Mixed lactic acid bacteria preparation for feed formulation

Abstract

To provide a mixed lactic acid bacteria preparation for feed formulation that enables superior fermentation performance under ambient temperature through the use of Pediococcus inopinatus, which shows high growth at low temperatures and exceptionally high fermentation capacity for feed rice and TMR in midwinter.SOLUTION: A mixed lactic acid bacteria preparation for feed formulation containing Pediococcus inopinatus strain IWT685 (NITE P-03829) and Lactococcus lactis strain SBS-0001 (NITE BP-1107) and / or Lactococcus lactis strain SBS-0001-S (NITE BP-04099).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a mixed lactic acid bacteria preparation for feed preparation, which contains the lactic acid bacteria Pediococcus inopinatus strain IWT685 (NITE P-03829) and the lactic acid bacteria Lactococcus lactis strain SBS-0001 (NITE BP-1107) and / or Lactococcus lactis strain SBS-0001-S (NITE BP-04099); feed or fermented feed containing these two or three lactic acid bacteria; and a method for preparing fermented feed using the mixed lactic acid bacteria preparation for feed preparation. [Background technology]

[0002] Japan's livestock industry is highly dependent on imported feed, and has been significantly affected by the unstable global situation in recent years. Further improving feed self-sufficiency is a major challenge to ensure a stable supply of livestock products. Research and development into fermented feed using domestically grown rice and feed rice is therefore underway, and it is being used in livestock farming. Advances in feed rice breeding have made it possible to harvest from the heat of summer to the depths of winter, extending the harvesting period and helping to spread out the busy season. Furthermore, because feed rice is characterized as a roughage crop, many consumers desire silage production, hoping for its palatability for dairy cows and its probiotic effects. For these reasons, new technologies that can be expected to enable silage production from the heat of summer to the depths of winter are desired. Furthermore, efforts are being made to promote the use of total mixed rations (TMR), which utilize domestically produced fermented feed and by-products from local food manufacturing, and there is a demand for technology that can stably store high-quality fermented feed even in cold regions and during cold seasons. Furthermore, these stable storage technologies are also in demand at feed production sites overseas.

[0003] Fermented feed, mainly silage, is a type of stored livestock feed prepared under anaerobic conditions by cleverly utilizing the power of lactic acid bacteria. Lactic acid bacteria are crucial for not only the fermentation quality but also the nutritional value and physiological metabolism of ruminant livestock. However, microorganisms such as butyric acid bacteria, aerobic bacteria, molds, and yeasts that coexist in fermented feed materials competitively inhibit the fermentation of lactic acid bacteria, resulting in a deterioration in the quality of the fermented feed and a loss of nutrients. Silage and fermented TMR prepared during cold seasons or in cold, high-altitude regions often undergo slow lactic acid fermentation, resulting in low lactic acid and high pH. During storage, harmful microorganisms such as butyric acid bacteria, coliform bacteria, and molds grow, resulting in a decrease in nutritional content, reduced livestock productivity due to harmful substances such as mycotoxins, and hygiene management issues, which must be resolved. Roll bale silage, made by forming and wrapping grass into a cylindrical shape, is often reused as fresh or fermented total mixed ration (TMR) after opening, and small-scale livestock farmers often have no choice but to store the silage under aerobic conditions for a certain period before feeding. Therefore, there is a high demand for technology to prevent spoilage due to secondary fermentation after opening. In particular, the recently developed high-sugar feed rice varieties "Tachisuzuka," "Tsukisuzuka," and "Tsukikotoka" are known to mature late or very late in cold regions, where the optimum harvest period is in a low temperature environment, resulting in slow lactic acid fermentation. This can lead to mold growth and the proliferation of yeast, which is thought to be one of the microorganisms responsible for secondary fermentation.

[0004] In response to these issues, the present inventors have discovered and reported that the lactic acid bacterium Pediococcus inopinatus has high proliferation ability at low temperatures and extremely high fermentation ability for feed rice and TMR in low-temperature environments (Patent Document 1). However, it has been newly confirmed that the lactic acid bacterium Pediococcus inopinatus grows slowly depending on the raw grass species and silage storage temperature conditions, and that in temperature ranges where other bacteria can grow vigorously, its effect in improving fermentation quality is weakened due to competition with them. On the other hand, it has been reported that the feed preparation lactic acid bacterium Lactococcus lactis generally grows quickly in pasture and produces the antibacterial substance nisin, thereby suppressing other contaminating bacteria even in feed that is highly contaminated, and significantly promoting feed fermentation (Patent Document 2). Specifically, the feed preparation lactic acid bacterium Lactococcus lactis is characterized by producing 40 IU or more of nisin when the sugar content in a grass broth medium is 0.1 to 0.4% by weight. Therefore, this nisin productivity is expected to improve fermented feed even in feed crops with low sugar content. However, the lactic acid bacterium Lactococcus lactis used in feed preparation has a problem in that it is poorly acid-resistant and is quickly killed by the acid it produces. Furthermore, although the commercially available product "Chikusa No. 1 Plus," a mixture of two lactic acid bacteria, can be used during the hot summer months, there are concerns about butyric acid fermentation in general feed rice silage, including staple rice, which contains more than 65% moisture, so further technological advances are desired. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2024-139726 [Patent Document 2] Patent No. 5931064 Summary of the Invention [Problem to be solved by the invention]

[0006] The objective of this invention is to provide a mixed lactic acid bacteria preparation for feed preparation that exhibits excellent fermentation properties when used at room temperature, using the lactic acid bacterium Pediococcus inopinatus, which has high proliferation properties at low temperatures and extremely high fermentation ability for feed rice and TMR even during the harsh winter. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems, they discovered that the lactic acid bacterium Pediococcus inopinatus has a characteristic that it has extremely high resistance to nisin Z compared to its resistance to nisin A. Taking advantage of this unique characteristic, they discovered that by using the lactic acid bacterium Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099), which produce nisin Z, in combination with the lactic acid bacterium Pediococcus inopinatus IWT685 strain (NITE P-03829), which has extremely high resistance to nisin Z, it can have excellent fermentation properties even at room temperature, thereby solving the above-mentioned problems.

