Novel lactic acid bacteria, lactic acid bacteria preparation for silage preparation, and silage preparation method

Abstract

To provide a novel lactic acid bacterium and a lactic acid bacterium preparation useful for silage preparation which can be raised even under a strong acidic condition by adding formic acid in silage preparation, and are capable of preparing preferable silage.SOLUTION: Lactobacillus paracasei that produces exopolysaccharide in which primary constituent sugar of exopolysaccharide are galactose, mannose and glucose.SELECTED DRAWING: Figure 13

Description

[Technical field]

[0001] The present invention relates to a lactic acid bacteria preparation for preparing silage, and a method for preparing silage using the same. [Background technology]

[0002] Silage is made by fermenting feed crops in a silo or other facility. In general, it refers to fermented green-cut grass (grass silage or grass silage). In other cases, it may be called silage by adding the name of the grain used as the raw material (e.g. corn silage, rice straw silage, etc.). For dairy cows, especially lactating cows, silage is an important staple food, and its quality has a major impact on dairy farm management. Lactic acid bacteria preparations containing lactic acid bacteria as the main ingredient are used to prepare good quality silage. Generally, lactic acid bacteria preparations and lactic acid bacteria additives for silage preparation contain lactic acid bacteria that have a strong ability to grow in pasture. Spraying this lactic acid bacteria on pasture and creating anaerobic conditions promotes lactic acid fermentation of the silage, suppresses the growth of butyric acid bacteria, mold, and yeast that cause abnormal fermentation, and enables the silage to be stored for a long time. On the other hand, when silage is made from grass or feed crops that are high in moisture and contain a lot of bacteria, harvested during periods of bad weather and lots of rain, the growth rate of the bacteria exceeds the growth rate of the added lactic acid bacteria. In this case, butyric acid fermentation often takes precedence over lactic acid fermentation, resulting in a decline in the quality of the silage. Silage that has been given priority over butyric acid fermentation produces a peculiar foul odor and may lose its value as feed because cattle will not eat it. A prior art for silage preparation using nisin-producing lactic acid bacteria that inhibit butyric acid fermentation is described in Patent Document 1. However, even if such lactic acid bacteria are used for silage preparation, it is difficult to inhibit butyric acid fermentation when silage is made from pasture grass or feed crops that have become highly moist due to rainfall, and only low-quality silage can be obtained. For this reason, various proposals have been made regarding silage preparation methods and lactic acid bacteria strains to be used.

[0003] In order to suppress the microorganisms that cause butyric acid fermentation, a method of spraying a strong acid on the silage raw material in advance is known. This method involves directly adding a strong acid, for example, formic acid, to the raw grass at 0.3 to 0.4% by mass to lower the pH and suppress the causative bacteria of butyric acid fermentation (Non-Patent Document 1). However, because it is a strong acid, there is a risk of burns and corrosion to machines and silos, and since it also stops the respiration of plants immediately after addition, there are problems such as oxygen remaining in the silo in pre-dried materials, which increases the risk of secondary fermentation. In addition, since some of the lactic acid bacteria added to promote the above-mentioned preferable lactic acid fermentation are killed by formic acid, it takes a long time to complete the silage. Furthermore, it is empirically known that yeast cannot be suppressed by adding formic acid. Therefore, if formic acid is only added, abnormal fermentation or secondary fermentation may occur, resulting in silage of reduced quality.

[0004] Patent Document 2 discloses a technique for preparing silage by adding 1,500 g of formic acid-resistant Lactobacillus plantarum and formate salts per ton of feed. It is described that the addition of Lactobacillus plantarum and formate salts was effective in reducing yeast and mold in the silage. The freeze-dried preparation of Lactobacillus plantarum was then incubated in an aqueous solution of sodium formate (30%, pH 6.25) for at least 4 hours and 10 minutes. 9 It has been reported that it maintains CFU / mL.

[0005] The invention described in Patent Document 3 aims to suppress harmful microorganisms by using the bacteriocin-producing lactic acid bacteria Enterococcus faecium NAS62 strain (NITE P-781) as a silage lactic acid bacterium, by utilizing the antibacterial action of the bacteriocin.

[0006] Patent Document 4 proposes an additive for preparing silage, which contains homofermentative lactic acid bacteria capable of preparing reuterin having antifungal activity, glycerol, and vitamin B12. Reuterin exhibits antibacterial properties against gram-positive bacteria, gram-negative bacteria, yeast, and mold. Patent Documents 3 and 4 are all inventions in which abnormal fermentation and secondary fermentation are suppressed by antibacterial substances produced by lactic acid bacteria. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 5931064 [Patent Document 2] Special Publication No. 05-047183 [Patent Document 3] JP 2011-041474 A [Patent Document 4] JP 2008-035728 A [Non-patent literature]

[0008] [Non-Patent Document 1] Northern Animal Journal Vol. 57, 243-248, 2013 [Non-Patent Document 2] Applied and Environmental Microbiology ,February 2017 Volume 83 Issue 3 e02702-16 Summary of the Invention [Problem to be solved by the invention]

[0009] Various lactic acid bacteria strains for silage preparation and lactic acid bacteria preparations using them have been proposed. However, the fact remains that none of them are necessarily satisfactory in terms of suppressing butyric acid fermentation, which has a significant effect on silage quality. In the future, inhibition of butyric acid bacteria growth and inhibition of secondary fermentation of silage may become major problems in feed preparation and total mixed ration (TMR) using silage as a raw material. Adding formic acid to keep the pH low is recommended as the first choice to suppress butyric acid fermentation and secondary fermentation of silage, but it is also well known that formic acid has the risk of killing lactic acid bacteria suitable for silage fermentation. An objective of the present invention is to provide novel lactic acid bacteria and a lactic acid bacteria preparation useful for silage preparation that can grow even under the strong acidic conditions caused by the addition of formic acid and enable the preparation of favorable silage. [Means for solving the problem]

[0010] The present invention comprises the following configuration. (1) Lactobacillus paracasei produces an exopolysaccharide whose main sugar components are galactose, mannose, and glucose. (2) Lactobacillus paracasei according to (1), in which the amount of exopolysaccharide produced is at least 83 mg per gram of dry mass of the cells. (3) Lactobacillus paracasei according to (1) or (2), wherein the exopolysaccharide does not contain any amino sugar as a constituent sugar and contains 50 to 60 mass% of glucose, 20 to 30 mass% of mannose, and 10 to 20 mass% of galactose based on the mass of the exopolysaccharide. (4) Lactobacillus paracasei according to any one of (1) to (3), which is resistant to growth inhibition by formic acid, ethyl alcohol and hydrogen peroxide. (5) Lactobacillus paracasei according to any one of (1) to (4) which can grow in an MRS medium containing one or more of 0.2% by mass of formic acid, 11% by mass of ethyl alcohol, and 150 ppm of hydrogen peroxide. (6) Lactobacillus paracasei according to any one of (1) to (5), wherein the Lactobacillus paracasei is SBS-0011 strain (NITE BP-03013). (7) A lactic acid bacteria preparation for preparing silage, comprising Lactobacillus paracasei according to any one of (1) to (6). (8) The lactic acid bacteria preparation for silage preparation according to (7) above, in any form selected from the group consisting of a liquid, a granule, and a powder. (9) A method for preparing silage, comprising spraying a preparation containing Lactobacillus paracasei according to any one of (1) to (6) onto silage raw materials and then fermenting the silage raw materials. (10) At least 1 × 10 per silage ingredient. 4 A method for preparing silage, comprising adding Lactobacillus paracasei according to any one of (1) to (6) at a concentration of CFU / g and fermenting the silage. (11) A method for preparing silage, comprising spraying an aqueous solution of formic acid containing at least 0.1% by mass of formic acid per silage raw material, and then adding Lactobacillus paracasei according to any one of (1) to (6) or a lactic acid bacteria preparation for silage preparation containing Lactobacillus paracasei according to any one of (1) to (6) to ferment the silage. (12) Silage, characterized by being fermented using Lactobacillus paracasei according to any one of (1) to (6). Effect of the Invention

