여 조사하였다. 배양 상자를 준비하고 약 5 mL의 멸균 증류수를 트레이에 부어서 수분을 유지하고 스트립을 트레이 위에 올려놓았다. 이후 상기 각 균주를 액체배지 (0.85% NaCl, Ref 20070, BioM

rieux, France)에 현탁하고 표준 탁도 농도 McFarland No 2.0 standard (BioM

rieux, France)를 맞추었다. 각 API 50CHL/B kit 에 100 μl씩 분주 및 현탁한 다음 37℃에서 48시간 동안 배양 (version 4.0 of API LAB plus; BioM

rieux, France) 후에 배지에 포함되어 있는 bromocresol purple 지시약에 의해 산이 생성되면 배지의 색이 노란색으로 변한다. Esculin test는 보라색에서 검은색으로 변하면 양성으로 기록하여 하기 표 2에 나타내었다. 대두박에 들어있는 당은 대부분 raffinose, stachyose 및 melibiose 이고, glucose, sucrose, fructose는 소량 포함된 것으로 알려져 있으며, 하기 표 2에서 보는 바와 같이, 49종의 당이 포함되어 있는 API 50CHL/B kit을 이용하고 당 이용성을 확인할 때 4종류 미생물은 galactose, glucose, fructose, mannose, mannitol, esculin, cellibiose, maltose, lactose, melibiose, sucrose, raffinose 등을 100% 이용한 것으로 나타났다.The sugar availability of the above four strains was API 50CHL/B (version 4.0 of API LAB plus, BioM

rieux, Mercy I'Etoile, France). A culture box was prepared, approximately 5 mL of sterile distilled water was poured into the tray to maintain moisture, and the strips were placed on the tray. Afterwards, each of the above strains was cultured in liquid medium (0.85% NaCl, Ref 20070, BioM

rieux, France) and adjusted to a standard turbidity concentration of McFarland No 2.0 standard (BioM).

rieux, France). Dispense and suspend 100 μl of each API 50CHL/B kit and then culture at 37°C for 48 hours (version 4.0 of API LAB plus; BioM

rieux, France), when acid is produced by the bromocresol purple indicator contained in the medium, the color of the medium changes to yellow. Esculin test was recorded as positive when it changed from purple to black, and is shown in Table 2 below. Most of the sugars in soybean meal are raffinose, stachyose, and melibiose, and small amounts of glucose, sucrose, and fructose are known to be contained. As shown in Table 2 below, the API 50CHL/B kit, which contains 49 types of sugars, is used. When checking sugar utilization, the four types of microorganisms were found to use 100% of galactose, glucose, fructose, mannose, mannitol, esculin, cellibiose, maltose, lactose, melibiose, sucrose, and raffinose.

Substrate L. fermentum DSHA L. salivarus Nam27 B. coagulans
NRR1207 B. coagulans S254 0 Control - - - - One Grycerol - - - +(50%) 2 Erythritol - - - - 3 D-Arabinose - - - - 4 L_Arabinose +(80%) + + + 5 D-Ribose + + + + 6 D-Xylose - - + +(20%) 7 L-Xylose - - - - 8 D-Adonitol - - - - 9 Methyl-βD xylopyranoside - - - - 10 D-Galactose + + +(90%) + 11 D-Glucose + + + + 12 D-Fructose + + + + 13 D-Mannose + + + + 14 L-Sorbose - - - - 15 L-Rhamnose - + +(10%) + 16 Dulcitol - - - - 17 Inositol - - - - 18 D-Mannitol + + + + 19 D-Sorbitol + + +(50%) +(50%) 20 Methyl-α D-Mannoryranoside - - - - 21 Methyl-α D-glucopyranoside - - +(80%) +(60%) 22 N-Acetyl glucosamine + + + + 23 Amygdaline + - + + 24 Arbutine + + + + 25 Esculine + + + + 26 Salicine + - + + 27 D-Cellobiose + + + + 28 D-Maltose + + + +(50%) 29 D-Lactose + + - + 30 D-Melibiose + + + + 31 D-Sucrose + + + +(60%) 32 D-Trehalose + + +(80%) +(50%) 33 Inuline - - - - 34 D-Melezitose + + - - 35 D-Rafiinose + + + + 36 Starch - - +(50%) +(50%) 37 Glycogene - - - - 38 Xylitol - - - +(20%) 39 Gentibiose + - + + 40 D-Turanose + +(50%) +(50%) +(50%) 41 D-Luxose - - - - 42 D-Tagatose - +(50%) - - 43 D-Fucose - - - - 44 L-Fucose - - - - 45 D-Arabitol - - - + 46 L-Arabitol - - - - 47 Gluconate + +(50%) +(10%) +(10%) 48 2keto-gluconate - - +(50%) + 49 5keto-gluconate + - +(70%) +(50%)

leaven

rieux, Mercy I’Etoile, France)을 사용하여 조사하였다. 배양 상자를 준비하고 약 5 mL의 멸균 증류수를 트레이에 부어서 수분을 유지한 다음 스트립을 트레이 위에 올려놓았다. 분리된 균주를 액체배지(0.85% NaCl, Ref 20070, BioM

rieux, France)에 현탁하고 표준 탁도 농도 McFarland No 2.0 standard (BioM

rieux, France)를 맞추었다. 각 API 50CHL/B kit 에 100 μl씩 분주 및 현탁 한 다음 29±2℃에서 72시간 동안 배양 후에 “0” 큐플을 음성 대조군으로 하고 혼탁도가 높은 큐플을 양성 반응으로 결과지에 기록하여 표 3에 나타내었다. 효모의 당 이용성을 확인할 때 효모에만 사용하는 API 20 C AUX kit을 이용하였다. 표 3의 결과에 의하면 사용한 2가지 효모는 19종 당 중에서 2-keto-D-gluconate, adonitol, xylitol, D-melezitose 외 모든 당을 이용하는 것으로 나타났다. The yeast Saccharomyces cerevisiae MNRU0927 and Saccharomyces The sugar availability of the two types of boulardi is API 20 C AUX (version 4.0 of API LAB plus, BioM

rieux, Mercy I'Etoile, France). A culture box was prepared, approximately 5 mL of sterile distilled water was poured into the tray to maintain moisture, and the strips were placed on the tray. The isolated strain was cultured in liquid medium (0.85% NaCl, Ref 20070, BioM

rieux, France) and adjusted to a standard turbidity concentration of McFarland No 2.0 standard (BioM).

