WO2023014072A1

WO2023014072A1 - Composition for increasing protein bypass rate and method for increasing protein bypass rate

Info

Publication number: WO2023014072A1
Application number: PCT/KR2022/011452
Authority: WO
Inventors: 이종화; 이혁기
Original assignee: 주식회사 피드업
Priority date: 2021-08-03
Filing date: 2022-08-03
Publication date: 2023-02-09
Also published as: KR20230020372A; KR102572213B1

Abstract

The present invention relates to a composition for increasing a rumen bypass rate of proteins, wherein the composition comprises a fermentate of soybeans fermented with fermenting bacteria including lactic acid bacteria and yeasts, and thus can undergo intensive digestive absorption into the small intestine after passing through the rumen of ruminants under a suppressed condition of digestive absorption in the rumen, and a composition and a method for reducing the greenhouse gas of ruminants, which can reduce methane production within the rumen of ruminants, discharge of nitrogenous excretion, and emission of nitrogen gas such as nitrous oxide and ammonia.

Description

Composition for increasing protein bypass rate and method for increasing protein bypass rate

The present invention relates to a composition for increasing the bypass rate with increased availability of proteins and carbohydrates, and a method for increasing the bypass rate of protein.

In the livestock sector, in order to achieve the goal of carbon neutrality by 2050, surplus nitrogen reduction is in progress through the distribution and expansion of feed to reduce environmental burden. In order to improve cattle productivity, the importance of supplying feed ingredients with high protein utilization is very high. To increase body weight and increase meat mass in Korean cattle, and to increase milk yield and To improve milk protein, the importance of supplying feed with high protein utilization is increasing. In addition, the importance of supplying feed raw materials that can reduce nitrogen manure excretion due to high protein utilization is increasing.

Ruminants use nitrogen from feed relatively inefficiently compared to other economic animals. For example, North American and Northern European dairy cows use nitrogen as milk on average 25% and 28%, respectively. The efficiency is lower, with only about 14% efficiency in weight gain from nitrogen in the feed. Eventually, nitrogen that is not transferred to animal body tissues or milk is released into feces and urine, contaminating soil, rivers, and air. Therefore, the supply of appropriate feed protein to ruminants and the technology to increase the nitrogen utilization rate of ruminants are important in terms of not only reducing feed costs but also reducing environmental pollution. Feed protein of rumen livestock can be classified into rumen degradable protein (RDP), rumen undegradable protein (RUP), and non-protein nitrogen (NPN).

Ammonia (NH3) emitted from livestock farming is a major cause of air and water pollution. Ammonia affects eutrophication, soil acidification, and fine dust increase. Ammonia is oxidized by soil microorganisms and converted into nitrous oxide (N2O), and nitrous oxide is one of the strongest greenhouse gases, and its effect on global warming is about 310 times higher than that of the same amount of carbon dioxide.

One example of the present invention is to provide a composition for increasing the rumen bypass rate of proteins, in which digestion and absorption can occur intensively in the small intestine after passing through the rumen in a state where digestion and absorption in the rumen of ruminants are inhibited. It is to do.

Another example of the present invention is to reduce methane production in the rumen of ruminants, reduce nitrogen manure excretion, and reduce emissions of nitrogen such as nitrous oxide and ammonia, which can reduce greenhouse gas emissions of ruminants, It is to provide a composition for reducing greenhouse gases in ruminants and a method for reducing greenhouse gases in ruminants.

One example of the present invention relates to a composition for increasing the rumen bypass rate of protein, including a fermented product of legumes fermented with fermenting bacteria including lactic acid bacteria and yeast.

Another example of the present invention relates to a feed containing the rumen bypass increase rate composition of protein according to an embodiment of the present invention.

Another example of the present invention is fermenting legumes with fermentation bacteria including lactic acid bacteria and yeast; And it relates to a method for producing a composition for increasing the rumen bypass rate of protein, including the step of heat-treating the fermented legumes, a method for producing a feed containing the composition, or a method for increasing the rumen bypass rate of protein.

Another example of the present invention relates to the use of fermented legumes fermented with fermentation bacteria including lactic acid bacteria and yeast for increasing the rumen bypass rate of protein.

Another example of the present invention relates to the use of fermented legumes fermented with fermenting bacteria including lactic acid bacteria and yeast for the preparation of a composition for increasing the rumen bypass rate of protein.

Another example of the present invention relates to a composition for reducing greenhouse gases in ruminants, including the rumen bypass increase rate composition of protein according to an embodiment of the present invention.

Another embodiment of the present invention relates to a method for reducing greenhouse gas emissions of ruminants, comprising the step of administering a rumen bypass increase rate composition of protein according to an embodiment of the present invention.

Another example of the present invention relates to the use of fermented legumes fermented with fermenting bacteria including lactic acid bacteria and yeast for reducing greenhouse gas emissions of ruminants.

Hereinafter, the present invention will be described in more detail.

One example of the present invention relates to a composition capable of improving productivity and reducing greenhouse gas emissions of ruminants due to high rumen bypass rate and high absorption rate in the small intestine. The composition according to one embodiment of the present invention includes a fermented product of soybeans fermented with a fermenting strain, and the fermenting strain may be a fermenting strain for forming an amine group of a protein and/or a fermenting strain for accelerating a Maillard reaction.

One example of the present invention relates to a bypass protein in which digestion and absorption in the small intestine can occur intensively after passage in a state in which digestion and absorption in the rumen of a ruminant animal are inhibited, and a feed containing the same. Specifically, fructose in legumes is fermented using lactic acid bacteria and yeast to make active sugar, suppress digestion and absorption in the rumen, and heat-treat the fermented product so that digestion and absorption are maximized in the small intestine. The bypass protein manufacturing process according to an example of the present invention causes a component change of legumes through fermentation without a separate additive to induce a Maillard reaction to effectively produce bypass protein.