[0011] The novel lactic acid bacteria provided by the present invention are lactic acid bacteria belonging to Lactobacillus paracasei that produce exopolysaccharides whose main constituent sugars are galactose, mannose, and glucose. Lactobacillus paracasei that produces the above-mentioned polysaccharides outside the bacteria has not been known, and is provided for the first time by the present invention. Moreover, the lactic acid bacteria Lactobacillus paracasei of the present invention has growth resistance against high concentrations of formic acid, and can grow in a culture solution containing 11% by mass of ethyl alcohol and a culture solution containing 150 ppm of hydrogen peroxide. Such lactic acid bacteria have not been provided so far. This novel lactic acid bacteria can grow even under strongly acidic conditions that suppress the growth of butyric acid bacteria when formic acid is sprayed on the grass used as a raw material for silage preparation. Therefore, even if formic acid is sprayed after the Lactobacillus paracasei of the present invention, the lactic acid bacteria will rapidly grow and promote lactic acid fermentation, suppressing butyric acid fermentation, and further consume and reduce the remaining oxygen, so the yeast will decrease and secondary fermentation will be suppressed in the silage obtained after fermentation is completed. As a result, the silage will satisfy the palatability of cows. In addition, the lactic acid bacteria preparation for silage preparation of the present invention quickly promotes lactic acid fermentation of silage raw materials even in any adverse environment. Even if abnormal fermentation occurs, the proliferation of the lactic acid bacteria of the present invention promotes a decrease in pH, and the abnormal fermentation is suppressed at an early stage. Furthermore, since it can be used in combination with formic acid, it can be used with silage raw materials such as rain-soaked grass and straw contaminated with cow manure that are at risk of butyric acid fermentation, making it possible to obtain homogeneous silage. In addition, since abnormal fermentation such as butyric acid fermentation does not occur, it is highly safe and improves the feeding ability of cows. It is also expected to increase milk production in dairy cows and weight gain in fattening cows. In particular, good silage can be obtained in hot and humid regions such as Japan and Southeast Asia. [Brief description of the drawings]

[0012] [Figure 1] 1 is a graph showing the results of a resistance test against hydrogen peroxide. [Diagram 2] 1 is a graph showing the results of a growth test using an MRS culture medium containing hydrogen peroxide and ethanol. [Diagram 3] 1 is a graph showing the results of a growth test using an MRS culture medium containing hydrogen peroxide and formic acid. [Figure 4] 1 is a graph showing the results of a growth test using an MRS culture medium containing 50 to 150 ppm of hydrogen peroxide, 0.1% formic acid, and 5 to 11% ethanol. [Diagram 5] 1 is a graph showing the results of a growth test using an MRS culture medium containing 50 to 150 ppm of hydrogen peroxide, 0.2% formic acid, and 5 to 11% ethanol. [Figure 6] 1 is a graph showing the results of a growth test using an MRS culture medium containing 50 to 150 ppm of hydrogen peroxide, 0.3% formic acid, and 5 to 11% ethanol. [Figure 7] These are images of the culture medium after culturing Lactobacillus paracasei SBS-0003 and Lactobacillus paracasei SBS-0011 strains overnight in a jar fermenter (hereafter referred to as "jar"). These are images of jars in which these lactic acid bacteria were cultured under the same conditions (left: SBS-0003 strain, right: SBS-0011 strain). The image on the right shows that the culture medium in which SBS-0011 strain was cultured has become cloudy due to polysaccharide production. [Figure 8] 1 is a graph showing the results of measuring the organic acid content and pH in quackgrass silage prepared using quackgrass and quackgrass silage prepared by adding the SBS-0011 strain obtained by the present invention and 0.2% formic acid. [Figure 9] 1 is a graph showing the results of measuring the organic acid content and pH of silage prepared by adding 0.3% formic acid to reed canary grass, silage prepared by adding SBS-0003 strain, which is a parent strain of the lactic acid bacteria strain of the present invention, and 0.2% formic acid to reed canary grass, and silage prepared by adding SBS-0011 strain obtained according to the present invention and 0.2% formic acid. [Figure 10] FIG. 1 is a graph showing the results of measuring the organic acid content and pH of reed canary grass silage prepared by adding a commercially available lactic acid bacteria preparation for silage, Cymaster AC, the SBS-0011 strain obtained according to the present invention, and 0.1% formic acid or 0.2% formic acid to reed canary grass. [Figure 11] FIG. 1 is a graph showing the results of measuring the organic acid content and pH of reed canary grass silage prepared by adding a commercially available lactic acid bacteria preparation for silage, Cymaster AC, the SBS-0011 strain obtained according to the present invention, and 0.1% formic acid or 0.2% formic acid to reed canary grass contaminated with cow dung. [Figure 12] FIG. 1 is a graph showing the results of measuring the organic acid content and pH of timothy-meadow fescue silage prepared by adding a commercially available lactic acid bacteria preparation for silage, Cymaster AC, the SBS-0011 strain obtained by the present invention, and 0.1% or 0.2% formic acid to a mixed raw material of timothy and meadow fescue. [Figure 13] FIG. 1 is a graph showing the results of measuring the organic acid content and pH of timothy-meadow fescue silage prepared by adding butyric acid fermentation lead liquor prepared from butyric acid fermented silage to a mixed raw material of timothy and meadow fescue, and then adding a commercially available lactic acid bacteria preparation for silage preparation, Cymaster AC, the SBS-0011 strain obtained by the present invention, and 0.1% formic acid or 0.2% formic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In this specification, "%" means "mass %" unless otherwise specified. The novel lactic acid bacteria of the present invention are lactic acid bacteria belonging to Lactobacillus paracasei, which produce exopolysaccharides whose main constituent sugars are galactose, mannose, and glucose, and can grow in a medium containing formic acid, ethyl alcohol, and hydrogen peroxide. This Lactobacillus paracasei is a lactic acid bacterium selected from the mutant strains of Lactobacillus paracasei SBS-0003 (NITE BP-1109), which is contained in the silage lactic acid bacteria preparation Cymaster AC (manufactured by Snow Brand Seed Co., Ltd.), a commercially available known lactic acid bacteria strain, irradiated with ultraviolet light to induce UV mutation. The SBS-0003 strain is an excellent lactic acid bacterium for silage. By inducing mutations in this lactic acid bacterium using ultraviolet light, producing polysaccharides outside the cell body from the mutated lactic acid bacterium, and selecting lactic acid bacteria with formic acid resistance, the novel Lactobacillus paracasei of the present invention can be obtained.

[0014] Mutation induction and selection are carried out as follows. The novel Lactobacillus paracasei of the present invention is prepared by growing the parent strain Lactobacillus paracasei SBS-0003 strain in 5 mL of MRS medium until it reaches the logarithmic growth phase, washing the bacteria three times with PBS on a petri dish, and then exposing the bacteria to UV light for about 50 seconds directly under a UV lamp in a clean bench. The bacteria are then cultured at 37°C for 3 hours in the dark using GYP medium, and the culture solution is inoculated at 1% by mass into five GYP media containing 0.4% by mass of formic acid, and then cultured overnight at 37°C. The surviving clones are then picked up, allowing the selection of clones with high growth ability. The UV lamp used was a germicidal lamp GL15 from Toshiba Lighting & Technology Corporation. The Lactobacillus paracasei of the present invention can be identified by exhibiting the following characteristics when cultured for 48 hours using the "API 20NE Bacterial Identification Kit System" in the "API Manual Kit / ID32 API Series" microbial identification test product sold by bioMérieux Japan Co., Ltd. Colony morphology: Compared to the parent strain SBS-0003, the colonies show a rough morphology. Sugar assimilation: The sugar assimilation of the parent strain SBS-0003 and the Lactobacillus paracasei strain SBS-0011 of the present invention after 48 hours of culture is shown in Table 1.