rieux, France). Dispense and suspend 100 μl of each API 50CHL/B kit, and then incubate at 29±2°C for 72 hours. The “0” cupule was used as a negative control, and the cupule with high turbidity was recorded as a positive reaction on the result sheet, as shown in Table 3. indicated. When checking the sugar availability of yeast, the API 20 C AUX kit, which is used only for yeast, was used. According to the results in Table 3, the two types of yeast used were found to use all sugars including 2-keto-D-gluconate, adonitol, xylitol, and D-melezitose among 19 types of sugars.

No Substrate S. cerevisiae MNRU0927 S. boulardi One Control - - 2 Glucose + (100%) + (100%) 3 Glycerol + (100%) + (100%) 4 2-keto-D-gluconate - - 5 L-arabinose + (100%) + (100%) 6 D-Xylose + (100%) + (100%) 7 Adonitol - - 8 Xylitol - - 9 D-galactose + (100%) + (100%) 10 Inositol - + (50%) 11 D-sorbitol - + (50%) 12 α-methyl-D-glucoside + (100%) + (100%) 13 N-acetyl-D-glucosamine + (100%) + (100%) 14 D-cellulose + (100%) + (100%) 15 D-lactose + (50%) + (100%) 16 D-maltose + (100%) + (100%) 17 D-sucrose + (100%) + (100%) 18 D-trehalose + (100%) + (100%) 19 D-melezitose - - 20 D-raffinose + (100%) + (100%)

Useful microorganisms include Lactobacillus fermentum DSHA, which the present inventor isolated as a strain with activity in decomposing anti-nutritional factors and proteins of soybean meal in order to select strains with excellent ability to produce acid as a metabolite and to produce enzymes that decompose stachyose and raffinose, which are anti-nutritional factors of soybean meal; Lactobacillus salivarus Nam27, Bacillus coagulans NRR1207, Bacillus coagulans S254, Saccharomyces cerevisiae The enzyme activity of MNRU0927 and Saccharomyces boulardi was measured.

1.3. soybean meal Fermentation characteristics investigation

Probiotic count measurement

The actual fermentation ability of soybean meal was confirmed based on enzyme analysis and sugar availability of the strains investigated above. 1,000 g of soybean meal and 450 g of sterilized distilled water (40°C) were mixed, 5% of each microorganism identified above was added, and the mixture was cultured at the optimal temperature for each microorganism for up to 48 hours as shown in Table 4 below.

Microorganisms Temperature (°C) Starter/soybean 1,000g L. fermentum DSHA 37℃ 5% L. salivarus Nam27 37℃ 5% B. coagulans NRR1207 40℃ 5% B. coagulans S254 40℃ 5% Saccharomyces cerevisiae MNRU0927 30℃ 5% Saccharomyces boulardi 30℃ 5%

At 0, 24, and 48 hours, 1 g of the culture sample was aseptically collected, dispensed into 9 mL of 0.85% NaCl solution, and diluted using the decimal dilution method to place lactic acid bacteria on MRS agar, Bacillus on TS agar, and yeast on PD agar. After inoculation, each was cultured at the optimal temperature to confirm, and the results are shown in Table 5 and Figure 1. As shown in the results in Table 5 and Figure 1, the number of viable bacteria for all six types of microorganisms increased rapidly during 24-hour fermentation, and decreased slightly after 48 hours, confirming that all six types of microorganisms were able to maintain a certain number of viable bacteria in soybean meal. .

Microorganism Viable cell count 0h 24h 48h L. fermentum DHSA 5.0×10 ⁷ 5.85×10 ⁸ 4.96×10 ⁸ L. salivarus Nam27 6.9×10 ⁶ 1.03×10 ⁹ 2.12×10 ⁸ B. coagulans NRR1207 2.37×10 ⁷ 1.25×10 ⁹ 1.8×10 ⁸ B. coagulans S254 1.89×10 ⁶ 2.23×10 ⁹ 5.62×10 ⁸ Saccharomyces boulardi 1.5×10 ⁶ 1.39×10 ⁸ 4.3×10 ⁷ Saccharomyces cerevisiae MNRU0927 1.24×10 ⁶ 1.38×10 ⁸ 6.3×10 ⁷

anti-nutritional factor Resolution measurement

1 g of fermented soybean meal fermented with each of the six types of microorganisms was mixed with 5 mL of 10% ethanol, stirred at 200 rpm for 60 minutes at 50°C, and then centrifuged at 4,000 rpm for 10 minutes. Then, to remove the protein, the supernatant was mixed with acetonitrile in a 2:3 ratio, evaporated for 12 hours, and then centrifuged at 9,000 rpm for 20 min using a centrifuge (Mega 17R, Hanil Science Industrial, Korea). The separated supernatant was taken, filtered using a 0.2 ㎛ membrane filter, and carbohydrates were analyzed using an HPLC system (Waters 2695 Separations Module, Waters Associates, USA). The detector used was a Refractive Index Detector (Waters Associates, USA), the column used SUPELCOGEL C-610H (38cm×7.8mm, Sigma-Aldrich Co., USA), the temperature of the column was maintained at 40°C, and the mobile phase was analyzed for 30 minutes at a flow rate of 0.5 mL/min. Quantitative analysis was performed using the analysis program Empower (Waters Associates, USA). Standard materials were purchased from Sigma-Aldrich Co. (USA) and used for analysis. The sugar utilization of soybean meal for each microorganism was confirmed by HPLC, and calculated using the standard sugar peak area, which is shown in Figure 2 and Table 6.