As used herein, “bypass” means passing through the rumen of a ruminant without being decomposed and being decomposed and absorbed in the intestine, and there are bypass fat and bypass protein products. Specifically, ruminants get most of the amino acids they need from proteins that have passed through the rumen, that is, bypass proteins, but in the case of legumes, which are the main raw materials of feed, especially soybean meal, which accounts for 60% of the world's vegetable protein feed ingredients, most of them come from the rumen. Decomposition takes place and only about 25% is bypassed, resulting in inefficiency. In the present specification, "bypass protein" means protein that has passed through the rumen, and "bypass rate" means the ratio of bypass protein among ingested proteins.

In one example of the present invention, the fermenting bacteria may include lactic acid bacteria and yeast. When legumes are fermented using lactic acid bacteria and yeast, both species are facultative anaerobe microorganisms, so they are suitable for anaerobic solid-phase fermentation, and have the advantage that co-culture is possible. In addition, when fermenting using lactic acid bacteria and yeast together, there is an advantage that the fermentation time can be greatly reduced by about half, and although the fermentation time is shortened, the protein content of the fermented product is greatly increased, and the protein is cut to form an amine group, It also has the advantage of generating reducing sugars. In addition, when lactic acid bacteria and yeast are used in combination as fermenting bacteria, there is an effect that the protein content of the fermented product is higher and the bypass rate is higher than in the case of fermentation with lactic acid bacteria alone. Specifically, as shown in the examples herein, the fermented product has high rumen non-degradable protein (RUP) and small intestine absorbable protein content due to the combined use of lactic acid bacteria and yeast, blood urea nitrogen concentration, methane gas production, fecal nitrogen excretion, and ammonia production , and the amount of nitrous oxide produced was significantly low, proving the synergistic effect by the combined use of lactic acid bacteria and yeast.

Among the components of legumes, for example, soybean meal, fructose represented by stachyose and raffinose and various carbohydrates are included, most of which are water-soluble and are a kind of anti-nutritional factor that inhibits digestion. The present invention can use lactic acid bacteria and yeast that effectively metabolize anti-nutritional factors that are irrelevant to the nutritional properties of legumes and rather degrade the performance as a feed. That is, it is possible to provide a composition for increasing the bypass rate that significantly reduces the fermentation time compared to fermentation of lactic acid bacteria alone and has a high protein content in the final product.

In particular, lactic acid bacteria are known to have excellent metabolizing ability for fructose. An enzyme called alpha-(1,6)galactosidase, which is commonly found in lactic acid bacteria, is contained in large amounts in soybean meal such as raffinose and stachyose. It is an enzyme that breaks down fructose. The content of raffinose and stachyose in soybean meal is more than 6% based on dry matter content. When one raffinose molecule is decomposed by alpha-galactosidase, it is decomposed into one molecule of each of three monosaccharides, such as galactose, fructose, and glucose. Meanwhile, stachyose is broken down into two galactose, one fructose, and one glucose molecules. In this way, the Maillard reaction can be promoted more effectively if lactic acid bacteria capable of secreting a large amount of alpha-galacosidase enzyme and converting fructose in soybean meal into more activated monosaccharides are used.

For example, the fermenting bacteria according to one embodiment of the present invention includes lactic acid bacteria and yeast, and the ratio of the number of cells of the lactic acid bacteria and the yeast is 100:1 to 1:100, 100:1 to 1:50, and 100:1 to 1:1, 50:1 to 1:50, 50:1 to 1:1, 20:1 to 1:20, 20:1 to 1:15, 20:1 to 1:10, 20:1 to 1 :5, 20:1 to 1:1, 20:1 to 5:1, 20:1 to 10:1, 15:1 to 1:20, 15:1 to 1:15, 15:1 to 1:10 , 15:1 to 1:5, 15:1 to 1:1, 15:1 to 5:1, 15:1 to 10:1, 10:1 to 1:20, 10:1 to 1:15, 10 :1 to 1:10, 10:1 to 1:5, 10:1 to 1:1, 10:1 to 5:1, 10:1 to 6:1, 10:1 to 7:1, 10:1 to 8:1, or 10:1 to 9:1.

For example, lactic acid bacteria inoculated into the fermentation raw material may be 10 ⁶ ~ 10 ⁸ cfu/g, and yeast may be 10 ⁵ ~ 10 ⁷ cfu/g.

The lactic acid bacteria are Enterococcus genus strains, Lactobacillus genus strains, Weissella genus strains, Leuconostoc genus strains, Streptococcus genus strains, and Lactococcus It may be one or more selected from the group consisting of genus strains.

The Enterococcus genus lactic acid bacteria include Enterococcus faecium and Enterococcus faecalis, and the Lactobacillus genus lactic acid bacteria include Lactobacillus plantarum, or Lactobacillus Lactobacillus acidophilus, etc., and lactic acid bacteria of the Weissella genus include Weissella koreensis or Weissella cibaria. In addition, lactic acid bacteria of the genus Leuconostoc include Leuconostoc citreum or Leuconostoc mesenteroides, and lactic acid bacteria of the genus Streptococcus include Streptococcus thermophilus thermophillus), and Lactococcus lactis is a lactic acid bacterium of the genus Lactococcus.

The yeast may be at least one selected from the group consisting of strains of the genus Saccharomyces, strains of the genus Tolula, strains of the genus Xanthophyllomyces, and strains of the genus Pichia. Yeasts of the genus Saccharomyces include Saccharomyces cerevisiae and Saccharomyces uvarum, yeasts of the genus Xanthophyllomyces include Xanthophyllomyces dendrorhous, yeasts of the genus Pichia include Pichia farinosa, and Torula yeasts include Cyberlindnera jadinii.

The fermented product included in the composition according to an embodiment of the present invention has a moisture content of 30 to 60% by weight, 30 to 50% by weight, 30 to 45% by weight, 30 to 40% by weight, 35 to 60% by weight, 35% by weight of the medium to 50% by weight, 35 to 45% by weight, 35 to 40% by weight, 40 to 60% by weight, 40 to 50% by weight, or 40 to 45% by weight.