[0015] [Table 1]

[0016] Lactobacillus paracasei SBS-0011 strain shows a phenotype that is clearly different from the wild SBS-0003 strain, such as lack of assimilation of 21st glucopyranoside and improved assimilation of 47th gluconate, compared to the parent strain SBS-0003. In addition, during jar culture in a medium containing corn steep liquor, it produces a large amount of polysaccharides in the culture solution. Therefore, the Lactobacillus paracasei of the present invention can be obtained more reliably by reselecting using the polysaccharide production in the jar culture solution as an indicator. It is clearly different from known Lactobacillus paracasei in that it produces extracellular polysaccharides whose main sugar components are galactose, mannose, and glucose during jar culture. Furthermore, no amino sugars are detected in this exopolysaccharide, and the exopolysaccharide contains 50 to 60 mass % of glucose, 20 to 30 mass % of mannose, and 10 to 20 mass % of galactose based on its mass. It can also grow in media containing high concentrations of formic acid, ethyl alcohol, and hydrogen peroxide. Based on these characteristics, it was determined that Lactobacillus paracasei SBS-0011 is a novel Lactobacillus paracasei.

[0017] The parent strain, SBS-0003, has been deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) as NITE BP-1109 (Deposit date: June 15, 2011). The lactic acid bacterium of the present invention, Lactobacillus paracasei SBS-0011 strain, has been internationally deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (Address: 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) as NITE BP-03013 (Deposit date: August 8, 2019).

[0018] Preferred culture conditions for growing the novel Lactobacillus paracasei of the present invention, which produces extracellular polysaccharides and is resistant to formic acid, ethyl alcohol, and hydrogen peroxide, may be any conditions under which normal lactic acid bacteria grow. For example, the strain may be inoculated into a grass broth medium at 0.1 to 1.0% by mass and allowed to stand at 25 to 35°C for 8 to 24 hours. Alternatively, GYP medium (manual for experiments on lactic acid bacteria, edited by Michio Ozaki (1992), published by Asakura Publishing Co.) or MRS medium (JC de Man, et al. (1960), Journal of Applied Bacteriology, Vol. 23, 130-135) can be used. There are no particular limitations on the culture conditions, but the culture can usually be carried out at pH 5.0 to 7.0, at 25 to 40°C, for 10 to 24 hours. The cultured Lactobacillus paracasei of the present invention can be added to silage materials or fermented feed materials as a lactic acid bacteria preparation for silage preparation in the form of a culture liquid or a concentrated liquid obtained by concentrating the bacteria. These liquids can also be stored as frozen products and thawed when needed. Furthermore, the cultured lactic acid bacteria can be freeze-dried, spray-dried, or fluidized bed dried together with an appropriate protective agent or base material to give a powdered lactic acid bacteria preparation for silage preparation.

[0019] The lactic acid bacteria preparation for silage preparation of the present invention may contain lactic acid bacteria used for silage preparation in addition to the Lactobacillus paracasei of the present invention that is resistant to formic acid, ethyl alcohol, and hydrogen peroxide.

[0020] Furthermore, the lactic acid bacteria preparation for silage preparation of the present invention may contain known enzymes and additives used in silage preparation, if necessary. Examples of the enzymes include cellulase and hemicellulase. Examples of the additives include stabilizers, antioxidants, emulsifiers, etc.

[0021] The lactic acid bacteria preparation for silage preparation of the present invention can obtain favorable silage when used in combination with formic acid spraying on silage raw materials that have a high moisture content and are at risk of abnormal fermentation such as butyric acid fermentation. Formic acid is sprayed at 0.1 to 0.3 mass% per silage raw material, and Lactobacillus paracasei resistant to formic acid, ethyl alcohol, and hydrogen peroxide of the present invention is sprayed at 1 x 10 4 CFU / g ~1×10 6 By adding CFU / g, it is possible to obtain favorable lactic acid fermented silage. In the case of low-moisture silage materials that are not at risk of abnormal fermentation, the Lactobacillus paracasei of the present invention, which is resistant to formic acid, ethyl alcohol, and hydrogen peroxide, is added at 1 x 10 4 CFU / g ~1×10 6 By adding lactic acid fermentation products at a concentration of CFU / g and then subjecting the products to sealed fermentation, it is possible to obtain silage with favorable lactic acid fermentation properties.

[0022] In the present invention, the lactic acid bacteria preparation for silage preparation can be used to prepare various silages and fermented feeds. The raw materials for silage and fermented feeds are not particularly limited as long as they are raw materials that are usually used as feeds, and examples of pasture and feed crops include alfalfa, clover, timothy, orchard grass, reed canary grass, quackgrass, Italian ryegrass, perennial ryegrass, tall fescue, meadow fescue, festulolium, Kentucky bluegrass, red top, guinea grass, rhodes grass, napier grass, oats, barley, rye, sorghum, sudan grass, barley, corn, and feed rice. Food manufacturing by-products include beer lees, tofu lees, tea lees, shochu lees, whiskey lees, beet pulp, bagasse, coffee lees, juice lees, kale lees, and starch lees. Agricultural by-products include rice straw, wheat straw, and non-standard vegetables. It can also be added to fermented TMR, which is prepared by mixing food manufacturing by-products such as soybean lees and malt grains, agricultural by-products, hay, concentrated feed, vitamin and mineral preparations and silage. It is desirable to adjust the moisture content of these silage and fermented feed materials to 40 to 90 mass % before use.

[0023] Methods for adding the lactic acid bacteria preparation include dissolving or suspending the Lactobacillus paracasei of the present invention or a lactic acid bacteria preparation containing the Lactobacillus paracasei of the present invention in water or the like and spraying the mixture onto the raw material, or sprinkling or mixing a powdered or granular lactic acid bacteria preparation onto the raw material.

[0024] Silage or fermented feed is fermented under anaerobic conditions after adding Lactobacillus paracasei or a lactic acid bacteria preparation of the present invention. The fermentation vessel is not particularly limited as long as it can maintain anaerobic conditions to a certain extent. For example, bunker silo, stack silo, trench silo, tower silo, underground silo, block silo, cup silo, roll bale, flexcon bag, and other vessels or devices generally used for silage preparation can be mentioned. Then, fermentation is carried out by leaving it at an outside temperature (about 5 to 30°C) for one week or more, preferably for two months.

[0025] Silage and fermented feed obtained using Lactobacillus paracasei or a lactic acid bacteria preparation of the present invention have good palatability and feed intake for cows because butyric acid fermentation is suppressed. In addition, ketosis does not occur. Furthermore, favorable fermentation continues even under relatively low to high temperature conditions. Furthermore, when the Lactobacillus paracasei or lactic acid bacteria preparation of the present invention is sprayed on the silage raw material after the formic acid spraying and then the silage is prepared, favorable fermentation proceeds, the growth of butyric acid bacteria is suppressed, and abnormal fermentation does not occur. Since the amount of oxygen remaining in the silage is lower than when only formic acid is added, the growth of yeast is suppressed, and as a result, even if the completed silage is opened from a sealed state to an aerobic environment, yeast growth does not occur, and there is no need to worry about secondary fermentation. Formic acid may be added after spraying the lactic acid bacteria preparation. It has been confirmed that the Lactobacillus paracasei lactic acid bacteria of the present invention can be sprayed after the addition of formic acid without any particular problems, and good silage fermentation proceeds. This is one of the major features of the present invention. In other words, unlike conventional lactic acid bacteria preparations, there is an advantage that the order of addition of formic acid can be different. In addition, it is resistant to hydrogen peroxide and ethanol, and its growth is not inhibited by peroxides or alcohol generated by abnormal fermentation, so it can continue to produce lactic acid and inhibit the growth of butyric acid bacteria and yeast that cause abnormal fermentation. EXAMPLES

[0026] Tests were performed to evaluate the capabilities of the lactic acid bacteria of the present invention using the Lactobacillus paracasei SBS-0011 strain (NITE BP-03013) of the present invention, and, as a comparative example, the Lactobacillus paracasei SBS-0003 strain (NITE BP-1109) isolated and established from silage held by Snow Brand Seed Co., Ltd., and type strains representing the characteristics and features of various lactic acid bacteria stored in the Japan Collection of Microorganisms (JCM) at the RIKEN BioResource Research Center, namely JCM8130 Lb. paracasei, JCM1164 Lb. coryniformis subsp. coryniformis, JCM1149 Lb. plantarum, and JCM1132 Lb. acidophilus.