Sugar (mg/g) Incubation time (h) Con.
0h L. fermentum L. salivatus B. coagulans NRR1207 B. coagulans S254 S. boulardi S. cerevisiae MNRU0927 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h 24h 48h Stachyose 17.23 18.15 19.69 10.17 12.70 0 0 0 0 1.23 1.23 1.30 0.53 Raffinose 16.02 0 0 13.44 11.91 12.79 5.96 14.01 11.51 11.20 10.82 12.54 12.59 Melibiose 23.07 9.26 5.24 7.07 3.98 6.98 1.97 7.89 4.64 6.42 4.82 7.45 6.83 Sucrose 21.87 10.28 8.90 7.98 6.08 6.43 7.05 7.41 6.45 6.52 7.37 8.42 9.40 Glucose 20.99 9.46 7.20 6.73 5.01 13.11 5.98 12.78 14.37 4.31 8.85 6.06 8.79 Fructose - 4.85 7.01 11.53 6.69 0.00 11.38 0 0 0 0 4.02 0.00

As shown in Figure 2 and Table 6, L. fermentum DSHA did not show a raffinose peak at 24 and 48 hours after fermentation, and the amount of stachyose slightly increased compared to 0 hours. Bacillus coagulans NRR1207 and Bacillus coagulans S254 did not show stachyose peak at 24 and 48 hours of fermentation. In the HPLC analysis, it is difficult to determine accurately because the peak positions of the stachyose and raffinose components are mixed, but it is believed that they were partially decomposed. Additionally, melibiose, sucrose, and glucose decreased, and fructose increased due to the decomposition of the polysaccharide raffinose. In L. salivarus Nam27, all sugars were reduced.

From the above results, it was found that B. coagulans NRR1207 completely decomposed stachyose, raffinose was decomposed from 16.02 mg/g to 5.96 mg/g, and melibiose, sucrose, and glucose were all significantly decomposed. Due to the decomposition of polysaccharides, 11.38 mg/g of fructose was produced. B. coagulans S254 was similar to B. coagulans S252 in its sugar utilization method, but had weak raffinose decomposition activity. with Saccharomyces cerevisiae MNRU0927 Saccharomyces boulardi Compared to the control, yeast hydrolyzed 17.23 mg/g of stachyose to 0.5 mg/g and also reduced mlibiose, sucrose, glucose, and fructose.

TLC analysis

Sugar decomposition of the fermented soybean meal was confirmed by Thin Layer Chromatography (TLC). Sugars were separated from fermented soybean meal using the same method as HPLC and used for TLC analysis. 5㎕ was stamped on the bottom of a Silica-Gel 60 F254 Aluminum sheet plate (Merck, Darmstadt, Germany), dried, and fully developed with isopropanol:water (80:20, v/v) solvent. A mixed solution of 1 g diphenylamine, 1 mL aniline, 5 mL of 85% H ₃ PO ₄ , and 50 mL acetone was spread on an aluminum sheet plate and heated at 120°C for 10 minutes, and the results are shown in Figure 3. The distance of the mobile phase was measured to obtain each fraction and ratio. As shown in Figure 3, L. fermentum DSHA, L. salivarus Nam27 , S. cerevisiae MNRU0927, S. The TLC results for fermented soybean meal using boulardi are Fog. It was consistent with the HPLC results of 2. In B. coagulans NRR1207, raffinose, melibiose, fructose, stachyose, and glucose spots disappeared after 48 hours of fermentation, and B. coagulans Sugar usability was superior to S254. S. boulardi and S . cerevisiae In MNRU1207, raffinose and melibiose were partially decomposed, and fructose, stachyose, and glucose spots disappeared.

< Example 2. Bacillus coagulans NRR1207 ( KACC92114P ) and Saccharomyces cerevisiae MNRU0927(KACC93265P) In mixed strains by soybean meal Fermentation and Characterization Investigation>

Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces to include the activities of β-galactosidase, N-acetyl-β-glucosamidase, and β-glucuronidase based on the sugar-decomposing ability of each of the above strains. cerevisiae Two strains, MNRU0927 (KACC93265P), were selected to determine whether their mixed strains were capable of sufficiently fermenting soybean meal.

Mix 1,000 g of soybean meal and 450 g of sterilized distilled water (40℃) and mix Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces . cerevisiae Each of the microorganisms of MNRU0927 (KACC93265P) and their mixed strains were added to a concentration of 5% microorganisms and cultured at 37°C for 48 hours. HPLC analysis was performed in the same manner as above, and is shown in Table 7.

Sugar (mg/g) Incubation time (h) Con.
0 h B. coagulans NRR1207 S. cerevisiae MNRU0927 BS+SC 24h 48h 24h 48h 24h 48h Stachyose 18.56 0 0 1.73 0.66 0 0 Raffinose 17.51 11.86 4.54 11.54 10.23 8.42 2.12 Melibiose 22.59 5.21 2.63 8.14 5.49 4.23 0.89 Sucrose 21.86 6.21 6.52 7.98 8.36 7.98 8.54 Glucose 21.45 14.23 6.25 6.00 8.05 5.43 8.69 Fructose - 0.00 10.42 5.26 1.23 4.22 1.12

remind As shown in Table 7, Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces cerevisiae MNRU0927 (KACC93265P) mixed strain showed high resolution in stachyose, raffinose, and melibiose compared to Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces cerevisiae MNRU0927 (KACC93265P) single strains. This means that anti-nutritional factors can be decomposed synergistically due to differences in the type and function of decomposition enzymes in Bacillus strains and yeast.