The fermented product included in the composition according to an example of the present invention is 20 to 40 ℃, 20 to 35 ℃, 20 to 30 ℃, 25 to 40 ℃, 25 to 35 ℃, 25 to 30 ℃, 30 to 40 ℃, 30 ℃ to 35°C, or 35 to 40°C.

The fermented product may have a high rumen undegradable protein (RUP) content, and/or a high intestinal absorption protein content. Accordingly, the composition according to one embodiment of the present invention may be a composition for increasing rumen bypass of proteins and/or a composition for increasing absorption of proteins in the small intestine.

The crude protein content of the fermented product may be 48.5% by weight or more, 49% by weight or more, 50% by weight or more, or 51% by weight or more. The crude protein content may be the crude protein content based on 8% by weight of water.

The rumen non-degradable protein refers to a protein that is not degraded in the rumen, for example, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, It may be a protein that does not degrade for 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. The rumen undegradable protein (RUP) content of the fermented product is 74 wt% or more, 75 wt% or more, 76 wt% or more, 77 wt% or more, 78 wt% based on 100 wt% of the total crude protein content of the fermented product. It may be more than 79% by weight, or more than 80% by weight.

The small intestine absorption protein or small intestine degradation protein is a protein that is not degraded in the rumen but is degraded and absorbed in the small intestine, for example, pepsin treatment for 1 hour, and / or small intestine enzyme solution 18 hours, 19 hours, 20 hours, 21 hours , proteins that are digested after 22, 23, or 24 hours of treatment. The small intestine enzyme solution may contain at least one of trypsin, chymotrypsin, amylase and lipase. The small intestine absorbed protein content of the fermented product may be 69% by weight or more, 70% by weight or more, 71% by weight or more, or 72% by weight or more based on 100% by weight of the total crude protein content of the fermented product.

The fermented product may be heat treated after fermentation. Specifically, heat treatment is required to create a brownish bond between activated sugars and proteins produced as a result of lactic acid fermentation, and through heat treatment, a Maillard reaction occurs in which amino groups of amino acids and carbonyl groups of reducing sugars condense in the fermented product. At this time, when more than necessary heat is applied, the bypass rate is high, but the digestibility in the small intestine is low, and when a small amount of heat is applied, the bypass rate is low. On the other hand, amino acids such as lysine, which are very important components for the quality of feed, are highly likely to be destroyed in the heat treatment process because they are relatively bad for heat. Therefore, heat treatment of the fermented product is a sensitive process that must be accurately controlled so that the minimum temperature and time required for the browning reaction are used. For the heat treatment of the fermented product, a device such as an extruder or an expeller or a device to which a roasting method is applied may be used. In roasting, which is the most commonly used method, devices that directly heat soybean meal dispersed by fins installed inside a cylinder-shaped reactor with flames are mainly used. In the present embodiment, a roasting heat treatment is used as an example, and a batch type rotary drum is used as a mechanical device for accurate control of temperature and processing time.

The heat treatment is 70 to 150 ℃, 70 to 145 ℃, 70 to 140 ℃, 70 to 130 ℃, 80 to 150 ℃, 80 to 145 ℃, 80 to 140 ℃, 80 to 130 ℃, 90 to 150 ℃, 90 to It may be performed at a temperature of 145 ° C, 90 to 140 ° C, or 90 to 130 ° C.

The heat treatment is 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, 5 to 35 minutes, 5 to 30 minutes, 5 to 25 minutes, 5 to 20 minutes, 5 to 15 minutes, 5 to 10 minutes after fermentation, 10 to 60 minutes, 10 to 50 minutes, 10 to 40 minutes, 10 to 35 minutes, 10 to 30 minutes, 10 to 25 minutes, 10 to 20 minutes, 10 to 15 minutes, 15 to 60 minutes, 15 to 50 minutes, 15 to 40 minutes, 15 to 35 minutes, 15 to 30 minutes, 15 to 25 minutes, 15 to 20 minutes, 20 to 60 minutes, 20 to 50 minutes, 20 to 40 minutes, 20 to 35 minutes, 20 to 30 minutes, 20 to 25 minutes, 25 to 60 minutes, 25 to 50 minutes, 25 to 40 minutes, 25 to 35 minutes, 25 to 30 minutes, 30 to 60 minutes, 30 to 50 minutes, 30 to 40 minutes, or 30 to 35 minutes may be performing

For example, the heat treatment may be performed at 90 to 100° C. for 10 to 60 minutes. In the present embodiment, heat treatment was performed at a temperature as low as possible in order to avoid problems that may occur due to excessive heat treatment.

The composition according to one embodiment of the present invention may include a fermented product produced by a fermentation process using lactic acid bacteria and yeast and a heat treatment process without adding additives such as enzymes or sugars at all. The manufacturing process of the fermented product is more economical and efficient than the conventional bypass soybean meal protein manufacturing process, and at the same time, it is possible to produce products with excellent efficiency and performance. As a method for this, (1) select lactic acid bacteria and yeast that effectively decompose fructose and protein in soybean meal during metabolism, (2) establish conditions for solid phase fermentation in which fructose is maximally decomposed into activated sugar form, ( 3) It includes a high-temperature heat treatment process that maximizes the Maillard reaction between soybean meal activated sugars and proteins. Specifically, the bypass ratio of proteins can be increased by promoting the Maillard reaction through yeast fermentation with lactic acid bacteria that effectively decompose fructose and proteins in soybean meal, such as Raffinose and Stachyose, into activated sugars in the metabolic process. The method for increasing the rumen bypass rate according to an example of the present invention uses only a fermentation process and heat treatment without additives such as enzymes or sugars, so it is possible to secure economic feasibility, and RUP (Rumen Undegradable Protein) is 74% or more in the small intestine of RUP. The digestion and absorption rate of the product is over 69%, and the product is competitive.