[0027] 1. Growth test on MRS medium A growth test was carried out on the parent strain SBS-0003 and the lactic acid bacterium strain SBS-0011 of the present invention in MRS medium and in MRS medium containing 0.2% formic acid. (1) Test method The culture solution obtained by culturing overnight at 37°C in 5 mL of MRS medium (Difco) was inoculated at 1% (V / V) into 5 mL of MRS medium (Difco) or MRS medium (pH 6.0) containing 0.2% formic acid, and then placed in an incubator at 37°C. 200 μL was sampled every 0, 3, 6, 9, 24, and 48 hours, and the absorbance (OD600) was measured at a wavelength of 600 nm using a 96-well microplate reader. This value was used as an index of growth.

[0028] (2) Results The growth results in MRS medium are shown in Table 2, and the growth results in MRS medium containing 0.2% formic acid are shown in Table 3.

[0029] [Table 2]

[0030] [Table 3]

[0031] As shown in Table 2, the growth rate of the SBS-0011 strain, which is a lactic acid bacterium of the present invention, in MRS medium was equivalent to that of the parent strain. On the other hand, in MRS medium containing 0.2% formic acid, the growth rate of the SBS-0011 strain was clearly superior after 24 to 48 hours of culture, as shown in Table 3. The SBS-0011 strain, which is a lactic acid bacterium of the present invention, grows rapidly even at a formic acid concentration of 0.2% (200 ppm). On the other hand, the SBS-0003 strain experienced delayed growth at the same formic acid concentration.

[0032] 2. Formic acid, ethanol, and hydrogen peroxide resistance tests (1) Preparation of medium for resistance testing In addition to formic acid, the growth resistance of the SBS-0011 strain, which is a lactic acid bacterium of the present invention, to ethanol and hydrogen peroxide, which are known to inhibit the growth of lactic acid bacteria, was tested. For comparison, the type strains of the various lactic acid bacteria exemplified above, JCM8130 Lb. paracasei, JCM1164 Lb. coryniformis subsp. coryniformis, JCM1149 Lb. plantarum, and JCM1132 Lb. acidophilus, were also tested. Each type strain was provided by the National Research and Development Agency, RIKEN.

[0033] An MRS medium containing formic acid, ethanol, and hydrogen peroxide was prepared so that the concentrations of formic acid (F), ethanol (E), and hydrogen peroxide (H) contained in the MRS medium were as shown in Table 4. Using this medium, a growth test of lactic acid bacteria in a culture solution containing formic acid, ethanol, and hydrogen peroxide was performed to confirm the characteristics of the SBS-0011 strain, which is a lactic acid bacterium of the present invention.

[0034] [Table 4]

[0035] (2) Growth test As described above, each lactic acid bacteria MRS culture solution after overnight culture at 37°C was inoculated into each medium at 1% (V / V), and the medium was then placed in an 8-tube PCR tube, which was then placed in an anaerobic jar (manufactured by Mitsubishi Gas Chemical Company, Inc.) and cultured aerobically at 37°C for 144 hours. The degree of growth of each lactic acid bacterium was measured by mixing 50 μL of the cultured bacterial solution and 50 μL of distilled water on a microplate, and then measuring the absorbance turbidity at a wavelength of 600 nm using a microplate reader. The absorbance turbidity value was expressed as the amount of growth of the lactic acid bacteria.

[0036] (3) Results 1) Resistance to hydrogen peroxide Figure 1 shows the results of the hydrogen peroxide resistance test. The growth of the lactic acid bacteria SBS-0011 strain of the present invention was hardly inhibited even in a medium containing hydrogen peroxide. On the other hand, the growth of the type strains was clearly inhibited as the concentration of hydrogen peroxide increased. In particular, JCM1164 and JCM1132 could hardly grow when the hydrogen peroxide concentration increased to 150 ppm.

[0037] 2) Tolerance to hydrogen peroxide and ethanol The results of the growth test using MRS culture medium containing hydrogen peroxide and ethanol are shown in FIG. 2. In a medium containing hydrogen peroxide and ethanol, the growth of the SBS-0011 strain, which is the lactic acid bacteria of the present invention, was hardly inhibited at ethanol concentrations of 5% and 8%. On the other hand, the growth of all the standard strains for comparison was significantly inhibited when the ethanol concentration was increased to 8%. Furthermore, the growth of all the lactic acid bacteria was inhibited at an ethanol concentration of 8% and a hydrogen peroxide concentration of 150 ppm. It was revealed that the coexistence of ethanol and hydrogen peroxide inhibits the growth of lactic acid bacteria. However, the growth of the lactic acid bacteria of the present invention was hardly inhibited even at an ethanol concentration of 8% and a hydrogen peroxide concentration of 100 ppm.

[0038] 3) Resistance to hydrogen peroxide and formic acid The results of the growth test using MRS culture medium containing hydrogen peroxide and formic acid are shown in Figure 3. In a medium containing hydrogen peroxide and formic acid, the growth of the SBS-0011 strain, which is a lactic acid bacterium of the present invention, was not inhibited up to a formic acid concentration of 0.3% and hydrogen peroxide concentration of 100 ppm. Even when the formic acid concentration was 0.3% and hydrogen peroxide concentration was 150 ppm, the inhibition was slight. On the other hand, the growth of the standard strain for comparison was inhibited depending on the increase in the formic acid and hydrogen peroxide concentrations.

[0039] 4) Resistance to hydrogen peroxide, ethanol, and formic acid The results of a growth test using an MRS culture medium containing 50-150 ppm hydrogen peroxide, 0.1% formic acid, and 5-11% ethanol are shown in Figure 4, the results of a growth test using an MRS culture medium containing 50-150 ppm hydrogen peroxide, 0.2% formic acid, and 5-11% ethanol in Figure 5, and the results of a growth test using an MRS culture medium containing 50-150 ppm hydrogen peroxide, 0.3% formic acid, and 5-11% ethanol in Figure 6. 4 to 6, in the presence of hydrogen peroxide, formic acid, and ethanol, the growth of the type strain of lactic acid bacteria is significantly inhibited, but the growth of the SBS-0011 strain, which is the lactic acid bacteria of the present invention, is not inhibited. That is, the growth of the lactic acid bacteria of the present invention is not inhibited even at concentrations of 150 ppm hydrogen peroxide, 11% ethanol, and 0.3% formic acid.

[0040] From the results of the above growth tests, it was revealed that the lactic acid bacteria strain SBS-0011 of the present invention has strong resistance to growth inhibition by hydrogen peroxide, ethanol, and formic acid, which is not present in conventional lactic acid bacteria type strains.

[0041] 3.Polysaccharide production test The lactic acid bacteria Lactobacillus paracasei SBS-0011 strain of the present invention exhibits strong resistance to growth inhibition by hydrogen peroxide, ethanol, and formic acid, which is not possessed by conventional lactic acid bacteria Lactobacillus paracasei standard strains. In addition, when comparing the Lactobacillus paracasei of the present invention with conventional lactic acid bacteria paracasei, it was confirmed that the lactic acid bacteria of the present invention have an extracellular polysaccharide, and further, the constituent sugar of the polysaccharide is clearly different from the constituent sugar of the polysaccharide possessed by the known Lactobacillus paracasei (see Non-Patent Document 2). It is considered that this difference in structure brings about strong resistance to growth inhibition by hydrogen peroxide, ethanol, and formic acid. An example of production and analysis of this extracellular polysaccharide will be shown and explained.