< Example 3. By adding enzymes soybean meal Fermentation and Characterization Investigation>

In the above experiment, the useful microorganisms did not have sufficient proteolytic activity as a result of the enzyme activity in all six types of microorganisms. Accordingly, an enzyme agent was added to increase the ratio of degradable and non-degradable proteins from soybean meal. Enzymes include alkaline protease and Maxa Feed Wheat (Xylanase, Amylase, Cellulase) from Lumis Co. (India) was used. Each enzyme activity was ≥2500 units/g. As shown in Table 8, 5 g of AP alkaline protease, 2 g of wheat Maxa Feed Wheat (more than 5000 units each of Xylanase, Amylase, and Cellulase), 450 g of 40°C sterilized distilled water, and 5% microbial starter were added to 1,000 g of soybean meal and incubated at 37°C for 48 hours. Cultured until. Samples were collected at 0, 4, 8, 12, 24, and 48 hours of fermentation and used in the experiment.

Materials Weight Soybean meal 1,000 g AP Alkalic protease 5g Maxa Feed Wheat (Xylanase, Amylase, Cellulase) Loomis Co. (India) 2g B. coagulans NRR1207 (5%) 50mL S. cerevisiae MNRU0927 (5%) 50mL Moisture 45% (40℃) 400g

pH

The pH of fermented soybean meal was measured using a pH meter (Mettler Toledo Seven Easy pH meter, Sigma-Aldrich, USA) and is shown in Table 9 and Figure 4.

Item pH fermentation time 0h 4h 8h 12h 24h 48h fermented soybean meal 6.46 6.31 5.89 5.47 4.92 4.80

As shown in Table 9 and Figure 4, the pH decreased from 6.46 to 4.80 depending on the fermentation time. This phenomenon is because lactic acid bacteria actively produce acid using the sugar component of soybean meal.

Probiotic count measurement

Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces cerevisiae in the fermented soybean meal The viable bacterial count of MNRU0927 (KACC93265P) was measured using the same method as the above-mentioned viable bacterial count measurement method and is shown in Table 10 and Figure 5. As shown in Table 10 and Figure 5, Bacillus coagulans NRR1207 (KACC92114P) increased from 3.0 × 10 ⁷ to 1.2 × 10 ⁹ depending on the fermentation time, and Saccharomyces cerevisiae MNRU0927 (KACC93265P) grew from 4.7×10 ⁶ to 9×10 ⁶ in 8 hours of fermentation and then decreased again.

microbe Viable cell count 0 h 4h 8h 12h 24h 48h B. coagulans NRR1207 3.0×10 ⁷ 2.6×10 ⁷ 1.56×10 ⁸ 4.77×10 ⁸ 1.38×10 ⁹ 1.2×10 ⁹ S. serevisiae 4.7×10 ⁶ 7.1×10 ⁶ 9.0×10 ⁶ 2.2×10 ⁶ 1.43×10 ⁶ 1.13×10 ³

protein breakdown

To investigate the degree of protein degradation of the fermented soybean meal, SDS-PAGE electrophoresis was performed according to the method of Livia (1982). 5 g of fermented soybean meal sample was mixed with 100 mL extraction buffer (0.05M Tris-HCl buffer pH=8.2) and extracted for 70 minutes by vortexing at 10-minute intervals in an ultrasonic bath at 40°C. Then, centrifuged at 9,000 rpm for 30 minutes and the supernatant was centrifuged at 1,000 rpm. After dialysis using a Dalton dialysis membrane, it was dried using a freeze dryer (IlShin Lab Co. Ltd, Siheung, Korea). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the dried sample, and the results are shown in Figure 6. As shown in Figure 6, the results of fermented soybean meal protein electrophoresis showed that proteins with high molecular weight were decomposed and decreased depending on the fermentation time, and as 12, 24, and 48 hours passed, protein decomposition occurred rapidly and the low molecular weight band appeared weak. Peptides with a molecular weight of 10 kDa or less are presumed to have escaped without appearing on electrophoresis.

glycolysis

To investigate the degree of sugar decomposition of the fermented soybean meal, HPLC and TLC were performed according to the above method. 5 g of the fermented soybean meal sample was mixed with 100 mL extraction buffer (0.05M Tris-HCl buffer pH=8.2) and extracted for 70 minutes while vortexed at 10-minute intervals in an ultrasonic bath at 40°C. Then, centrifuged at 9,000 rpm for 30 minutes to obtain the supernatant. After dialysis using a 1,000 dalton dialysis membrane, it was dried using a freeze dryer (IlShin Lab Co. Ltd, Siheung, Korea). HPLC and TLC were performed on the dried sample, and the results are shown in Table 11, Figure 7 (HPLC), and Figure 8 (TLC). As shown in Table 11 and Figure 7 (HPLC) and Figure 8 (TLC), the amounts of stachyose and melibiose decreased significantly with soybean meal fermentation time, and the amounts of sucrose, glucose, and fructose also decreased. In the case of raffinose, the decrease was not significant on HPLC, but as a result of TLC below, it was determined that raffinose, like stachyose and melibiose, was mostly hydrolyzed at 24 and 48 hours after fermentation.