In one example of the present invention, legumes may include at least one selected from the group consisting of soybeans, rapeseeds, palm, peas, kidney beans, corn, mung beans, red beans, soybeans, lupines, and oil meal thereof there is. Soybean meal, a representative feed ingredient in legumes, is a very important protein source, accounting for 60% of the world's vegetable protein feed ingredients based on production volume. It has the downside of not being able to. Cattle get most of the amino acids they need from microbial proteins made in the fermentation process in the rumen or proteins that have passed through the rumen, that is, bypass proteins. Various methods of processing soybean meal have been studied to increase the bypass ratio in ruminants. Heat treatment or treatment with lignosulfonate or formaldehyde is mainly used. Recently, a method of coating with vegetable oil has also been developed. . Among them, the process of heat-treating soybean meal is the most tried, which uses the Maillard reaction (Maillard reaction) that occurs during heating of proteins and sugars in soybean meal. That is, when the carbonyl group of sugar and the amino group of amino acid are combined by heat, most of the digestion occurs in the small intestine without being attacked by microbial enzymes that cause digestion in the rumen. A method of applying heat at a high temperature for a short time using an extruder or adding reactive sugar such as xylose and then heat-treating is used. A process for accelerating the Maillard reaction by heat treatment after treatment has also been developed.

On the other hand, processes in which soybean meal is treated with enzymes or fermented with microorganisms are industrialized to remove anti-nutritional factors such as trypsin inhibitors by non-heating methods. In the latter case, fungi such as Aspergillus or Bacillus It is common to use bacteria such as Some use an economical fermentation process using an anaerobic process using lactic acid bacteria. In the case of lactic acid bacteria, it is known that they actively decompose oligo-saccharides, one of the anti-nutritional factors of soybean meal, during the metabolic process.

Accordingly, the inventors of the present invention developed a method that can increase the bypass ratio of proteins by promoting the Maillard reaction through fermentation with yeast and lactic acid bacteria that effectively decompose fructose and proteins in soybean meal, such as Raffinose and Stachyose, into activated sugars and peptides in the metabolic process. process was developed.

Another example of the present invention relates to a feed, including a composition for increasing the rumen bypass rate of protein according to an embodiment of the present invention. The feed may be feed for ruminants.

In the present specification, “ruminant” means an animal having a ruminant stomach among animals belonging to the order Utilus, and includes, for example, one or more species selected from the group consisting of Bovine, Camel, Deer, Deer, and Giraffe. it may be

The bovine family may include, for example, at least one selected from the group consisting of Korean cattle, dairy cows, yaks, bison, buffalo, impala, gaur, water buffalo, goats, sheep, goats, and antelopes.

The Camelidae may include, for example, one or more species selected from the group consisting of dromedaries, bactrian camels, llamas, alpacas, guanacos, and vicuñas.

The deer family may include, for example, at least one selected from the group consisting of deer, reindeer, roe deer, elk, elk, and moose.

The deer family may include, for example, one or more species selected from the group consisting of deer and mouse deer.

The giraffe family may include, for example, at least one member selected from the group consisting of giraffes and okapis.

Another example of the present invention is fermenting legumes with fermentation bacteria including lactic acid bacteria and yeast; And a method for producing a composition for increasing the rumen bypass rate of protein, comprising heat-treating the fermented legumes, or a method for producing a feed containing the composition.

Another example of the present invention is fermenting legumes with fermentation bacteria including lactic acid bacteria and yeast; And it relates to a method for increasing the rumen bypass rate of protein, comprising the step of heat-treating the fermented legumes.

Another example of the present invention relates to a composition for reducing greenhouse gases in ruminants, including a fermented product of legumes fermented with fermenting bacteria including lactic acid bacteria and yeast. In the present example, as a result of administering the fermented product obtained by fermenting legumes using a combination of lactic acid bacteria and yeast to ruminants, blood urea nitrogen decreased, methane gas and ammonia production decreased, and manure nitrogen excretion decreased. Therefore, the composition according to one embodiment of the present invention and the feed containing the same have an effect of reducing greenhouse gas emissions of ruminants. Accordingly, another embodiment of the present invention relates to a method for reducing greenhouse gas emissions of ruminants, comprising administering the composition to the ruminants. The greenhouse gas may be methane gas and/or nitrous oxide.

A composition according to an example of the present invention may induce one or more of the following properties (1) to (6):

(1) Reduction in blood urea nitrogen concentration, for example, fasting blood urea nitrogen concentration compared to the control group is 90% or less, 80% or less, or 75% or less (the control group is a group administered with unfermented legumes or legumes fermented with lactic acid bacteria alone) can),

(2) Decreased methane production in the rumen, e.g., less than 95%, less than 90%, less than 85%, less than 80%, less than 70%, less than 60%, less than 55%, less than 50% compared to the control group , or 45% or less (the control group may be a group treated with unfermented legumes, fermented legumes for unit animals, or legumes fermented with lactic acid bacteria alone),

(3) Reduction of ammonia production in the rumen, for example, ammonia concentration compared to the control group is 99% or less, 98% or less, 97% or less, 96% or less, 95.5% or less, 90% or less, 85% or less, 80% or less, 70% or less, 60% or less, or 50% or less (the control group may be a group treated with non-fermented legumes, fermented legumes for unit animals, or legumes fermented with lactic acid bacteria alone),

(4) decrease in manure nitrogen excretion, for example, 99% or less, 98% or less, 97% or less, or 96% or less in manure nitrogen excretion compared to the control group (the control group is not administered with the composition according to an embodiment of the present invention) may be military),

(5) Increase in retained nitrogen, for example, residual nitrogen is greater than 1 times, 1.05 times or more, or 1.1 times or more compared to the control group (the control group may be a group not administered with the composition according to an embodiment of the present invention) there is).