[0042] (1) Cultivation of Lactobacillus paracasei for polysaccharide production After activating the stock strains of Lactobacillus paracasei SBS-0011 and the conventional Lactobacillus paracasei SBS-0003, each lactic acid bacteria strain was inoculated into 5 mL of MRS medium at 1% by volume and pre-cultured at 37°C for 24 hours. Next, 1% by volume of the pre-cultured culture solution was inoculated into 50 mL of a jar fermenter (hereinafter referred to as "jar") culture medium, and cultured for 24 hours at 37° C. After the culture, 1% by volume of this culture solution was inoculated into 2.5 L of a jar culture medium (composition shown in Table 5 below) whose pH had been adjusted to 5.5, and cultured overnight at 37° C., 60 rpm, and pH 5.5 using a jar culture apparatus. The cultivation was terminated when it was confirmed that the pH did not decrease for 30 minutes after stopping the pH adjustment device, and then the operation of the device was stopped.

[0043] [Table 5]

[0044] Figure 7 shows an image of the state of the culture solution after the end of cultivation. As shown in Figure 7, cloud-like, hazy or colloidal floating matter can be observed in the culture solution of Lactobacillus paracasei SBS-0011 strain. In other words, it was observed that exopolysaccharides were being produced. On the other hand, the culture solution of the control Lactobacillus paracasei SBS-0003 strain was entirely turbid.

[0045] (2) Recovery of cultured cells After the jar culture was completed, each culture solution was collected and centrifuged at 3500 rpm and 4° C. for 15 minutes to separate the culture supernatant and precipitate. The precipitate was collected and washed with distilled water to obtain bacterial cells. The cells were washed with distilled water, freeze-dried, and then the dried product was collected and pulverized. The mass of the collected dried product was measured and used as the mass of the cells.

[0046] (3) Analysis of carbon (C) and nitrogen (N) in the bacterial body To clarify the main components of the bacterial cells, the nitrogen and protein analyzer TruMac CN manufactured by LECO was used for analysis. The analytical method was according to the instruction manual that came with the device.

[0047] (4) Separation and recovery of exopolysaccharides contained in dried cells 1 g of bacterial powder was weighed out. To remove soluble impurities contained in the powder, the powder was washed with 10 mL of PBS and then centrifuged at 12,000 g and 4° C. for 15 minutes. The precipitate was collected, washed again with 10 mL of PBS, and centrifuged again at 12,000 g and 4° C. for 15 minutes. Next, to separate and elute the polysaccharide, 10 mL of 1 M NaOH was added to the precipitate, and the mixture was shaken overnight at 4°C and 60 rpm in a shaking incubator. The sodium hydroxide-containing solution was then centrifuged at 12,000 g and 4° C. for 15 minutes to recover the supernatant. The recovered supernatant was mixed with an equal volume of ice-cold ethanol (10 mL) and allowed to stand at 4° C. for 30 minutes to precipitate the alkali-soluble polysaccharide. This solution was centrifuged at 12,000 g and 4° C. for 35 minutes, and the resulting precipitate was collected. The collected precipitate was dissolved in 20 to 50 mL of distilled water, and then trichloroacetic acid was added to make the concentration 10% by volume. After thorough stirring, the mixture was allowed to stand for 30 minutes at 4° C. Then, the mixture was centrifuged at 12,000 g and room temperature for 10 minutes, and the supernatant was collected. The collected supernatant was added with 2 volumes of ethanol, left to stand at 4°C for 30 minutes, then centrifuged at 12,000g and 4°C for 10 minutes, the resulting precipitate was collected, and degassed and dried in a desiccator. 10 mL of distilled water was added to the entire dried matter to dissolve it, and low molecular weight compounds of 5 kDa or less were removed using Vivaspin 5K (manufactured by Cytiva) to remove monosaccharides and oligosaccharides. The remaining liquid from which the low molecular weight substances had been removed was transferred to an Eppendorf tube and dried using a centrifugal evaporator. The dried product obtained by the above procedures was treated as an exopolysaccharide of Lactobacillus paracasei, and its constituent sugars were analyzed.

[0048] (5) Analysis of Lactobacillus paracasei exopolysaccharides 1) Acid hydrolysis The total amount of the obtained exopolysaccharide was dissolved in 20 μL of ultrapure water and subjected to sugar analysis by acid hydrolysis. 5 μL of the ultrapure water solution was taken, 5 μL of 8 M trifluoroacetic acid was added to it, stirred, briefly centrifuged, and then kept at 100° C. for 3 hours, cooled in air, and briefly centrifuged again. Then, the mixture was evaporated to dryness using a centrifugal evaporator. 40 μL of isopropanol was added to the dried product, stirred, briefly centrifuged, and the solvent was evaporated to dryness using a centrifugal evaporator. 40 μL of pyridine / methanol (10 / 90, v / v) was added to the dried product, stirred, briefly centrifuged, and then 10 μL of acetic anhydride was added, stirred, and briefly centrifuged again. The centrifuge tube was removed from its lid, covered with aluminum foil and left at room temperature for 30 minutes, and then evaporated to dryness using a centrifugal evaporator.

[0049] 2) Labeling of sugars that make up polysaccharides 5 μL of ultrapure water was added to the exopolysaccharide analysis sample after the acid hydrolysis treatment that had been evaporated to dryness, and then 20 μL of pre-prepared ABEE reagent (GlyScope ABEE labeling kit: Cosmo Bio Co., Ltd.: preparation method according to the attached manual) was removed from the freezer and dissolved, and then added. The mixture was stirred and briefly centrifuged. Next, this solution was kept at 80°C for 60 minutes, air-cooled, and then centrifuged to allow the liquid to settle to the bottom of the centrifuge tube. The lid was removed, and 200 μL of ultrapure water and 400 μL of chloroform were added. The solution was mixed by stirring using a vortex mixer for 30 seconds, and centrifuged again at 20,000 g for 1 minute to separate the supernatant and precipitate. The supernatant was collected, 400 μL of chloroform was added, and the mixture was stirred and mixed using a vortex mixer for 30 seconds, and then centrifuged at 20,000 g for 1 minute to separate the precipitate from the supernatant. The supernatant was filtered through a membrane filter (mesh size 0.22 μ). The filtrate was used as a sample for high performance liquid chromatography (HPLC) analysis to analyze the constituent sugars.

[0050] 3) HPLC analysis of sugar components The HPLC analysis conditions were as follows: Eluent A: 0.2 M potassium borate buffer (pH 8.9) Eluent B: 100% acetonitrile Column equilibration conditions Eluent A: 97% (initial condition) Eluent B: 3% (initial conditions) Flow rate: 0.7mL / min Conditions for solution B (major monosaccharides: glucose, xylose, galactose, arabinose, rhamnose) 0~2 min 3~10%(separation) 2~7 min 10~21% (separation) 7~8 min 40% (wash) 8~10 min 3% (initial condition) Flow rate: 0.7mL / min Column temperature: 50℃ Autosampler temperature: 10℃ Absorption wavelength: 305nm Fluorescence wavelength: 360nm Sample injection volume: 2μL

[0051] (5) Results 1) Amount of bacteria recovered The bacterial cell mass of the lactic acid bacteria Lactobacillus paracasei SBS-0011 strain of the present invention was 6.54 g. On the other hand, the bacterial cell mass of the control Lactobacillus paracasei SBS-0003 strain was 5.33 g. Although the bacterial counts in the culture solution were almost the same for both strains, the bacterial cell mass of the SBS-0011 strain was more than 20% higher than that of the SBS-0003 strain.

[0052] 2) Analysis of carbon (C) and nitrogen (N) in the bacterial body The results of C and N analysis in the cells are shown in Table 6 below.