[0008] Specifically, the present invention provides the following: 1. A mixed lactic acid bacteria preparation for feed preparation containing Pediococcus inopinatus strain IWT685 (NITE P-03829) and Lactococcus lactis strain SBS-0001 (NITE BP-1107) and / or Lactococcus lactis strain SBS-0001-S (NITE P-04099). 2. Feed containing Pediococcus inopinatus strain IWT685 (NITE P-03829) and Lactococcus lactis strain SBS-0001 (NITE BP-1107) and / or Lactococcus lactis strain SBS-0001-S (NITE P-04099). 3. A fermented feed containing Pediococcus inopinatus strain IWT685 (NITE P-03829) and Lactococcus lactis strain SBS-0001 (NITE BP-1107) and / or Lactococcus lactis strain SBS-0001-S (NITE BP-04099). 4. A method for preparing fermented feed, characterized by using the mixed lactic acid bacteria preparation for feed preparation described in 1. and fermented feed material. 5. The method for preparing fermented feed according to 4., wherein the fermented feed material is feed rice. [Effects of the Invention]

[0009] The mixed lactic acid bacteria preparation for feed preparation of the present invention maintains the characteristics of the lactic acid bacteria Pediococcus inopinatus IWT685 strain (NITE P-03829), such as its excellent low-temperature growth ability and the ability to promote fermentation by increasing the production of organic acids such as lactic acid even in low-temperature environments, enabling the production of high-quality livestock feed.However, when used in combination with Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099), the growth ability is significantly improved, making it possible to quickly achieve a significant decrease in pH, an increase in lactic acid production, and suppression of butyric acid production. The mixed lactic acid bacteria preparation for preparing feed of the present invention has excellent fermentation properties even at room temperature, and is therefore very useful in practical situations. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a mixed lactic acid bacteria preparation for feed preparation that exhibits excellent fermentation characteristics when used at room temperature, using the lactic acid bacterium Pediococcus inopinatus, which has high proliferation properties at low temperatures and extremely high fermentation ability for feed rice and TMR even during the harsh winter. The present invention also relates to a feed or fermented feed containing the mixed lactic acid bacteria, and a method for preparing a fermented feed using the mixed lactic acid bacteria preparation for preparing a feed and a fermented feed material, for example, feed rice.

[0011] The present invention will be described in detail below. A. Pediococcus inopinatus strain IWT685 The Pediococcus inopinatus IWT685 strain of the present invention is a lactic acid bacterium that has excellent low-temperature growth ability, growth ability in acidic regions, and also has the ability to inhibit the growth of yeast. The IWT685 strain was deposited at the Patent Microorganisms Deposit Center of the National Institute of Technology and Evaluation on February 16, 2023, and its deposit number is NITE P-03829. The mycological properties of the Pediococcus inopinatus IWT685 strain of the present invention include "properties suitable for producing high-quality fermented feed," and specifically, its excellent low-temperature growth ability. Here, excellent low-temperature growth performance refers to acid production ability and viability, and is essential for producing high-quality fermented feed containing large amounts of organic acids such as lactic acid in a low-temperature environment. In the present invention, low temperature means 0°C or higher and 10°C or lower. A temperature range of 0°C or higher and 5°C (±1°C) or lower is more preferable as low temperature in the present invention. Furthermore, excellent acid production ability in the present invention means having efficient lactic acid fermentation ability and highly metabolic L-lactic acid production ability. Furthermore, the Pediococcus inopinatus IWT685 strain of the present invention has the ability to inhibit the growth of harmful microorganisms such as aerobic bacteria, coliform bacteria, yeasts and molds during the fermentation process of fermented feed.

[0012] The Pediococcus inopinatus IWT685 strain of the present invention is a lactic acid bacterium that has excellent low-temperature growth ability as well as growth ability in an acidic range, where the acidic range in the present invention means a range of pH 5 or lower. Butyric acid fermentation is one of the major causes of the deterioration of the quality of fermented feed. In order to obtain high-quality fermented feed, it is important to suppress the activity of butyric acid bacteria as much as possible, and one method of suppressing butyric acid fermentation is known to be keeping the pH below 4.2. The type strain of lactic acid bacteria Pediococcus inopinatus, JCM12518 T " is significantly inhibited in its growth at pH 4.5, making it difficult to avoid the risk of butyric acid fermentation. However, the Pediococcus inopinatus IWT685 strain of the present invention has excellent growth performance in the acidic range, making it possible to quickly lower the pH of fermented feed to 4.2 or less. As a result, it is a lactic acid bacterium with excellent performance in inhibiting butyric acid fermentation and stabilizing the quality of fermented feed.

[0013] B. Lactococcus lactis strain SBS-0001 The Lactococcus lactis SBS-0001 strain of the present invention is a lactic acid bacterium that has the ability to grow and ferment even in the presence of feed ingredients with a low sugar content, and is characterized by rapid proliferation in feed ingredients. This characteristic is due to the SBS-0001 strain's production of the antibacterial substance nisin Z. Even in feed ingredients with a low sugar content or containing many contaminating bacteria, this nisin Z productivity suppresses other contaminating bacteria, allowing the fermentation of the feed to progress significantly. The SBS-0001 strain was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation on June 15, 2011, and its accession number is NITE BP-1107.