(6) Improve the efficiency of nitrogen utilization in the body, for example, increase the efficiency of nitrogen utilization in the body by 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.01% or more, 3.02% or more, or 3.03% improvement of 3.04% or more, 3.05% or more, or 3.06% or more (the control group may be a group administered with a feed not containing the composition according to an embodiment of the present invention).

The nitrogen utilization rate in the body may be calculated by Equation 1 below:

[Equation 1]

Nitrogen utilization efficiency in the body (%) = (Nitrogen utilization rate compared to the control group) / (Nitrogen intake) X 100

The present invention can contribute to reducing manure odor by reducing ammonia gas (NH3) and reducing greenhouse gases such as nitrous oxide (N2O) by reducing methane and nitrogen excretion in the rumen of ruminants, and increasing protein utilization in ruminants to increase productivity and nitrogen reduction can solve the greenhouse gas problem.

The present invention provides rumen bypass protein to increase the small intestine absorption rate, reduce ammonia generated by excessive protein decomposition by ruminant microorganisms, and reduce the amount of nitrogen excreted as manure, thereby minimizing the use of unnecessary protein sources and increasing productivity, nationally. It is possible to provide feed (low-protein, low-temperature gas feed) that reduces environmental burden to help solve environmental problems.

The composition for increasing the bypass rate according to an embodiment of the present invention can reduce greenhouse gases by reducing the amount of methane generated in the rumen and the amount of nitrogen in feces.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

실시예 1: 유산균 및 효모 병용에 의한 콩류 발효물 제조Example 1: Preparation of legume fermented product by lactic acid bacteria and yeast combination

(1) 유산균 및 효모 병용에 의한 콩류 발효(1) Fermentation of legumes by the combination of lactic acid bacteria and yeast

Legumes were fermented using a combination of lactic acid bacteria and yeast as fermenting bacteria, and the crude protein content of the fermented product was measured. In this Example, soybean meal was used as an example of legumes, and the type of lactic acid bacteria used for fermentation of soybean meal can be used without limitation, but Enterococcus faecium or Lactococcus lactis 1.0x10 ⁶ As a result of comparing the number of bacteria 24 hours after inoculation of /g, E. faecium was 6.5 x 10 ⁸ /g and Lactococcus lactis was 2.5x 10 ⁷ /g. When using Enterococcus strains, more efficient soybean meal fermentation is possible did Thus, in this Example, fermentation was performed using Enterococcus pesium as a lactic acid bacterium.

Fermentation was carried out for 24 hours by adding 500 g of seed liquid (moisture content of 480 g) to 1000 g of soybean meal (moisture content of 120 g) in an incubator pre-adjusted at 30°C or 35°C. Cells used for fermentation were 10 ⁷ cfu/g of lactic acid bacteria (Enterococcus faecium KCTC13566BP) and 10 ⁶ cfu/g of yeast (Saccharomyces cerevisiae).

(2) 발효 콩류의 열처리(2) Heat treatment of fermented legumes

For the heat treatment of the fermented product, a DMD dryer (double mixing dryer) type dryer is installed. It has a continuous structure in which double paddles are installed inside the drum, the temperature is constantly controlled with a double jacket, and raw materials are continuously injected and discharged. The fermented product after heat treatment was treated from 10 minutes to less than 60 minutes by adjusting the heat treatment time according to the product temperature (130 ℃ 10 minutes; 120 ℃ 15 minutes; 110 ℃ 20 minutes; 90 ℃ 35 minutes). Thereafter, the temperature was limited to 60 ° C. and dried until the moisture content was 12% or less to obtain a fermented soybean product (sample name: Example 1) by a combination of lactic acid bacteria and yeast.

비교예 1: 유산균 단독에 의한 콩류 발효물 제조Comparative Example 1: Production of legume fermented product by lactic acid bacteria alone

Fermentation was performed in the same manner as in (1) of Example 1, except that 10 ⁷ cfu/g of lactic acid bacteria (Enterococcus faecium KCTC13566BP) was used alone as the fermenting bacteria. The obtained fermented product was heat-treated and dried in the same manner as in Example 1 (2) to obtain a fermented soybean product by lactic acid bacteria alone (Sample name: Comparative Example 1).

실시예 2: pH 및 조단백 함량 측정Example 2: Measurement of pH and crude protein content

After completion of fermentation and before heat treatment, twice the amount of distilled water by weight was added to the fermented product, stirred for 5 minutes, and then the pH was measured, the crude protein content was measured by the Kjeldahl method, and the water content of the sample was measured by 8 weight It was corrected on a % basis and shown in Table 1.

구분division	발효온도(℃)Fermentation temperature (℃)	pHpH	조단백질(%) (수분 8중량% 기준)Crude protein (%) (based on 8% by weight of water)
실시예 1Example 1	3030	5.65.6	50.350.3
실시예 1Example 1	3535	5.45.4	51.051.0
비교예 1Comparative Example 1	3030	5.55.5	47.847.8
비교예 1Comparative Example 1	3535	5.65.6	48.248.2

As shown in Table 1, it was confirmed that lactic acid was produced by fermentation of lactic acid bacteria and the pH of the medium was lowered. % was high.

실시예 3: RUP 및 소장 흡수 단백질 함량 측정Example 3: Measurement of RUP and small intestine absorbed protein content

For RUP (rumen non-degradable protein) analysis of each sample, in vitro analysis using a protease solution was performed. Intestinal absorbed proteins were analyzed by digestion in pepsin (pH 2) for 1 hour and enzyme solution (pH 9.0) supplemented with trypsin, chymotrypsin, amylase and lipase for 24 hours. Table 2 shows the RUP content and small intestine absorbed protein content based on 100% by weight of total crude protein (CP) content (% CP).