[0053] [Table 6]

[0054] As is clear from the C / N ratio, the C content of the SBS-0011 strain was increased by more than 20% compared to the SBS-0003 strain.

[0055] 3) Constituent sugars of recovered polysaccharides The types of constituent sugars obtained by HPLC analysis and the contents (mg) per mass (g) of recovered cells are shown in Table 7. Table 8 shows the ratios (%) of the constituent sugars per exopolysaccharide.

[0056] [Table 7]

[0057] [Table 8]

[0058] As shown in Table 7, it was found that Lactobacillus paracasei SBS-0011 strain possesses exopolysaccharides whose main sugar components are galactose, mannose, and glucose. It was also found that the exopolysaccharides do not contain amino sugars such as galactosamine and glucosamine. Furthermore, as shown in Table 8, it was found that Lactobacillus paracasei SBS-0011 strain enhanced resistance to growth inhibition by hydrogen peroxide, ethanol, formic acid, etc. by increasing the amount of exopolysaccharides produced without changing the composition ratio of the exopolysaccharides produced.

[0059] (6) Discussion From the results of the weight of the collected cells and the C / N ratio of the cells, it was revealed that the Lactobacillus paracasei SBS-0011 strain of the present invention has more than 20% extracellular polysaccharide per dry mass of the cells. In addition, as a result of analyzing the constituent sugars of the polysaccharide, the polysaccharide has galactose, mannose, and glucose as its main constituent sugars. The SBS-0003 strain, which does not produce polysaccharides extracellularly, is inhibited in growth by hydrogen peroxide, ethanol, and formic acid, but the SBS-0011 strain is not inhibited in growth. In other words, it is considered that the polysaccharide produced extracellularly is protected from harmful components such as hydrogen peroxide, ethanol, and formic acid by covering the cells as a capsule. In addition, since no Lactobacillus paracasei is known to produce polysaccharides mainly composed of galactose, mannose, and glucose extracellularly, it was revealed that the Lactobacillus paracasei of the present invention is a novel lactic acid bacterium.

[0060] 3. Silage preparation test using SBS-0011 strain (1) Using the lactic acid bacteria strain SBS-0011 of the present invention, a silage preparation test was carried out using quackgrass and reed canarygrass, two types of grass that tend to have poor fermentation quality and therefore pose problems in silage preparation. (1) Preparation of pouch silage using quackgrass 1) Test method Quackgrass was harvested at the optimum time for silage preparation from the beginning of heading to the heading stage, cut into 1 cm pieces using a cutter, and divided into 300 g portions. The lactic acid bacteria SBS-0011 strain of the present invention was added to the test plots, and then formic acid was sprayed. A spray bottle was used to add lactic acid bacteria and formic acid. Silage was prepared by wrapping with vinyl film. Specifically, 100g of grass after spraying with lactic acid bacteria was placed in each pouch (Asahi Kasei Pax Corporation, standard bag Hiryu N-9), and silage was made by degassing and sealing the bag using a commercial tabletop sealing packaging machine (SQ-303W). Each treatment area was replicated three times. The silage was stored for two months at 25℃, and the main organic acid content and pH of the silage were measured after two months to confirm the fermentation status. Sensory evaluation of odor, etc. was also performed by a specialist evaluator. The amount of lactic acid bacteria added to the silage was 0.5 × 10 5 The number of pieces was set at 1g of grass, and the amount of formic acid added was set at 0.2%.

[0061] Organic acid analysis and pH measurements are as follows: After opening, 30 g of the silage was weighed into a Stoma Filter (Central Science Trading Co., Ltd.), and 90 mL of sterilized distilled water was added using an autoclaved (121°C, 15 min) measuring cylinder, followed by kneading for 1 minute with a Masticator S (Central Science Trading Co., Ltd.). After kneading, the silage was left to stand overnight at 4°C to transfer the water-soluble components in the silage to the water fraction. After leaving it to stand overnight, it was filtered using No. 5A filter paper from ADVANTEC. The filtrate was used for pH measurement and organic acid analysis. The pH was measured using a pH meter. For the organic acid analysis, the filtrate was diluted 10-fold with pure water and then filtered through a 0.22 μL filter (Millex GP, manufactured by Merck Millipore) to prepare a measurement sample. Organic acids were analyzed by HPLC using the post-column method using LC-20AD, LC-30AD, SIL-30AC, CTO-20A, and SPD-M20A manufactured by Shimadzu Corporation. The column for separating organic acids was TSKgel OApak-A manufactured by TOSOH, the column temperature was 40°C, and the flow rate for both solution A and solution C was 0.8 mL / min. The mobile phase (liquid A) was a 75 mM sulfuric acid solution, and the reaction solution (liquid C) was a BTB solution (0.1 mM BTB, 7.5 mM Na2HPO4). The analysis was performed by the post-column method. In addition, calibration curves were prepared for lactic acid, acetic acid, butyric acid, and formic acid. For each organic acid, calibration curves were prepared using standard solutions of 0.1 mass%, 0.05 mass%, and 0.01 mass%.

[0062] 2) Test results The organic acid content and pH in the silage are shown in Table 9 and FIG.

[0063] [Table 9]

[0064] As shown in Table 9 and Figure 8, the silage made only from quackgrass had a high butyric acid content and a strong butyric acid odor, resulting in low-quality silage with a foul odor. The pH was also high at 5.2, clearly indicating that the silage was of extremely low quality. On the other hand, the silage to which the lactic acid bacteria strain SBS-0011 of the present invention was added and then sprayed with formic acid was extremely high quality silage, with a high lactic acid content and a pH of 3.6. It was found that the butyric acid-producing bacteria normally present in quackgrass were suppressed by spraying with formic acid, and the lactic acid bacteria SBS-0011 strain was found to grow well and produce lactic acid.

[0065] (2) Preparation of pouch silage using reed canary grass 1) Test method Reed canary grass was harvested at the optimum time for silage preparation from the beginning of heading to the heading stage, cut into 1 cm lengths using a cutter, and divided into 300 g portions. Depending on the test plot, the lactic acid bacteria SBS-0011 strain of the present invention or SBS-0003 strain for comparison was added, and then formic acid was sprayed. Lactic acid bacteria and formic acid were added using a spray bottle. Silage was made by the bottle silage method. The amount of silage made was 700g per bottle. There were three replicates per treatment. The silage was stored at 25℃ for one month, and after opening, the main organic acid contents and pH of the silage were measured in the same manner as for quackgrass. Sensory evaluation of odor, etc. was also conducted by a specialist evaluator. The amount of lactic acid bacteria added to the silage was 1×10 4 ~1×10 5 The number of pieces was 1g of grass, and the amount of formic acid added was 0.2%.

[0066] 2) Test results The organic acid content and pH in the silage are shown in Table 10 and FIG.

[0067] [Table 10]

[0068] As shown in Table 10 and Figure 9, the silage prepared by spraying only formic acid on reed canary grass did not undergo butyric acid fermentation, but it was a silage with a low lactic acid content. In addition, the pH was high at 4.0, and it was a low-quality silage with a risk of secondary fermentation. The silage treated with SBS-0003 and 0.2% formic acid had a low pH and showed relatively good fermentation conditions, but the amount of lactic acid produced was lower than that of normal silage. On the other hand, reed canary grass silage to which the lactic acid bacteria SBS-0011 strain of the present invention was added and then sprayed with 0.2% formic acid was an extremely high quality silage with a high lactic acid content and a pH of 3.6 to 3.7. The silage also had a pleasant aroma. It was found that butyric acid-producing bacteria normally present in reed canary grass were suppressed by spraying 0.2% by mass of formic acid per silage material. It was also found that the lactic acid bacteria SBS-0011 strain grew smoothly and produced lactic acid without any delay in fermentation or death due to formic acid.