Carbohydrate Fermentation time (h) 0 4 8 12 24 48 Stachyose (mg/g) 17.23 16.24 14.73 13.34 6.14 5.01 Raffinose (mg/g) 16.02 16.46 16.58 17.05 16.27 14.20 Melibiose (mg/g) 23.07 19.99 10.90 8.59 8.01 7.44 Sucrose (mg/g) 21.87 19.73 13.89 11.43 10.93 12.17 Glucose (mg/g) 20.99 16.93 17.39 13.19 8.31 9.42 Fructose (mg/g) 0.00 8.78 17.33 15.85 15.42 13.19

Organic acid production

The degree of organic acid production in the fermented soybean meal was investigated. 1 g of the fermented soybean meal was mixed with 10% ethanol mL, stirred at 200 rpm for 60 minutes at 50°C, and then centrifuged at 4,000 rpm for 10 minutes. To remove proteins, the supernatant was mixed with Acetonitrile in a 2:3 ratio, evaporated for 12 hours, and then centrifuged at 9,000 rpm for 20 min using a centrifuge (Mega 17R, Hanil Science Industrial, Korea). The separated supernatant was taken, filtered using a 0.2 ㎛ membrane filter, and organic acids were analyzed using an HPLC system (Waters 2695 Separations Module, Waters Associates, USA). The detector used was a Refractive Index Detector (Waters Associates, USA), the column used SUPELCOGEL C-610H (38cm×7.8mm, Sigma-Aldrich Co., USA), the temperature of the column was maintained at 40°C, and the mobile phase was analyzed for 30 minutes at a flow rate of 0.5 mL/min and is shown in Figure 9. Quantitative analysis was performed using the analysis program Empower (Waters Associates, USA). Standard materials were purchased from Sigma-Aldrich Co. (USA) and used for analysis. As shown in Figure 9, as a result of organic acid analysis of fermented soybean meal, lactic acid increased significantly with fermentation time. In the case of acetic acid, it slightly decreased at 24 and 48 hours of fermentation, and folic acid (phytic acid) increased significantly at 48 hours. In the case of citric acid, tartaric acid, and succinic acid, there were no significant changes throughout the fermentation process.

decomposability protein (DIP) and non-degradable protein( UIP ) measurement

The degree of organic acid production in the fermented soybean meal was investigated. Degradable intake protein (DIP) was analyzed according to the method of Licitra et al. (1996). 0.5 g of the fermented soybean meal was taken in a 125 ml Erlenmeyer flask, 50 ml of borate phosphate buffer (pH 6.8) was added, and 10% sodium was added. 1 ml of azide solution was added. After standing at room temperature for 3 hours, it was filtered through Whatman filter paper 541, and the residue on the filter paper was washed twice with distilled water. The filter paper was placed in a protein digestion tube, decomposed, distilled, and titrated, and the protein on the filter paper was subtracted from the total protein to calculate the soluble protein according to the formula below.

For undegradable intake protein (UIP), about 0.2 g of a sample dried at room temperature was placed in an Erlenmeyer flask, mixed with 40 ml of borate-phosphate buffer (pH 6.7), and incubated in a 39°C water bath for 1 hour. Then, add 10 ml of fresh protease solution and shake gently (protease solution is Streptomyces griseus, Sigma Chemical Co, Ltd.). The sample was filtered through Whatman 541 after 18 hours and washed with 250 ml distilled water. Nitrogen residue was measured by the Kjeldahl method. Crude protein degradability was calculated as a percentage of total CP as follows.

%CP resolution = [(initial CP - CP after incubation)/initial CP] × 100.

Undigested Intake Protein (UIP) %UIP = 100 - %CP Resolution

As a result, the crude protein (CP) of soybean meal after 48 hours of fermentation using the strain and enzyme composition of the present invention was 63.45%, of which degradable protein was 18.45% and non-degradable protein was 17.62%. Crude protein is composed of non-protein nitrogen (NPN) such as urea, amino acids, and amines and true protein, while true protein is composed of degradable protein (DIP) and non-degradable protein (UIP). There is a need to further increase degradable protein (DIP) and non-degradable protein (UIP).

< Example 4. Using useful microorganisms and enzymes soybean meal , of rice wine marmalade Investigation of fermentation characteristics>

Based on the results of fermented soybean meal using the above useful microorganisms and enzymes, rice wine marmalade was added to soybean meal to determine whether true proteins and amino acids could be increased through fermentation using the strain and enzyme composition of the present invention.

The ratio of soybean meal (SBM) and rice distiller's dried grain (RDGG) was prepared as shown in Table 12 below and fermented under the same conditions as in the above example. Useful microorganisms include Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces , which have excellent sugar decomposition ability. cerevisiae MNRU0927 (KACC 93265P) was selected, and the enzymes were alkaline protease and Maxa Feed Wheat (Xylanase, Amylase, Cellulase) from Lumis Co. (India) was used.

Materials Weight(w/v) Rice ginseng meal (RDGG): Soybean meal (SBM) A (RDGG 50 : SBM 50) 1 kg (500 g : 500 g) B (RDGG 60 : SBM 40) 1 kg (600 g : 400 g) C (RDGG 70:SBM 30) 1 kg (700 g : 300 g) D (RDGG 80 : SBM 20) 1 kg (800 g : 200 g) E (RDGG 90:SBM 10) 1 kg (900 g : 100 g) Alkaline Protease + Maxa Feed Wheat (Xylanase, Amylase, Cellulase) Lumis Co. (India) 5g Bacillus coagulans NRR1207 5% 50mL Saccharomyces cerevisiae MNRU0927 5% 50mL Sterile distilled water (40℃) 45% (400 g = 500 g - 100 mL of microbial culture)

Rice ethanol and soybean meal were mixed in the same ratio as Table 12, and 450 g of sterilized distilled water adjusted to 40°C and 5% useful microorganism starter were added to 1 kg of sample and cultured at 34°C for 48 hours.

pH

The pH of the mixed fermentation product according to the ratio of rice ethanol and soybean meal was measured using a pH meter (Mettler Toledo Seven Easy pH meter, Sigma-Aldrich, USA) and is shown in Figure 10. As shown in Figure 10, the soybean meal 50:50 mixing ratio (sample A) decreased over fermentation time as the lactic acid bacteria grew well. On the contrary, the pH of sample E (rice ethanol 90: soybean meal 10) increased slightly. Sample E had a low pH from the beginning of fermentation, which is because rice ethanol itself is a by-product of producing alcohol and produces a lot of acid during fermentation. It is believed that this is because it was done.