구분division	발효온도(℃)Fermentation temperature (℃)	RUP (%CP)RUP (%CP)	소장흡수 단백질 (%CP)Small Intestine Absorbed Protein (%CP)
실시예 1Example 1	3030	7979	72.272.2
실시예 1Example 1	3535	8080	72.172.1
비교예 1Comparative Example 1	3030	7272	68.168.1
비교예 1Comparative Example 1	3535	7373	68.368.3

As shown in Table 2, the fermented product of Example 1 fermented using lactic acid bacteria and yeast in combination showed an increase in RUP content by about 10% and an absorption of about 4% more in the small intestine after heat treatment. Therefore, it was confirmed that the fermented product of Example 1 fermented by using lactic acid bacteria and yeast in combination did not decompose in the rumen of ruminants, increased the bypass rate, and had a high absorption rate in the small intestine.

실시예 4. 바이패스 콩류의 혈중 요소 질소 감소 효과Example 4. Blood urea nitrogen reduction effect of bypass legumes

The fermented soybean product of Example 1, the fermented soybean product of Comparative Example 1, or soybean meal (Sajo No. 5-more than 46% crude protein, Sajo Daerim) was administered to 6 animals in each group (aged 40 to 45 months) at an amount of 350 g/kg each for 6 weeks. After feeding Holstein castration), blood was collected from a vein and blood urea nitrogen (BUN) concentration and total protein concentration were analyzed and shown in Table 3.

항목item	비교예 1Comparative Example 1		대두박soybean meal		실시예 1Example 1
항목item	공복spandrel	3시간3 hours	공복spandrel	3시간3 hours	공복spandrel	3시간3 hours
BUN (mg/dL)BUN (mg/dL)	10.6010.60	12.4812.48	11.1511.15	15.1615.16	7.837.83	9.839.83
Total protein (g/dL)Total protein (g/dL)	8.928.92	10.3510.35	9.259.25	9.509.50	9.489.48	9.849.84

As shown in Table 3, in the case of the fermented legumes of Example 1, the concentration of BUN 3 hours after administration was 9.83 mg/dL, which is lower than the fasting BUN concentration of the fermented legumes or soybean meal feed of Comparative Example 1. showed Compared to the BUN concentration 3 hours after administration, it was 2.65 mg/dL lower than that of the fermented soybean product of Comparative Example 1 (about 21.2% decrease) and 5.33 mg/dL lower than that of the soybean meal feeder (about 35.2% decrease). Therefore, the fermented soybean product according to one embodiment of the present invention is widely used as a metabolic protein, and the effect of reducing the nitrogen source discharged into manure was remarkably excellent.

실시예 5. 바이패스 콩류의 메탄 대사 개선 효과Example 5. Effect of improving methane metabolism in bypass legumes

(1) 실험재료 준비(1) Preparation of experimental materials

The rumen gastric juice was collected using 3 Korean cattle equipped with rumen fistulas reared at the animal breeding farm attached to Sunchon National University. After filtering to remove feed particles, the temperature was maintained at 39° C. using a constant temperature water bath.

(2) 메탄가스 감소 효과(2) Methane gas reduction effect

The fermented soybean product of Example 1, the fermented soybean product of Comparative Example 1, fermented soybean meal and soybean meal for unit animals were prepared, each sample was put into a 160mm serum bottle at a total volume of 1.0% (DM), and gastric juice and buffer solution were mixed. 100 ml was dispensed at a ratio of 1:3, covered with a butyl rubber stopper and an aluminum cap, and cultured at 100 rpm in a shaking incubator at 39 ° C. During the entire process of dilution and filtration of gastric juice, gastric juice was bubbling with N ₂ to maintain the gastric juice in an anaerobic state. Fermented soybean meal for unit animals is for animals with a single stomach, such as pigs or chickens. It is a product used as a substitute for fish meal by removing anti-nutritional factors from soybean meal to increase digestion and absorption. It is a product manufactured by drying under conditions that prevent the Mayer reaction by not exceeding ℃.

Table 4 shows the total amount of gas produced for each sample. As shown in Table 4, the fermented soybean meal for monogastric animals showed a significantly high gas production amount, and the fermented product of Example 1 showed a significantly low gas production amount because the decrease in protein degradation rate in the rumen inhibited ruminal microbial fermentation. It is presumed to have been seen. In Table 4, SEM is the standard error of the mean, and different subscripts for a, b, and c in the same row show significant differences (p<0.05).

시간hour	무 처리구radish treatment	대두박soybean meal	단위동물용 발효대두박for unit animals fermented soybean meal	비교예1Comparative Example 1	실시예 1Example 1	SEMSEM	P valueP value
3h3h	7.000^b ^7.000b	12.333^a 12.333 ^a	11.400^a 11.400 ^a	12.153^a 12.153 ^a	12.233^a 12.233 ^a	0.4780.478	0.00010.0001
6h6h	9.000^c 9.000 ^c.	23.500^a 23.500 ^a	22.833^a ^22.833a	20.867^b ^20.867b	20.667^b ^20.667b	0.5710.571	<.0001<.0001
12h12h	11.500^d 11.500 ^d	33.167^a 33.167 ^a	30.333^b § ^30.333b	28.920^c ^28.920c	27.000^c 27.000 ^c.	0.4460.446	<.0001<.0001
24h24h	19.333^d ^19.333d	53.333^b ^53.333b	62.333^a 62.333 ^a	44.358^c ^44.358c	42.333^c ^42.333c	1.5861.586	<.0001<.0001

In order to analyze the methane generated over time in the rumen fermentation process, it was collected using a 6ml vacuum tube (vacutainer). Methane analysis was performed using gas chromatography (Gas Chromatography, Agilent technolgies HP 5890). The detector uses TCD, column Carboxen 1006PLOT capillary column 30m×0.53mm (Supelco), and the analysis conditions are oven temperature 35℃, injection part temperature 200℃, detector temperature of the injection port part 200℃, and mobile phase gas N ₂ at 3ml/min. It was flowed and analyzed the amount generated by culture time.