[0069] 4. Silage preparation test using SBS-0011 strain (2) A lactic acid bacteria preparation for silage preparation using the lactic acid bacteria SBS-0011 strain of the present invention and its parent strain SBS-0003 strain, trade name "Cymaster AC" (manufactured by Snow Brand Seed Co., Ltd.), was used to carry out a silage preparation test using reed canary grass as a raw material.

[0070] (1) Preparation of bottle silage 1) Test method Reed canary grass (variety: Paraton) cultivated on the grounds of Rakuno Gakuen University was harvested on May 27th and cut into 1cm lengths at the Hokkaido Research Farm of Snow Brand Seed Co., Ltd., and bottle silage was prepared. The reed canary grass harvested was in a young growth stage before ear development. The amount of reed canary glass packed into the bottle was 670g. The test plots were the following four plots. Each treatment plot was repeated three times, and significance tests were performed using the Tukey method. A.No additives B.Thighmaster AC C.SBS-0011 + 0.1% formic acid D.SBS-0011 + 0.2% formic acid

[0071] According to the product instructions, the prescribed amount of lactic acid bacteria was added via spray to Cymaster AC, as well as the attached fiber-degrading enzyme. In the test group where the SBS-0011 strain was added, the amount of lactic acid bacteria added was 0.5 × 10 5 The concentration was 1 g of grass per piece, and the amount of fiber-degrading enzyme added to the Cymmaster AC was half that of the Cymmaster AC test plot. It was opened about two months later on July 23rd, and after opening, pH and organic acid analyses were carried out in the same manner as for quackgrass.

[0072] 2) Test results The organic acid contents and pH in the silage are shown in Table 11 and FIG.

[0073] [Table 11]

[0074] As shown in Table 11 and Figure 10, the silage made only with reed canary grass had a high butyric acid content and a strong butyric acid odor, resulting in low-quality silage with a foul odor. The pH was also high at 5.1, making it clear that the silage was of extremely low quality. There was no significant difference in moisture content. Silage made using commercially available Cypressmaster AC tended to have a slightly higher pH, and butyric acid was also detected. On the other hand, the silage to which the lactic acid bacteria SBS-0011 strain of the present invention was added and then sprayed with formic acid had a high lactic acid content, and by adding 0.1% or 0.2% formic acid after the addition of SBS-0011 strain, butyric acid fermentation could be completely inhibited, resulting in an extremely high-quality silage with a pH of 4.1 or 3.9. In other words, it was possible to obtain silage with less butyric acid than that obtained with Cymaster AC. In particular, when applied to reed canary grass, it was found that the normally present butyric acid-producing bacteria were inhibited by spraying with formic acid. It was also revealed that the lactic acid bacteria SBS-0011 strain grew smoothly and produced lactic acid.

[0075] 5. Silage preparation test using SBS-0011 strain (3) A silage preparation test was carried out assuming that the silage raw material was contaminated with cow dung. That is, a silage raw material was sprayed with fecal juice prepared by filtering cow dung through gauze, and a silage preparation test was carried out using reed canary grass as a raw material, using a lactic acid bacteria preparation for silage preparation using the lactic acid bacteria SBS-0011 strain of the present invention and its parent strain SBS-0003 strain: product name "Tsaimaster AC" (manufactured by Snow Brand Seed Co., Ltd.), in the same manner as in the reed canary grass silage test in 4 above.

[0076] (1) Preparation of bottle silage 1) Preparation of fecal juice Fresh cow feces was collected from Japanese black cattle calves raised at the Snow Brand Seed Company's research farm, and the liquid filtered through double gauze was used as the fecal juice.

[0077] 2) Test method Reed canary grass (variety: Paraton) under the same conditions as above was treated with fecal juice at 1×10 5 The contaminated silage raw material was prepared by spraying 1000 units of general bacteria / g. Specifically, fresh feces was collected from Japanese beef calves 3 days before silage preparation, and the general bacteria (anaerobic conditions) contained in the feces were measured, and the amount of addition was determined based on the number of bacteria. The general bacteria were measured using NLi medium (a medium made by Difco with 0.5% lithium chloride added) by the pour mixture method. The incubation temperature was 37°C, and the number of bacteria was calculated by counting the colonies obtained after incubation for 2 days. The collected feces was stored at 4°C until it was used for silage preparation. The mass of the contaminated reed canary glass packed in each test bottle was 670 g. The test plots were the following four plots in the same manner. Each treatment plot was repeated three times, and significance tests were performed using the Tukey method. A.No additives B.Thighmaster AC C.SBS-0011 + 0.1% formic acid D.SBS-0011 + 0.2% formic acid

[0078] According to the product instructions, the prescribed amount of lactic acid bacteria was added via spray to Cymaster AC, as well as the attached fiber-degrading enzyme. In the test group where the SBS-0011 strain was added, the amount of lactic acid bacteria added was 0.5 × 10 5 The concentration was 1 g of grass per piece, and the amount of fiber-degrading enzyme added to the Cymmaster AC was half that of the Cymmaster AC test plot.

[0079] The containers were opened after two months, and pH and organic acid analyses were carried out after opening as in the quackgrass test.

[0080] 3) Test results The organic acid contents and pH in the silage are shown in Table 12 and FIG.

[0081] [Table 12]

[0082] As shown in Table 12 and Figure 11, the silage made only from reed canary grass was contaminated with feces, and the butyric acid content increased, resulting in a low-quality silage with a strong butyric acid odor and a foul odor. The pH was also high at 5.1, making it clear that the silage was of extremely low quality. In silage made using commercially available Cystar AC, lactic acid fermentation was predominant, but a decrease in lactic acid content and an increase in butyric acid content were observed compared to silage without manure contamination. It was found that lactic acid fermentation was more predominant in the silage made according to the present invention than in the silage made using commercially available Cysmaster AC, and that the silage was more resistant to contamination by manure and other substances. On the other hand, the silage obtained by adding the lactic acid bacteria SBS-0011 of the present invention to the feces-contaminated silage material and spraying with formic acid had a high lactic acid content, and butyric acid fermentation could be suppressed by spraying with 0.1% or 0.2% formic acid after adding the SBS-0011 strain. The pH was maintained at a low value of 4.2, making it an extremely high quality silage. Even when the silage raw material was contaminated with butyric acid-producing bacteria or feces that are normally present in reed canarygrass, the application of formic acid suppressed butyric acid fermentation and secondary fermentation, and the application of Lactobacillus subtilis SBS-0011 strain produced a favorable lactic acid fermented silage.

[0083] 6. Silage preparation test using two types of grass Silage preparation tests were conducted using timothy and meadow fescue, which are representative pasture grasses, to evaluate the effects of the present invention. In this test, two tests were conducted: "A. Normal test," in which silage was prepared under normal conditions, and "B. Butyric acid fermentation Reed liquid addition test," in which butyric acid fermentation Reed liquid containing lactic acid bacteria prepared from silage that had been butyric acid fermented in advance was sprayed, to evaluate the effects of the lactic acid bacteria of the present invention and the effects of adding formic acid.

[0084] A. Regular Test 1) Test method Timothy (variety: Horizon) and meadow fescue (variety: Cosmopolitan) cultivated in the fields of Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm were harvested and cut into 1 cm lengths using a cutter at Snow Brand Seed Co., Ltd.'s Hokkaido Research Farm to prepare bottle silage. Timothy was used with even ears, while meadow fescue was used with some seeds and late-harvested grass. The ratio of timothy to meadow fescue in the field was 30% timothy and 70% meadow fescue. The weight of grass packed in the bottle was 600 g. The test plots were the following four plots. Each treatment plot was repeated three times, and significance tests were performed using the Tukey method. A.No additives B.Thighmaster AC C.SBS-0011 + 0.1% formic acid D.SBS-0011 + 0.2% formic acid

[0085] According to the product instructions, Cymaster AC was sprayed to add the amount of lactic acid bacteria specified in the instructions, and the attached fiber-degrading enzyme was also added (150g of the enzyme attached to the Cymaster product was added per 10 tons of fresh grass). In the test group where the SBS-0011 strain was added, the amount of lactic acid bacteria added was 0.5 × 10 5 The concentration was 1 g of grass per piece, and the amount of fiber-degrading enzyme added to the Cymmaster AC was half that of the Cymmaster AC test plot. Lactic acid bacteria, enzymes, and formic acid were added using a commercially available small-volume sprayer.