Probiotic count measurement

Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces in the mixed fermentation product according to the ratio of rice ethanol and soybean meal cerevisiae The viable bacterial count of MNRU0927 (KACC93265P) was measured using the same method as the above-mentioned viable bacterial count measurement method and is shown in FIG. 11. As shown in Figure 11 , the number of useful microorganisms in sample A (50 rice ethanol: 50 soybean meal) was the highest at 1.04 × 10 ¹⁰ at 24 hours after fermentation and at 48 hours after fermentation. It decreased slightly to 2.17×10 ⁹ . S. serevisiae MNRU0927 showed the highest E (rice 90: large 10) at 4.89×10 ⁷ at 24 hours of fermentation and 2.2×10 ⁸ at 48 hours of fermentation.

protein breakdown

To investigate the degree of protein degradation in the mixed fermentation product according to the ratio of rice ethanol and soybean meal, SDS-PAGE electrophoresis was performed in the same manner as above. Mix 5 g of rice ethanol and soybean meal mixed fermentation sample with 100 mL extraction buffer (0.05M Tris-HCl buffer pH=8.2), extract in an ultrasonic bath at 40°C for 70 minutes while vortexing at 10-minute intervals, and then centrifuge at 9,000 rpm for 30 minutes. The supernatant was dialyzed using a 1,000 dalton dialysis membrane, dried using a freeze dryer (IlShin Lab Co. Ltd, Siheung, Korea), and then dissolved in buffer and subjected to SDS-PAGE. The results are shown in Figure 12. It was. As shown in Figure 12, compared to Control (0 hours), the proteins of Sample A and Sample B were almost completely decomposed, and as the ratio of rice ethanol was increased, protein hydrolysis did not work well. It is believed that the pH of rice wine marmalade is too low and the activity of proteolytic enzymes (alkaline protease, etc.) has decreased.

glycolysis

HPLC and TLC were performed according to the above method to investigate the degree of sugar decomposition of the mixed fermentation product according to the ratio of rice ethanol and soybean meal. 5 g of mixed fermentation sample according to the ratio of rice ethanol and soybean meal was mixed with 100 mL extraction buffer (0.05M Tris-HCl buffer pH=8.2) and extracted for 70 minutes while vortexing at 10-minute intervals in an ultrasonic water bath at 40°C at 9,000 rpm. After centrifugation for 30 minutes, the supernatant was dialyzed using a 1,000 dalton dialysis membrane and then dried using a freeze dryer (IlShin Lab Co. Ltd, Siheung, Korea). HPLC and TLC were performed on the dried sample, and the results are shown in Figures 13 to 15. As shown in Figures 13 and 14 (HPLC), the anti-nutritional factors stachyose and raffinose were greatly reduced in Sample A, with stachyose from 7.02 mg/g at 0 hours of fermentation to 1.76 mg/g at 48 hours, and raffinose from 13.23 mg. /g decreased from 3.13 mg/g, melibiose decreased from 9.39 mg/g to 0, and fructose 2.09 mg/g was produced at 48 hours of fermentation. It is believed that as the ratio of rice wine marmalade increases, the sugar hydrolyzing ability decreases. In sample E, the amounts of all sugars except glucose did not change significantly.

In addition, as shown in Figure 15 (TLC), stachyose and raffinose were decomposed in samples A and B, which was consistent with the HPLC results in which stachyose and raffinose were not decomposed as the amount of rice ethanol was increased.

Organic acid production

The degree of organic acid production in the mixed fermentation product according to the ratio of rice ethanol and soybean meal was investigated. 1 g of mixed fermentation of rice ethanol and soybean meal was mixed with 5 mL of 10% ethanol, stirred at 200 rpm for 60 minutes at 50°C, and then centrifuged at 4,000 rpm for 10 minutes. To remove proteins, the supernatant was mixed with Acetonitrile in a 2:3 ratio, evaporated for 12 hours, and then centrifuged at 9,000 rpm for 20 min using a centrifuge (Mega 17R, Hanil Science Industrial, Korea). The separated supernatant was taken, filtered using a 0.2 ㎛ membrane filter, and carbohydrates were analyzed using an HPLC system (Waters 2695 Separations Module, Waters Associates, USA). The detector used was a Refractive Index Detector (Waters Associates, USA), the column used SUPELCOGEL C-610H (38cm×7.8mm, Sigma-Aldrich Co., USA), the temperature of the column was maintained at 40°C, and the mobile phase was analyzed for 30 minutes at a flow rate of 0.5 mL/min. It is shown in Figure 16. Quantitative analysis was performed using the analysis program Empower (Waters Associates, USA), and standard materials were purchased from Sigma-Aldrich Co. (USA) and used for analysis. As shown in Figure 16, as a result of organic acid analysis of the mixed fermentation of rice ethanol and soybean meal, the amount of lactic acid and acetic acid in sample A (50% soybean meal: 50% rice ethanol) significantly increased as the fermentation time increased. In sample B (40% soybean meal: 60% rice ethanol), the amount of lactic acid and acetic acid increased significantly, similar to sample A. In sample C (30% soybean meal: 70% rice ethanol), acetic acid increased rapidly as the fermentation time increased, and in sample D (20% soybean meal: 80% rice ethanol), lactic acid slightly increased and acetic acid increased rapidly. In sample E (10% soybean meal: 90% rice ethanol), lactic acid slightly decreased and acetic acid slightly increased. Citric acid and tartaric acid did not change significantly throughout the entire fermentation process compared to the control.