[0014] C. A mutant strain of Lactococcus lactis SBS-0001 The Lactococcus lactis SBS-0001-S strain of the present invention is a lactic acid bacterium in which freeze-thaw resistance has been imparted to the SBS-0001 strain. Compared to the SBS-0001 strain, the yield of bacteria in the freeze-dried powder after factory cultivation is higher, allowing for a reduction in the number of times the strain needs to be cultured. In addition, compared to the SBS-0001 strain, the growth rate and nisin productivity at pH 5.0 are improved, and the strain also has performance equivalent to or better than the SBS-0001 strain in terms of feed fermentation quality. The SBS-0001-S strain was domestically deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE) on March 13, 2024, under the accession number NITE P-04099. Subsequently, a request was made to transfer the strain to an international deposit under the Budapest Treaty, and a certificate of receipt for the original deposit was issued on March 21, 2025, for the international deposit accession number NITE BP-04099.

[0015] (mixed lactic acid bacteria preparation for feed preparation) The mixed lactic acid bacteria preparation for preparing feed of the present invention contains the Pediococcus inopinatus strain IWT685 and the Lactococcus lactis strain SBS-0001 and / or the Lactococcus lactis strain SBS-0001-S. The mixing weight ratio of the Pediococcus inopinatus IWT685 strain and the Lactococcus lactis SBS-0001 strain and / or the Lactococcus lactis SBS-0001-S strain is not particularly limited, but it is preferable to mix the Pediococcus inopinatus IWT685 strain in the range of 1 to 99% by weight and the Lactococcus lactis SBS-0001 strain and / or the Lactococcus lactis SBS-0001-S strain in the range of 99 to 1% by weight. It is more preferable to mix the Pediococcus inopinatus IWT685 strain in a range of 10 to 90% by weight with the Lactococcus lactis SBS-0001 strain and / or the Lactococcus lactis SBS-0001-S strain in a range of 90 to 10% by weight, and it is even more preferable to mix the Pediococcus inopinatus IWT685 strain in a range of 40 to 60% by weight with the Lactococcus lactis SBS-0001 strain and / or the Lactococcus lactis SBS-0001-S strain in a range of 60 to 40% by weight.

[0016] The form of the mixed lactic acid bacteria preparation for feed preparation of the present invention is not particularly limited. For example, the Pediococcus inopinatus IWT685 strain and the Lactococcus lactis SBS-0001 strain and / or the Lactococcus lactis SBS-0001-S strain may be freeze-dried into a powder form, mixed with excipients or the like to form a solid, filled into a capsule, or in the form of a liquid ampoule. The freeze-dried preparation form is preferred. The mixed lactic acid bacteria preparation for use in preparing feed can be added directly to the raw materials of fermented feed, but it is preferable to use it dissolved in water or the like. The amount of the mixed lactic acid bacteria preparation for feed preparation of the present invention is preferably 1.0E+2 to 1.0E+6 bacterial count (1.0E+2 to 1.0E+6 CFU / g, where CFU stands for colony-forming unit) of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001 and / or Lactococcus lactis SBS-0001-S per gram of raw material. More specifically, the amount of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001 and / or Lactococcus lactis SBS-0001-S added is more preferably 1.0E+3 to 1.0E+5 bacterial count (1.0E+3 to 1.0E+5 CFU / g).

[0017] (Feed, fermented feed and preparation method thereof) The feed of the present invention, particularly the fermented feed such as silage, contains the Pediococcus inopinatus strain IWT685 and the Lactococcus lactis strain SBS-0001 and / or the Lactococcus lactis strain SBS-0001-S. The method for preparing the fermented feed such as silage will be explained below. In the present invention, when producing fermented feed such as silage, the growth ability is significantly improved by adding the mixed lactic acid bacteria preparation for feed preparation of the present invention to the fermented feed raw material, so that not only can high-quality fermented feed be obtained even in a low-temperature environment, but also a decrease in pH and an increase in lactic acid content can be achieved quickly even in a room-temperature environment. In the present invention, it is essential to add the mixed lactic acid bacteria preparation for use in preparing feed of the present invention to fermented feed ingredients. The method of addition is preferably such that the mixed lactic acid bacteria preparation for use in preparing feed of the present invention is uniformly distributed throughout the raw material, preferably uniformly. For example, the mixed lactic acid bacteria preparation for use in preparing feed of the present invention can be suspended in water and sprayed, or mixed and stirred. The mixed lactic acid bacteria preparation for use in preparing feed of the present invention is preferably added at a bacterial count level of 1.0E+2 to 1.0E+6 (1.0E+2 to 1.0E+6 CFU / g) per gram of raw material. More specifically, it is more preferable to add the mixed lactic acid bacteria preparation for use in preparing feed of the present invention at a bacterial count level of 1.0E+3 to 1.0E+5 (1.0E+3 to 1.0E+5 CFU / g). The mixed lactic acid bacteria preparation for use in preparing feed of the present invention is preferably added before the start of feed fermentation, but may also be added during the feed fermentation process. The fermentation can be carried out under the same conditions as normal feed fermentation, i.e., under anaerobic conditions at ambient temperature for 30 days or more, such as the normal method using a silo, the roll bale silage method, or the flexible container bag method. The fermented feed obtained by fermentation with the addition of the mixed lactic acid bacteria preparation for feed preparation of the present invention is a high-quality fermented feed with a low pH and a high lactic acid content, and is suitable in that the growth of harmful microorganisms such as coliform bacteria, yeast, and mold is suppressed during the fermentation process.Specific examples of the fermented feed produced include silage and TMR fermented feed. [Example]