Table 5 shows the amount of methane produced through rumen fermentation in mM/ml concentration. In terms of methane production, the fermented product of Example 1 was significantly the lowest in 12-hour and 24-hour culture. Specifically, the bypass fermented product of Example 1 showed methane reduction effects of 35.9% and 45.5%, respectively, compared to soybean meal in 12-hour and 24-hour cultivation. The fermented product of Comparative Example 1 showed methane reduction effects of 31.2% and 38.8%, respectively, compared to soybean meal after 12 hours and 24 hours of cultivation. In the case of fermented soybean meal for monogamous animals, compared to soybean meal, it showed a reduction effect of 18.2% in 12-hour culture, but increased by 30.8% in 24-hour culture. In Table 5, SEM is the standard error of the mean, and different subscripts for a, b, and c in the same row show significant differences (p<0.05).

TimeTime	무 처리구radish treatment	대두박soybean meal	단위동물용 발효대두박for unit animals fermented soybean meal	비교예1Comparative Example 1	실시예1Example 1	SEMSEM	P valueP value
3h3h	0.4730.473	2.3872.387	5.5405.540	2.1202.120	1.9201.920	0.8370.837	0.2820.282
6h6h	0.513^b ^0.513b	3.603^ab ^3.603ab	5.170^a 5.170 ^a	4.673^a 4.673 ^a	4.433^a 4.433 ^a	0.8420.842	0.0880.088
12h12h	3.523^c ^3.523c	10.670^a 10.670 ^a	8.727^ab 8.727 ^ab	7.340^b ^7.340b	6.840^b ^6.840b	0.5520.552	0.0010.001
24h24h	4.177^d ^4.177d	13.995^ab 13.995 ^ab	18.307^a 18.307 ^a	8.562^cd 8.562 ^CD	7.623^cd 7.623 ^CD	1.4411.441	0.0010.001

(3) 암모니아 농도분석(3) Ammonia concentration analysis

Cultivated in the same manner as in (2) above, and ammonia concentration analysis was calculated by measuring OD (absorbance) at a wavelength of 630 nm of a spectrophotometer (Manufactured by Biochrom Ltd, CB40Fj, England) according to the method of Chaney and Marbach (1962). Specifically, NH ₃ reacts with hypochlorite in an alkaline solution to form NH ₂ Cl, which sequentially reacts with phenol to produce a blue indophenol dye. If sodium nitroprusside or MnS04 as a catalyst is added during the reaction to speed up the reaction, the reaction can usually be completed within 10 minutes. The indophenol produced in this reaction is stable and is a good example of an analytical method through a chemical reaction. Indophenol absorbs photons at a wavelength of 630 nm.

Ammonia concentrations through rumen fermentation are shown in Table 6 as mM/L concentrations. As shown in Table 6, the concentration of ammonia (NH ₃ -N) in the 24-hour culture period was significantly higher in the fermented soybean meal for unit animals (p<0.05). The bypass fermented soybean meal of Example 1 had a significantly low ammonia concentration, showing that protein degradation did not occur in the rumen and digestion proceeded in the small intestine. In Table 6, SEM is the standard error of the mean, and different subscripts for a, b, and c in the same row show significant differences (p<0.05).

TimeTime	무 처리구radish treatment	대두박soybean meal	단위동물용 발효대두박Fermented soybean meal for unit animals	비교예1Comparative Example 1	실시예1Example 1	SEMSEM	P valueP value
3h3h	14.06214.062	12.19712.197	16.03916.039	13.12113.121	13.01213.012	1.1301.130	0.3690.369
6h6h	15.75815.758	12.49812.498	16.65716.657	15.65815.658	15.29815.298	1.3791.379	0.3920.392
12h12h	19.33719.337	14.36814.368	20.53220.532	19.42119.421	17.43317.433	1.3911.391	0.1200.120
24h24h	23.277^b ^23.277b	24.235^b ^24.235b	38.527^a 38.527 ^a	20.128^b ^20.128b	19.128^b ^19.128b	2.8802.880	0.0130.013

실시예 6. 바이패스 콩류의 질소 대사 개선 효과Example 6. Effect of improving nitrogen metabolism in bypass legumes

Eight 14.8-month-old Hanwoo castrated cattle were prepared and placed in 8 cages, divided into two groups, a control group and an experimental group, and fed with general feed according to Table 7 or feed containing the fermented product of Example 1. Water was freely fed in a drinking tank, and roughages were fed at 08:00 and 14:00 every day. Individual feeding was carried out through feed baskets while hanging on the stanchion, and the remaining amount was measured using a scale at the end of each feeding. Concentrated feed was freely fed using a DeLaval automatic feeder, and individual intake was measured and recorded daily using Delpro software. Weight measurement was performed before breakfast feed, and all feces and urine were collected at 9:00 every morning to record the amount of excretion. Samples of 10% for feces and 5% for urine were collected and stored frozen. After 5 days, samples from each sample were mixed, collected for analysis, and stored frozen. After the end of the 24-day experiment, after washout for 2 days to verify the experiment result, the control group and the experimental group were changed to each other and the same experiment was performed.

성분ingredient	대조군 (Low RUP)Control (Low RUP)	실험군 (High RUP)Experimental group (High RUP)
Flaked cornFlaked corn	22.0022.00	27.0027.00
Corn gluten feedCorn gluten feed	19.0019.00	7.007.00
Fine wheatFine wheat	17.4017.40	15.0015.00
Corn DDGSCorn DDGS	12.0012.00	17.0017.00
Palm Kernel MealPalm Kernel Meal	8.408.40	20.0020.00
Ground alfalfa pelletGround alfalfa pellet	7.007.00	0.000.00
MolassesMolasses	4.504.50	5.005.00
Soy hullsSoy hulls	2.802.80	0.000.00
LimestoneLimestone	2.302.30	2.502.50
Fine cornFine corn	1.101.10	1.101.10
Rice branRice bran	1.001.00	1.001.00
Mineral premixMineral premix	0.850.85	0.850.85
UreaUrea	0.600.60	0.000.00
Extruded linseedExtruded linseed	0.500.50	0.000.00
GlycerinGlycerin	0.300.30	0.300.30
SaltSalt	0.200.20	0.200.20
Vitamin premixVitamin premix	0.050.05	0.050.05
실시예 1Example 1	0.000.00	3.003.00
TotalTotal	100.00100.00	100.00100.00

RUP = rumen-undegradable protein; DDGS = distillers dried grains with solubles.