antibacterial activity

The antibacterial effect of the mixed fermentation product according to the ratio of rice ethanol and soybean meal was confirmed. Listeria monocytogenes ( Listeria monocytogenes) monocytogenes 530) strain was used to measure antibacterial activity using the paper disc agar diffusion method. 1 g of mixed fermentation product with different ratios of rice ethanol and soybean meal was mixed with 5 mL of distilled water, reacted in a shaking incubator at 50°C for 60 minutes, filtered using a mesh net, and the filtrate was centrifuged at 13,000 rpm for 15 minutes to obtain the supernatant. It was filtered using a 0.2 um syringe filter (Satorius Stedin Biotech, Germany) and then used as a sample. Listeria monocytogenes 530 was inoculated into LB broth and cultured at 30°C for 24 hours, then inoculated onto LB agar. A sterilized paper disc (8 mm) was placed on the medium, 100 μl of sample was injected, and cultured for 48 hours to determine whether a clear zone was formed. was confirmed and shown in Figure 17. As shown in Figure 17, the fermentation sample was Listeria monocytogenes 530 ( aeruginosa ) had an antibacterial effect, and sample A had the highest antibacterial effect. From Sample B to Sample E (the larger the mixing ratio of rice ethanol), the antibacterial effect decreased, and Sample E (10% soybean meal: 90% rice ethanol) showed no effect. The clear zone size created in the fermentation sample in Sample A was 0.12 mm at 24 hours and 0.35 mm at 48 hours, in Sample B it was 0.1 mm at 24 hours and 0.26 mm at 48 hours, and in Sample C it was 0.07 mm at 24 hours, 48 hours was 0.14 mm, and no clear zone occurred in samples D and E.

antioxidant activity

The antioxidant effect of the mixed fermentation product according to the ratio of rice ethanol and soybean meal was confirmed. 1 g of the fermented soybean meal and fermented rice ethanol mixture was mixed with 5 mL of 10% ethanol, mixed with extraction buffer (0.05M Tris-HCl buffer pH=8.2) at 50°C, and extracted for 70 minutes while vortexing at 10-minute intervals in an ultrasonic water bath at 40°C. Then it was centrifuged at 9,000 rpm for 30 minutes. After mixing 0.2 mL of the supernatant with 0.8 mL of 0.2 mM DPPH, an ABTS radical scavenging ability test reagent, the mixture was left in the dark at 25°C for 10 minutes, and the absorbance was measured at 490 nm using an ELIZA reader, as shown in Figure 18. As shown in Figure 18, RDDG 50% + SBM 50% (A) was 29.93% at 0 hours of fermentation, while it rapidly increased with fermentation time to 50.33% at 24 hours and 58.44% at 48 hours. Compared to 0 hours, the fermentation mixture increased by about 100% at 48 hours. RDDG 60%+SBM 40% (B) was 36.61.% at 0 hours of fermentation, while it was 60.25% at 24 hours and 62.15% at 48 hours, which increased rapidly as fermentation time elapsed, compared to 0 hours at 48 hours. The fermentation mixture increased by approximately 85%. RDDG 70%+SBM 30% (C) was 36.51% at 0 hours of fermentation, while it increased rapidly with fermentation time to 60.53% at 24 hours and 57.20% at 48 hours, with 48 hours of fermentation compared to 0 hours. The mixture increased by approximately 78%. RDDG 80%+SBM 20% (D) was 45.28% at 0 hours of fermentation, while it was 60.25% at 24 hours and 62.54% at 48 hours, which increased rapidly with the passage of fermentation time, compared to 0 hours at 48 hours of fermentation. The mixture increased by approximately 69%. RDDG 90%+SBM 10% (E) was 54.53% at 0 hours of fermentation, while it was 65.20% at 24 hours and 63.68% at 48 hours, which increased rapidly with the passage of fermentation time, compared to 0 hours at 48 hours of fermentation. The mixture increased by approximately 45.75%. As the fermented rice ethanol content increased, the antioxidant effect increased in a concentration-dependent manner at 0 hours, which was believed to be influenced by the metabolites of the rice ethanol. On the other hand, the rate of increase in antioxidant activity decreased after 48 hours of fermentation. This is believed to be due to the increase in the number of viable lactic acid bacteria and yeast in the mixed fermentation in which the amount of soybean meal was increased and the amount of rice ethanol was reduced, and the metabolites produced accordingly affected the antioxidant effect.

5 types of treatment (A: RDDG 50%+SBM 50%, B: RDDG 60%+SBM 40%, C: RDDG 70%+SBM 30%, D: RDDG 80%+SBM 20%, E: RDDG 90%+ Among SBM 10%), RDDG 50% + SBM 50% (A) showed the highest antioxidant effect with an increase of about 100% compared to 0 hours.

Constituent amino acids and decomposability Protein (DIP), non-degradable protein( UIP ) measurement

The constituent amino acids, degradable protein (DIP), and non-degradable protein (UIP) of the mixed fermentation product with the rice ethanol and soybean meal ratio of 50:50 were measured. The conditions for analyzing the amino acid content are shown in Tables 13 and 14. The constituent amino acids were analyzed for 18 components using the ninhydrin post column reaction method using ion exchange chromatography by modifying the AOAC method. For amino acid composition analysis, 0.2 g of the sample was placed in a decomposition tube, 10 ml of 6 N HCl was added, nitrogen gas was injected, and the sample was hydrolyzed at 110°C for 24 hours. The filtrate was concentrated using a vacuum concentrator, adjusted to 50 ml with 0.2M sodium citrate buffer, and then filtered through a 0.45 μm nylon syringe filter and used as an analysis sample. Performic acid oxidation method was used for sulfur-containing series methionine and cysteine, and alkaline hydrolysis method was used for tryptophan.

Items Conditions machine Hitachi, L-8900 column Ion exchange column (#2622PH column)
4.6×60mm mobile phase Buffer set (PH-SET KANTO) detect UV/Vis (440, 570㎚) flow rate Ninhydrin; 0.35㎖/min
Buffer ; 0.40㎖/min Injection amount 20 μL

Items Conditions machine Waters Isocratic 600 pump, 486 UV/VIS detector column CAPCELL C18 (4.6x250㎜) mobile phase 0.0085M Sodium acetate:methanol = 95:5 detect UV 280㎚ flow rate 1.0㎖/min Injection amount 20㎕

Degradable protein (DIP) and undegradable protein (UIP) were measured using the same method as described above, and the constituent amino acids and degradable protein (DIP) and undegradable protein (UIP) results are shown in Table 15.