[0018] The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited to these examples. Example 1 Isolation of Lactococcus lactis SBS-0001-S strain and characterization of the strain (1) Isolation of strains Lyophilized cells of the SBS-0001 strain were cultured statically in MRS (de Man, Rogosa, Sharpe) liquid medium (Difco) at 37°C for 16 hours, and 1 mL of the culture medium was dispensed into an Eppendorf tube and centrifuged at 3000 rpm for 15 minutes at 4°C using a micro high-speed refrigerated centrifuge (Tomy Kogyo Co., Ltd., MX-307). The supernatant was then discarded. The bacterial pellet was suspended in 1 mL of 10% glycerol solution and frozen at -20°C for 24 hours. After 24 hours, it was thawed at room temperature, serially diluted, and plated on MRS agar medium, followed by anaerobically culturing at 37°C for 24 hours. Anaerobic conditions were created by placing an Anaerobic Jar (Mitsubishi Gas Chemical Company, Inc.) inside the jar. Ten strains of the resulting colonies were selected and inoculated again into MRS liquid medium using a platinum loop, cultured at 37°C for 16 hours, centrifuged in the same manner as above, suspended in a 10% glycerol solution, and frozen at -20°C for 24 hours. After 24 hours, the strains were thawed at room temperature, and the bacterial counts of the 10 strains were measured by serial dilution. From the plate containing the strains with the highest bacterial counts, 10 more strains were selected. This method was repeated two more times, and the final 10 strains obtained were cultured in MRS medium at 37°C for 16 hours, centrifuged using the same method, suspended in 1 mL of 10% glycerin, and freeze-dried together in the Eppendorf tube. The freeze-drying was carried out using Kyowa Vacuum Co., Ltd.'s RLEII-103, and the conditions were as follows: pre-freezing at -50°C, followed by raising the shelf temperature to 60°C over 450 minutes, and then further drying at the same temperature for 2 days. Ten freeze-dried strains were subjected to serial dilution to determine the bacterial count. Four strains with high viability were serially diluted. Single colonies of each strain were picked from the MRS agar plate with a platinum loop and inoculated into MRS liquid medium. The cultures were then cultured at 37°C for 16 hours. The cells were then centrifuged and collected in the same manner as above. The cells were then suspended in a 10% glycerin solution and stored at -80°C. The serial dilutions were carried out by adding 1 g of freeze-dried lactic acid bacteria powder and sterile distilled water to a 100 mL measuring flask, adding the diluted solution, and suspending the mixture thoroughly. Test tubes containing 9 mL of sterile distilled water were capped with steel caps and autoclaved (121°C, 15 minutes). This procedure was repeated to dispense 1 mL of the above-described suspended lactic acid bacteria solution. The four strains were scraped from -80°C stocks with a platinum loop and inoculated into MRS liquid medium. Cultures were then conducted at 37°C for 16 hours. Five mL of each culture medium from the four strains was mixed to prepare a 20 mL mixed lactic acid bacteria solution. Ten mL of this lactic acid bacteria solution was then inoculated into 2.0 L of medium (2% glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), 1% SK yeast extract (Nippon Paper Industries, Ltd.), 1% Hypopolypton N (Shiotani MS Co., Ltd.), 0.02% magnesium sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.01% manganese sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.01% ferrous sulfate (Fujifilm Wako Pure Chemical Industries, Ltd.), 0.1% polyoxyethylene sorbitan monooleate, and 1% sodium chloride), and the mixture was cultured in a jar fermenter (Takasugi Seisakusho, TS-M3L). The culture conditions were 25°C, 16 hours, and pH 5.5. The alkaline solution used to maintain the pH was 40% sodium hydroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). After the culture was completed, the mixture was centrifuged at 4°C, 6500 rpm for 15 minutes (using a Hitachi himac20GII rotor R9A manufactured by Hitachi, Ltd.), the supernatant was discarded, and a protective agent (1.5% monosodium glutamate manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 15.0% lactose manufactured by the same company, and 0.6% calcium carbonate manufactured by the same company) was added in an amount of 1 / 20 of the culture medium, mixed, and spread thinly on an aluminum plate, followed by freeze-drying under the same conditions as above. The freeze-dried cells were crushed and placed in an aluminum bag (AL-E, manufactured by Nippon Seisan Co., Ltd.) and stored at 37°C for 6 months. After that, the cells were inoculated into MRS liquid medium and cultured at 37°C for 16 hours. The colonies obtained by serial dilution on MRS agar medium were purified and designated the SBS-0001-S strain.

[0019] (2) Strain characteristics The SBS-0001 and SBS-0001-S strains were cultured in jar fermenters using the same method as for obtaining the strains, and after centrifugation, the cells were dispersed in four types of protective agents and freeze-dried using the same method, and the viable cell count was investigated using the serial dilution method. The viable cell counts (CFU / g) of SBS-0001 and SBS-0001-S strains, and the ratio of the viable cell count of SBS-0001-S to that of SBS-0001, are shown in Table 1 below as "survival." The protective agents A to D in Table 1 are as follows: Protectant A: Monosodium glutamate 1.5%, lactose 15.0%, calcium carbonate 0.6% Protectant B: Lactose 15.0%, Calcium Carbonate 0.6% Protectant C: Monosodium glutamate 1.5%, lactose 15.0% Protectant D: Lactose 15.0% The bacterial counts of the SBS-0001 strain and the SBS-0001-S strain in the culture medium were 1.39E+10 CFU / mL and 1.56E+10 CFU / mL, respectively.

[0020] [Table 1]

[0021] As shown in Table 1, it was confirmed that the survival rate of the SBS-0001-S strain after freeze-drying was significantly improved by 2.3 to 27.3 times compared to the parent strain SBS-0001.