[0086] After two months, the containers were opened and pH and organic acid analysis was carried out in the same manner as in the test for quackgrass, using HPLC in the same manner as in the test for quackgrass shown above.

[0087] 2) Test results The organic acid contents and pH of the silage in each test plot are shown in Table 13 and FIG.

[0088] [Table 13]

[0089] As shown in Table 13 and Figure 12, the mixed silage of timothy and meadow fescue, which are representative pasture grasses, was sprayed with 0.1% or 0.2% formic acid, and the pH was lowered by adding the SBS-0011 strain, resulting in a high-quality silage that did not contain butyric acid. On the other hand, no lactic acid was detected in the test area without addition. This is thought to be because the pH was high at the beginning of fermentation, which caused butyric acid bacteria to grow and the lactic acid produced was consumed. As a result, the silage was a defective product with a strong odor. The silage with the addition of Cymaster AC also showed a favorable fermentation state and was a favorable silage, but the pH was slightly higher than that of the test area sprayed with 0.1% or 0.2% formic acid and added with SBS-0011 strain. Furthermore, when the lactic acid bacteria of the present invention were used, secondary fermentation of the silage did not occur after opening, which revealed that the silage could be left for a long period of time after opening.

[0090] B. Butyric acid fermentation lead liquid addition test In order to confirm the effect of the lactic acid bacteria of the present invention on contaminated raw materials, pasture grass to which a solution containing the causative bacteria of butyric acid fermentation (butyric acid fermentation lead solution) had been added in advance was used as the raw material, and the effect of the lactic acid bacteria SBS-0011 strain of the present invention was tested.

[0091] (1) Test method 1) Preparation of butyric acid fermentation reed liquor (hereinafter referred to as "reed liquor") The reed solution was prepared by placing 30 g of butyric acid fermented silage (reed silage) in a stoma filter (Central Scientific Trading Co., Ltd.), adding 30 mL of sterile distilled water, and kneading for 1 minute using a Masticator Type S (Central Scientific Trading Co., Ltd.). The number of lactic acid bacteria in the lead solution was determined by serial dilution method. The number of lactic acid bacteria in this lead solution is 5.5 × 10 7 The results were CFU / mL. This was sprayed at 2.0 mL / kg onto cut timothy and meadow fescue, which are silage raw materials.

[0092] 2) Silage preparation The same mixed grass of timothy and meadow fescue in a ratio of 3:7 as in "A. Conventional Test" above was used. The weight of grass packed in the bottle was 600 g, the same as in "A. Regular Test." The test plots were the following four plots. Each treatment plot was repeated three times, and significance tests were performed using the Tukey method. A.No additives B.Thighmaster AC C.SBS-0011 + 0.1% formic acid D.SBS-0011 + 0.2% formic acid

[0093] For the test sections B. Cymaster AC, C. SBS-0011 + 0.1% formic acid, and D. SBS-0011 + 0.2% formic acid, the above additions were carried out in the same manner as in “A. Regular test.”

[0094] After two months, the containers were opened and pH and organic acid analysis was performed using HPLC, as in the above quackgrass silage test.

[0095] 3) Test results The organic acid contents and pH in the silage are shown in Table 14 and FIG.

[0096] [Table 14]

[0097] As shown in Table 14 and Figure 13, the mixed silage of timothy and meadow fescue, which are representative pasture grasses, became high quality with an extremely low pH and no butyric acid by spraying 0.1% or 0.2% formic acid and adding the SBS-0011 strain. In particular, when the spray concentration of formic acid was 0.2%, no butyric acid was detected. In addition, the pH was 3.9 when 0.1% formic acid was sprayed, and 3.7 when 0.2% formic acid was sprayed. The reason for such a low pH was thought to be due to the synergistic effect of the spray of formic acid and the lactic acid bacteria of the present invention.

[0098] On the other hand, in the non-additive test section, no lactic acid was detected in the "A. Normal test". This was thought to be because the pH was high at the beginning of fermentation, causing the butyric acid bacteria to grow, and the lactic acid produced by the lactic acid bacteria was consumed as a result. The resulting silage was a defective product with a strong foul odor caused by butyric acid. The silage to which Cymaster AC was added showed a favorable fermentation state with a high lactic acid content, but the butyric acid fermentation also progressed at the same time due to the butyric acid bacteria in the reed liquor, and the final silage was not favorable. The pH was also high at 4.6. From the results of "A. Conventional test" and "B. Butyric acid fermentation lead liquid addition test" above, it was confirmed that by spraying the lactic acid bacteria of the present invention on the grass used as silage in advance and then spraying formic acid at a concentration of 0.1 to 0.2%, the growth of butyric acid bacteria can be inhibited, and even grass contaminated with butyric acid bacteria can be made into desirable silage. It was also revealed that when the lactic acid bacteria of the present invention are used, secondary fermentation does not occur in silage after opening, so that the silage can be left for a long period of time after opening.

[0099] 7. Summary of silage preparation tests The lactic acid bacteria SBS-0011 strain of the present invention is a lactic acid bacterium with strong resistance to formic acid. Therefore, by adding the lactic acid bacteria SBS-0011 strain of the present invention to grass as a silage raw material, spraying formic acid to lower the pH, inhibit the growth of butyric acid bacteria, and ferment, high-quality silage can always be obtained. In addition, since lactic acid is continuously produced, the growth of butyric acid bacteria and yeast that cause abnormal fermentation can be inhibited. Furthermore, secondary fermentation was not observed after opening the silage, and it was revealed that the rapid decrease in pH and the continuous production of lactic acid have a high effect of inhibiting the growth of yeast and the like that cause secondary fermentation.

Claims

1. The main constituent sugars of the exopolysaccharide are galactose, mannose, and glucose, and the SBS-0011 strain (NITE BP-03013) is a Lactobacillus paracasei strain that produces an exopolysaccharide.

2. The Lactobacillus paracasei according to claim 1, wherein the exopolysaccharide does not contain amino sugars as its constituent sugars and contains 50 to 60% by mass of glucose, 20 to 30% by mass of mannose, and 10 to 20% by mass of galactose per mass of the exopolysaccharide.

3. 3. The Lactobacillus paracasei according to claim 1 or 2, which is resistant to growth inhibition by formic acid, ethyl alcohol and hydrogen peroxide.

4. The Lactobacillus paracasei according to any one of claims 1 to 3, which can grow in an MRS medium containing any one or more of 0.2% by mass of formic acid, 11% by mass of ethyl alcohol, and 150 ppm of hydrogen peroxide.

5. A lactic acid bacteria preparation for preparing silage, comprising the Lactobacillus paracasei according to any one of claims 1 to 4.

6. The lactic acid bacteria preparation for silage preparation according to claim 5, which is in any form selected from the group consisting of a liquid preparation, a granule preparation, and a powder preparation.

7. A method for preparing silage, comprising spraying a preparation containing Lactobacillus paracasei according to any one of claims 1 to 4 onto silage raw materials, followed by fermentation.

8. At least 1 x 10 per silage ingredient ４ A method for preparing silage by adding Lactobacillus paracasei according to any one of claims 1 to 4 at CFU / g and fermenting the silage.

9. A method for preparing silage, comprising spraying an aqueous solution of formic acid containing 0.1 to 0.3% by mass of formic acid per silage raw material, and then adding a lactic acid bacteria preparation for preparing silage containing Lactobacillus paracasei according to any one of claims 1 to 4 or Lactobacillus paracasei according to claim 5, and fermenting the silage.

10. A silage fermented using the Lactobacillus paracasei according to any one of claims 1 to 4.

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