Item RDDG+SBM (0 h, %DM) RDDG+SBM (48 h, %DM) Crude protein 52.66% Crude protein 44.83% DIP 19.97 48.26 UIP 18.04 28.68 Aspartic acid 1.68 2.54 Threonine 0.67 1.02 Serine 0.87 1.30 Glutamic acid 2.81 4.37 Proline 0.88 1.33 Glycine 0.71 1.08 Alanine 0.80 1.23 Valine 0.80 1.18 Isoleucine 0.69 1.00 Leucine 1.35 1.98 Tyrosine 0.51 0.77 Phenylalanin 0.85 1.27 Histidine 0.39 0.61 Lysine 0.86 1.28 Arginine 1.03 1.55 Cysteine 0.25 0.50 Methionine 0.15 0.38 Tryptophan 0.17 0.24

Crude protein is composed of non-protein nitrogen (NPN) such as urea, amino acids, and amines, and true protein, and true protein is composed of degradable protein (DIP) and non-degradable protein (UIP). As shown in Table 15, comparing the control group (fermentation 0 h) and the test group (fermentation 48 h), the crude protein content decreased by 7.83% from 52.66% based on dry matter content in the control group to 44.83% in the test group, which is due to the fermentation process. This is thought to be due to the decomposition of heavy proteins and use by useful microorganisms as a source of nutrients. Degradable intake protein in the crude protein was 19.97% in the non-fermented product as a control, but it increased by 28.29% to 48.26% in the 48-hour fermented product as a test group, which is “NPN + soluble true protein + protein decomposed during fermentation.” Because it is composed of, it was produced at very high levels in a mixture of fermented soybean meal and fermented rice ethanol. In addition, undegradable intake protein increased by 10.64% to 28.68% in the 48-hour fermented sample as the test group, compared to 18.04% in the non-fermented sample as the control group. This is because it consists of “some components of NPN + unsoluble true protein.” It was produced at a very high level in the mixed fermentation of rice ethanol and soybean meal. In other words, it was confirmed that the metabolic protein, which is the sum of degradable and non-degradable proteins, which was 38.01% in the crude protein of the non-fermented sample, more than doubled to 76.94% after fermentation.

Additionally, it was confirmed that all 18 types of amino acids analyzed were increased in the test group (fermentation 48 h) compared to the control group. This is thought to be due to the increase in the amount of amino acids due to protein decomposition by proteolytic enzymes, which are metabolites of useful microorganisms used during fermentation.

Through the above results, Bacillus coagulans NRR1207 (KACC92114P) and Saccharomyces cerevisiae of the present invention The mixed fermentation of rice ethanol and soybean meal using MNRU0927 (KACC93265P) mixed strain and enzyme composition significantly reduces the antibacterial factors of soybean meal and increases the ratio of constituent amino acids and degradable protein (DIP) to non-degradable protein (UIP) to supplement livestock farming. It was confirmed that it was suitable as feed.

Rural Development Administration National Academy of Agricultural Sciences Microbial Bank (KACC) KACC92114P 20151215 Rural Development Administration National Academy of Agricultural Sciences Microbial Bank (KACC) KACC93265P 20160930

Claims (9)

A supplementary feed composition for livestock containing a fermented product cultured by inoculating a mixture of rice ethanol and soybean meal with mixed strains of Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae ( Saccharomyces cerevisiae MNRU0927, KACC 93265P) In
The supplementary feed composition for livestock is fermented by further comprising an enzyme agent including alkaline protease, xylanase, amylase, and cellulase in the rice ethanol and soybean meal mixture. A supplementary feed composition for livestock, characterized in that it is water. delete According to paragraph 1,
The rice ethanol and soybean meal mixture is a supplementary feed composition for livestock, characterized in that rice ethanol and soybean meal are mixed at a weight ratio of 50 to 90:10 to 50. According to paragraph 1,
The supplementary feed composition for livestock is added to the rice ethanol and soybean meal mixture.
Mixed strains of Bacillus coagulans NRR1207, KACC92114P) and Saccharomyces cerevisiae MNRU0927, KACC 93265P;
Enzyme agents including alkaline protease, xylanase, amylase, and cellulase; A supplementary feed composition for livestock, characterized in that it is a fermented product administered and cultured for 24 to 48 hours. According to paragraph 1,
The supplementary feed composition for livestock is a mixture of rice ethanol and soybean meal containing Bacillus coagulans (Bacillus coagulans NRR1207, KACC92114P) 2×10 ⁷ ~2×10 ⁸ cell/g and Saccharomyces cerevisiae ( Saccharomyces cerevisiae MNRU0927, KACC 93265P) ) Supplementary feed composition for livestock, characterized in that 1×10 ⁶ ~1×10 ⁷ cell/g was inoculated and cultured. According to paragraph 1,
The supplementary feed composition for livestock farming is characterized in that the ratio of degradable protein (DIP) and undegradable intake protein (UIP) in crude protein is 75% or more. According to paragraph 1,
The supplementary feed composition for livestock is characterized in that it has antibacterial activity against Listeria monocytogenes 530. According to paragraph 1,
The supplementary feed composition for livestock farming is characterized in that it has antioxidant activity. For 100 parts by weight of the rice ethanol and soybean meal mixture, 0.5 parts by weight of alkaline protease, 0.2 parts by weight of mixed enzyme of xylanase, amylase and cellulase, Bacillus coagulans NRR1207, KACC92114P and Saccharomyces cerevisiae. cerevisiae Mixing 10 parts by weight of the mixed strain (MNRU0927, KACC 93265P) and 40 parts by weight of water;
Incubating the mixture at 34-37°C for 24-48 hours; and
A method of producing a supplementary feed composition for livestock, comprising: collecting and drying the culture.