[0022] <Example 2> Test to confirm the synergistic effect of the mixed lactic acid bacteria preparation for feed preparation of the present invention The effects of using Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S strains in combination as a mixed lactic acid bacteria preparation for feed preparation of the present invention were confirmed in detail. (Test lactic acid bacteria) Pediococcus inopinatus strain IWT685 Lactococcus lactis strain SBS-0001-S As the mixed lactic acid bacteria preparation for feed preparation of the present invention, a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain was used. (Test Method) The raw materials were the staple rice varieties "Daichi no Hoshi" and "Emimaru" harvested from the fields of a cooperative farm in Shinshinotsu. The raw materials were chopped to a theoretical cutting length of 2 cm using a mount cutter (IHI Agritech Cylinder Cutter SCR1900), then frozen and stored at -30°C. The chopped materials were then re-thawed and mixed in equal amounts of each variety. The growth and fermentation characteristics of the silage prepared using the method described below were investigated. Silage was prepared by wrapping in vinyl film. Specifically, a bacterial solution was prepared by cultivating each test lactic acid bacteria in 700 g of raw material so that the total bacterial count of each test lactic acid bacteria was 1.0E+5 CFU / g. After spraying, 100 g of grass was placed in each pouch bag (Hiryu N-9, manufactured by Asahi Kasei Pax Corporation), and the pouches were vacuum-sealed using a commercial tabletop sealing and packaging machine (SQ-303W, manufactured by Asahi Kasei Pax Corporation) to produce silage. The storage temperature was 30°C, and the storage period was 18 days. The number of replications was 3. The organic acid content was analyzed by weighing 30 g of opened silage into a Stoma Filter (Central Scientific Trading Co., Ltd.), adding 90 mL of sterilized distilled water using an autoclaved (121°C, 15 minutes) measuring cylinder, and kneading the silage for 1 minute using a Masticator S (Central Scientific Trading Co., Ltd.). After kneading, the silage was left to stand overnight at 4°C to allow the water-soluble components in the silage to migrate to the water fraction. After standing overnight, the sample was filtered using filter paper (Advantec Toyo Co., Ltd., quantitative filter paper No. 5A). The filtrate was used to measure pH and analyze the organic acid content. pH was measured using a pH meter (Horiba, Ltd., benchtop pH / water quality analyzer F-73).

[0023] (Analysis method) For the analysis of organic acid content, the filtrate was diluted 10 times with pure water and then filtered through a filter with a pore size of 0.22 μm (Millex GP, manufactured by Merck Millipore) to prepare a measurement sample. The organic acid content was analyzed by high-performance liquid chromatography (HPLC) using LC-20AD, LC-30AD, SIL-30AC, CTO-20A, and SPD-M20A (all manufactured by Shimadzu Corporation) with a post-column method. The column used for organic acid separation was TSKgel OApak-A (manufactured by Tosoh Corporation), with a column temperature of 40°C and a flow rate of 0.8 mL / min for both solutions A and C. The mobile phase (solution A) was a 75 mM sulfuric acid solution, and the reaction solution (solution C) was a bromothymol blue solution (0.1 mM BTB, 7.5 mM Na2HPO4). In addition, calibration curves for lactic acid, acetic acid, and butyric acid were prepared. Lactic acid was quantified by preparing a calibration curve using standard solutions of 0.1 wt%, 0.05 wt%, and 0.01 wt%. Acetic acid was quantified by preparing a calibration curve using standard solutions of 0.05 wt%, 0.025 wt%, and 0.005 wt%. Butyric acid was quantified by preparing a calibration curve using standard solutions of 0.01 wt%, 0.005 wt%, and 0.001 wt%. The pH values ​​and organic acid contents (wt% fresh) are shown in Table 2 below. In addition, Table 2 shows that there is a difference between the different lowercase letters "a," "b," and "ab" at a 5% significance level (<0.05) using the Tukey method.

[0024] [Table 2]

[0025] As shown in Table 2, after 18 days, the area treated with Pediococcus inopinatus IWT685 alone had a higher pH, lower lactic acid content, higher acetic acid content, and higher butyric acid content than the area treated with the combined use of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S, a specific example of a mixed lactic acid bacteria formulation for feed preparation of the present invention. After 18 days, the Lactococcus lactis SBS-0001-S strain-only treatment group had a higher pH, lower lactic acid content, higher acetic acid content, and higher butyric acid content than the group treated with the combined use of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain, a specific example of a mixed lactic acid bacteria formulation for feed preparation of the present invention. On the other hand, the treatment area with a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain, which is a specific example of a mixed lactic acid bacteria preparation for feed preparation of the present invention, showed a lower pH, a higher lactic acid content, a lower acetic acid content, and in particular, undetectable butyric acid content compared to the untreated treatment area, the IWT685 treatment area, and the SBS-0001-S treatment area. Butyric acid bacteria are strict anaerobic bacteria, and are known to begin to function when the silo becomes completely anaerobic after the proliferation of lactic acid bacteria, which are generally facultative anaerobes.In addition, they are highly sensitive to pH, and it is believed that their proliferation stops at pH levels of 4.2 or lower. In this test, the silage was made by deaerating and sealing it, so there was a certain degree of anaerobic conditions from the beginning, but it was confirmed that there was no change in the significance of lactic acid bacteria, which are facultative anaerobes, at the start. In other words, when attempting to inhibit the growth of butyric acid bacteria by lowering the pH, it is extremely important to lower the pH quickly before the butyric acid bacteria become active. As shown in Table 2, in the treatment area with a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain, which is a specific example of a mixed lactic acid bacteria preparation for feed preparation of the present invention, the pH dropped to 4.36 on the 18th day, which is the early opening date, and in addition, no butyric acid was detected, making it extremely useful compared to the treatment areas with each strain alone.