The results of analysis of nitrogen intake and manure excretion are shown in Table 8. Nitrogen intake through feed was the same at 160 g/d in both control and experimental groups, but excretion of manure nitrogen was 5 g/d lower in the experimental group than in the control group, and nitrogen utilization (N retention), which means accumulation as protein in the body, was also tested It was 4.9 g/d higher in the old one. Therefore, the bypass protein according to an example of the present invention shows that the efficiency of nitrogen utilization in the body is improved by about 3.063%. From 6 months to 30 months of breeding, about 953 kg of crude protein is required, which is 153 kg when converted into nitrogen, 76.5 kg when converted into nitrous oxide, and 23.7 tons when converted into carbon dioxide. 3.063% of 23.7 tons is 726 kg, and through the increase in the utilization efficiency of nitrogen by the bypass protein according to an example of the present invention, it has the effect of reducing about 726 kg CO ₂ per head until the 30-month slaughter of Hanwoo geese, in Korea When applied to 1.27 million heads of Korean cattle under breeding, it has the effect of reducing 922,000 tons of CO ₂ .

Therefore, the bypass protein according to an example of the present invention reduces the nitrogen content excreted in manure and reduces the generation of ammonia gas and nitrous oxide gas, thereby having an advantageous effect on eco-friendly livestock farming and increasing the nitrogen utilization rate of ruminants. The effect of improving nitrogen utilization efficiency and reducing environmental burden was confirmed.

구분division	대조군 (Low RUP)Control (Low RUP)	실험군 (High RUP)Experimental group (High RUP)	SEMSEM	p value p value
질소 섭취량 (g/d)Nitrogen intake (g/d)	160160	160160	1.641.64	0.770.77
질소 배설량 (g/d)Nitrogen excretion (g/d)	119119	114114	2.212.21	0.100.10
잔류 질소 (retained N) (g/d)Retained N (g/d)	40.940.9	45.845.8	2.422.42	0.0970.097

Claims

A composition for increasing the rumen bypass rate of protein, comprising a fermented product of legumes fermented with fermenting bacteria including lactic acid bacteria and yeast.
The composition according to claim 1, wherein the lactic acid bacteria and/or yeast form an amine group of proteins contained in the legumes and/or promote Maillard reaction of the legumes.
The composition of claim 1, wherein the rumen undegradable protein (RUP) content of the fermented product is 74% by weight or more based on 100% by weight of the total crude protein content of the fermented product.
The composition according to claim 1, wherein the composition is a composition for increasing absorption of protein in the small intestine, and the content of protein absorbed in the small intestine of the fermented product is 69% by weight or more based on 100% by weight of the total crude protein content of the fermented product.
The composition according to claim 1, wherein the fermented product has a crude protein content of 49% by weight or more.
The composition of claim 1, wherein the fermented product is heat-treated after fermentation.
The composition of claim 1, wherein the fermented product is heat-treated at a temperature of 80 to 150 ° C after fermentation.
The composition of claim 1, wherein the fermented product is heat-treated for 5 to 60 minutes after fermentation.
The composition of claim 1, wherein the fermented product is fermented at a temperature of 20 to 40 °C.
The method of claim 1, wherein the legumes include at least one selected from the group consisting of soybean, rapeseed, palm, pea, kidney bean, corn, mung bean, red bean, soybean, lupine, and oil meal thereof phosphorus, composition.
The method of claim 1, wherein the lactic acid bacteria are Enterococcus genus strains, Lactobacillus genus strains, Weissella genus strains, Leuconostoc genus strains, Streptococcus genus strains, And at least one member selected from the group consisting of Lactococcus genus strains, composition.
The method of claim 1, wherein the yeast is one selected from the group consisting of Saccharomyces genus strain, Tolula genus strain, Xanthophyllomyces genus strain, and Pichia genus strain. A composition that is more than species.
A feed comprising the composition according to any one of claims 1 to 12.
The feed according to claim 13, wherein the feed is feed for ruminants.
The feed according to claim 14, wherein the ruminant includes at least one member selected from the group consisting of bovine, camel, deer, deer, and giraffe.
Fermenting legumes with fermenting bacteria including lactic acid bacteria and yeast; and

Including the step of heat-treating the fermented legumes,

Method for producing a composition for increasing the rumen bypass rate of protein.
Fermenting legumes with fermenting bacteria including lactic acid bacteria and yeast; and

Including the step of heat-treating the fermented legumes,

A method for increasing the rumen bypass rate of proteins.
A composition for reducing greenhouse gas emissions of ruminants, comprising the composition according to any one of claims 1 to 12.
19. The composition of claim 18, wherein the composition induces one or more of the following properties (1) to (6) when administered to a ruminant:

(1) Decreased blood urea nitrogen concentration;

(2) reduced methane production in the rumen;

(3) reduction of ammonia production in the rumen;

(4) reduction of manure nitrogen excretion;

(5) an increase in retained nitrogen, and

(6) Improving the utilization efficiency of nitrogen in the body.
A method for reducing greenhouse gas emissions of ruminants, comprising administering a composition according to any one of claims 1 to 12 to ruminants.
21. The method of claim 20, wherein the greenhouse gas is methane gas and/or nitrous oxide.