[0026] Example 3 Confirmation test of nisin resistance in Pediococcus inopinatus strain IWT685 (Test Method) Nisin A was prepared by dissolving a nisin preparation with known activity (containing 2.5% nisin, manufactured by Sigma) in water, and the nisin activities were adjusted to 250, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 IU / mL for use in the test. Nisin Z was prepared by culturing Lactococcus lactis SBS-0001-S strain in 10 mL of MRS (de Man, Rogosa, Sharpe) medium (manufactured by Difco) at 37°C for 2 days, centrifuging at 12,000 rpm for 5 minutes, and sterilizing the supernatant with a 0.22 μm pore size filter (Millex GP, manufactured by Merck Millipore). The culture supernatant before dilution was used as the ×1 solution, and serial dilutions were made up to ×2, ×4, ×8, ×16, ×32, and ×64 using sterile water, and these were used. Nisin activity was measured using antibacterial activity against nisin-sensitive strains of lactic acid bacteria as an index. A penicillin cup (8 mm in diameter) was placed on a plate of 1.5% agar MRS medium, and Lactobacillus delbrueckii subsp. bulgaricus type strain JCM1002 was used as an indicator strain. T A 0.75% agar MRS medium containing 0.1% of the strain's culture medium was layered on top. After the agar solidified, the penicillin cup was removed and used as an antibacterial test plate. 100 μL of the culture supernatant, which had been passed through a 0.22 μm pore size filter (Millex GP, Merck Millipore), was added to the circular wells, with the penicillin cup placed to prevent the agar from flowing in. After 24 hours of anaerobic incubation at 37°C, the diameter of the inhibition zone for the indicator bacteria was measured, and the activity of Nisin Z was calculated by applying this to a calibration curve prepared from the inhibition zone length of Nisin A, which has known activity. 100 mL of the above nisin solution and 2×MRS solution were mixed in a 96-well microplate, and the following bacterial strains were inoculated at 1%. After culturing at 30° C. for 3 days, the OD600 was measured. The sensitivity to nisin was determined by determining the nisin activity at which the OD600 of the control group, in which water was added instead of nisin solution, was 100%, and this was taken as the highest nisin concentration at which growth was possible. The results are summarized in Table 3 below. (Test strain) Pediococcus inopinatus type strain JCM12518 T KK Pediococcus inopinatus strain IWT685 Pediococcus damnosus type strain JCM5886 T KK Pediococcus dextrinicus type strain JCM5887 T strain (currently renamed to Rapidilactobacillus dextrinicus due to genus change) Pediococcus pentosaceus type strain JCM5890 T KK Pediococcus acidilactici type strain JCM8797 T KK

[0027] [Table 3]

[0028] As shown in Table 3, the type strain of Pediococcus inopinatus, JCM12518 T The strain and the Pediococcus inopinatus strain IWT685 were found to be highly sensitive to nisin A and highly resistant to nisin Z. This study has revealed for the first time that this characteristic is specific to the Inopinatus species, and not to the Pediococcus genus in general.

[0029] Example 4 Test 1: Confirmation of silage quality of staple rice varieties using the mixed lactic acid bacteria preparation for feed preparation of the present invention (Test lactic acid bacteria) As the mixed lactic acid bacteria preparation for feed preparation of the present invention, a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain was used. The commercially available product used was Chikusa No. 1 Plus (manufactured by Snow Brand Seed Co., Ltd.). (Test Method) The treatments were either no additives, Chikusa No. 1 plus, or a mixed formulation of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S. Staple rice (Emimaru) was used as the silage material, and was chopped using a mount cutter (IHI Agritech cylinder cutter SCR1900) to a theoretical cutting length of 2 cm. Chikusou No. 1 Plus was sprayed onto 400g of raw materials using a small spray bottle in the specified amount according to the product's instructions. For the mixed formulation, a lactic acid bacteria mixed powder prepared by mixing each freeze-dried bacterial powder was dissolved in water to give a concentration of 1.0E+5 CFU / g of raw material, and then sprayed onto 400g of raw material using a small spray bottle. After the addition, 100 g of the mixture was placed in each pouch (Hiryu N-9, manufactured by Asahi Kasei Pax Corporation), and the silage was prepared by vacuum sealing using a commercial tabletop sealing and packaging machine (SQ-303W, manufactured by Asahi Kasei Pax Corporation) and stored at 25°C for two months. The experiment was repeated three times.

[0030] (Analysis method) The organic acid content was analyzed by weighing 30 g of opened silage into a Stoma Filter (Central Scientific Trading Co., Ltd.), adding 90 mL of sterilized distilled water using an autoclaved (121°C, 15 minutes) measuring cylinder, and kneading the silage for 1 minute using a Masticator S (Central Scientific Trading Co., Ltd.). After kneading, the silage was left to stand overnight at 4°C to allow the water-soluble components in the silage to migrate to the water fraction. After standing overnight, the sample was filtered using filter paper (quantitative filter paper No. 5A, manufactured by Advantec Toyo Co., Ltd.). The filtrate was used for pH measurement, VBN analysis, and organic acid content analysis. pH was measured using a pH meter (Horiba, Ltd., benchtop pH / water quality analyzer F-73). For the organic acid content analysis, the filtrate was diluted 10-fold with pure water and then filtered through a filter with a pore size of 0.22 μm (Millex GP, manufactured by Merck Millipore) to obtain a measurement sample. The lactic acid content (wt % fresh), acetic acid content (wt % fresh), and butyric acid content (wt % fresh) were measured in the same manner as in the organic acid content analysis by HPLC described in Example 2.

[0031] VBN (Volatile Basic Nitrogen) stands for volatile basic nitrogen, as described in the literature (Self-Sufficient Feed Utilization Research Group (ed.) (2009) Third Edition, Roughage Quality Evaluation Guidebook, Japan Grassland Livestock Seed Association, Tokyo, p. 68), and it has been found that in silage, most of it is ammonia. The VBN value was calculated using the steam distillation method. Specifically, 20 mL of the filtrate was placed in a Kjeldahl distillation apparatus (VAP500C, Gerhardt Japan), alkali (35% NaOH solution) was added to make it alkaline, and distillation was carried out for approximately 4 to 5 minutes. The evaporated ammonia was recovered in 40 mL of 3% boric acid solution, and the recovered solution was titrated with 1 / 10 N sulfuric acid solution. The titer was taken as B mL, and the VBN value (mg / 100 g) was calculated using the following formula, taking into account the 4-fold dilution ratio when preparing the filtrate in the "Analysis Method" above. [Calculation formula] VBN value = (1.401 x B) / filtrate volume (20 mL) x 4 x 100 / 1000

[0032] The V2 score is an index that evaluates the silage fermentation quality by scoring it, and is calculated using the following formula. As shown in the calculation formula below, a large number of points are allocated to the ammonia and butyric acid content. It is generally known that excessive intake of butyric acid and ammonia not only has adverse effects on cows (such as the onset of ketosis), but also has a significant impact on palatability. For this reason, dairy farmers prefer silage with low levels of these substances, i.e., a high V2 score. [Calculation formula] V2=A'+B'+C' About "A'" VBN value is A If A is 20 or less, A' = 50 If A is greater than 20 and less than 200, A' = 50 + (100 - 5A) / 18 If A is greater than 200, A'=0 About "B'" Acetic acid content (%) + propionic acid content (%) = B If B is 0.2 or less, B'=10 If B is greater than 0.2 and less than or equal to 1.5, B' = (150 - 100B) / 13 If B is greater than 1.5, then B'=0 The content (%) of volatile fatty acids higher than butyric acid (specifically, the total content of butyric acid, caproic acid, and valeric acid, but most of it is butyric acid) is defined as C. If C is between 0 and 0.5, C' = 40-80C If C is greater than 0.5, C'=0 To determine moisture content, the sealed pouches containing the silage were opened, and after taking the samples required for organic acid content analysis and pH analysis, three replicates of the silage were placed together in tared paper bags (ASKUL, square bottom paper bags No. 12). The fresh weight (fresh weight of the silage) was measured, and the bag was then exposed to a 60°C dryer for two days. The weight after removal (dry weight + bag weight) was taken as the dried weight and calculated using the following formula. [Calculation formula] Moisture (weight %) = (fresh weight - (weight after drying - tare)) ÷ fresh weight x 100 The pH value, organic acid content (lactic acid, acetic acid, butyric acid), VBN value (mg / 100g raw material), moisture content (%), and V2 score for each treatment are summarized in Table 4.

[0033] [Table 4]

[0034] As shown in Table 4, silage of staple rice varieties prepared using a mixed preparation of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S, which is a specific example of a mixed lactic acid bacteria preparation for feed preparation according to the present invention, tended to have a lower pH, a higher lactic acid content, and lower acetic acid and butyric acid contents than the commercially available product, Chikusa No. 1 Plus. It was also confirmed that the VBN value was significantly lower and the V2 score was also significantly higher. These results demonstrate that the mixed lactic acid bacteria preparation for feed preparation of the present invention achieves a significant decrease in pH, an increase in lactic acid production, and an early suppression of acetic acid and butyric acid production compared to known techniques, and exhibits excellent fermentation characteristics even at room temperature. In addition, Table 4 shows that there is a difference between the different lowercase letters "a," "b," and "c" at a 5% significance level (<0.05) using the Tukey method.

[0035] <Example 5> Test 2: Confirmation of silage quality of staple rice varieties using the mixed lactic acid bacteria preparation for feed preparation of the present invention (Test lactic acid bacteria) Lactococcus lactis strain SBS-0001-S Pediococcus inopinatus strain IWT685 As the mixed lactic acid bacteria preparation for feed preparation of the present invention, a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain was used. (Test Method) The treatments were: no addition, Lactococcus lactis SBS-0001-S strain alone, Pediococcus inopinatus IWT685 strain alone, and a mixture of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S strains. The silage raw material was a staple rice variety (Sorayutaka) harvested from the fields of a farmer cooperating in the trial in Shinshinotsu, and was shredded using a mount cutter (IHI Agritech cylinder cutter SCR1900) to a theoretical cutting length of 2 cm. The growth and fermentation characteristics of the silage prepared by the following method were investigated. Silage was prepared by wrapping with vinyl film. Specifically, a culture solution of each test lactic acid bacteria was prepared by cultivating it in 700 g of raw material so that the total bacterial count of each test lactic acid bacteria was 1.0E+5 CFU / g. After spraying, 100 g of the raw material was placed in each pouch bag (Hiryu N-9, manufactured by Asahi Kasei Pax Co., Ltd.), and the bag was deaerated and sealed using a commercial tabletop sealing packaging machine (SQ-303W, manufactured by Asahi Kasei Pax Co., Ltd.) to create silage. The storage temperature was 25°C, and the storage periods were 1 day, 3 days, 7 days, 1 month, and 2 months. The number of replicates was 1 for storage periods of 1, 3, and 7 days, and 3 for storage periods of 1 month and 2 months, and evaluation was based on the average value. The analysis of the organic acid content and confirmation of the pH were carried out in the same manner as in Example 2. The moisture content was determined by opening the bag, taking the samples required for organic acid content analysis and pH analysis, and then placing three replicates of the silage into tared paper bags (ASKUL, square bottom paper bags No. 12). The fresh weight (fresh weight of the silage) was measured, and the bag was then exposed to a 60°C dryer for two days. The weight after removal (dry weight + bag weight) was considered the dried weight, and calculated using the following formula. [Calculation formula] Moisture (weight %) = (fresh weight - (weight after drying - tare)) ÷ fresh weight x 100 The pH values ​​and organic acid content analysis results after storage for 1 day, 3 days, and 7 days are shown in Table 5 below, the pH values, organic acid content analysis results, and moisture content after storage for 1 month are shown in Table 6 below, and the pH values, organic acid content analysis results, and moisture content after storage for 2 months are shown in Table 7 below. In addition, Tables 6 and 7 show that there is a difference between different lowercase letters "a," "b," "c," "d," "ab," and "bc" at the 5% significance level (<0.05) using the Tukey method.

[0036] [Table 5]

[0037] [Table 6]

[0038] [Table 7]

[0039] As shown in Table 5, the silage of staple rice varieties prepared using a mixed preparation of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S, a specific example of a mixed lactic acid bacteria preparation for feed preparation according to the present invention, showed a decrease in pH and accumulation of lactic acid from the early stages of storage, at 1, 3, and 7 days. The fermentation quality after storage for 1 and 2 months was also significantly lower than that of other treatments, with a high lactic acid content and a low butyric acid content, confirming that the silage was of good quality, with a pH of 4.2 or less. In other words, it was revealed that the mixed lactic acid bacteria preparation for feed preparation of the present invention achieves a significant decrease in pH, an early increase in lactic acid production, and an early suppression of butyric acid production, and exhibits excellent fermentation characteristics even at room temperature. On the other hand, in the SBS-0001-S strain-only treatment area, compared to the untreated area and the IWT685 strain-only treatment area, an early decrease in pH and accumulation of lactic acid content were observed in the early storage periods of 1, 3, and 7 days. However, after 1 and 2 months of storage, the silage did not become of high quality with a pH of 4.2 or less, and butyric acid fermentation was also observed. Furthermore, in the IWT685 strain-only treatment area, a decrease in pH and accumulation of lactic acid were observed in the early stages of storage, 1, 3, and 7 days, compared to the untreated treatment area. However, compared to the SBS-0001-S strain-only treatment area, the rate of decrease in pH and accumulation of lactic acid was slower, and the fermentation quality after 1 and 2 months of storage did not result in high-quality silage with a pH of below 4.2, and butyric acid fermentation was also observed.

[0040] Example 6 Fermentation quality test of general feed rice using the mixed lactic acid bacteria preparation for feed preparation of the present invention (Test lactic acid bacteria) As the mixed lactic acid bacteria preparation for feed preparation of the present invention, a mixed preparation of Pediococcus inopinatus IWT685 strain and Lactococcus lactis SBS-0001-S strain was used. The commercially available product used was Chikusa No. 1 Plus (manufactured by Snow Brand Seed Co., Ltd.). (Test Method) The treatments were: no additives, Chikusa No. 1 Plus product (manufactured by Snow Brand Seed Co., Ltd.), and a mixed formulation of Pediococcus inopinatus strain IWT685 and Lactococcus lactis strain SBS-0001-S. Roll bale silage was prepared using a chopped whole crop harvester (manufactured by Takakita Co., Ltd.) and a self-propelled wrapping machine (manufactured by Takakita Co., Ltd.) on August 10-11, 2021. The silage raw material was the forage rice variety "Yumeaoba." The mixed formulation was prepared by mixing equal amounts of each freeze-dried powder of lactic acid bacteria and adding it to the silage raw material using a pressure-type sprayer (HS-503W, Koshin Co., Ltd.) to achieve 1.0E+5 CFU / g raw material. The specified amount of Chakusa No. 1 Plus was added using the pressure-type sprayer according to the product's instructions. The silage was stored outdoors for 30 days. (Analysis method) The moisture content of the sample was determined by placing the silage immediately after opening into a tare-measured paper bag (High Bag H20 unbleached plain paper bag manufactured by Pack Takeyama Co., Ltd.), measuring the weight before drying (fresh weight of silage + tare weight), drying at 50°C for at least one week, and then measuring the weight after drying (weight of dried silage + tare weight) using the following formula. The results are shown in Table 8 below. [Calculation formula] Moisture (%) = (Weight before drying - Weight after drying) ÷ (Weight before drying - Tare weight) x 100 The pH measurement and organic acid content analysis of the samples were carried out as follows. 100 g of silage was refrigerated and soaked in 500 mL of distilled water for 24 hours, and the extract obtained by filtering (quantitative filter paper No. 5A, manufactured by Advantec Toyo Co., Ltd.) was used for the study. The pH value was measured using a pH meter (D-71, manufactured by Horiba, Ltd.), and the lactic acid content (wt% fresh), acetic acid content (wt% fresh), and butyric acid content (wt% fresh) were measured using a high-performance liquid chromatograph (LC-2030C, manufactured by Shimadzu Corporation). The results are shown in Table 8 below. In addition, Table 8 shows that there is a difference between the different lowercase letters "a" and "b" at a 5% significance level (<0.05) using the Tukey method. VBN was analyzed according to the method described in Example 4, and the fermentation quality was evaluated by calculating the V2 score. The analytical conditions for high performance liquid chromatography are as follows. Column: HPX-87H (BIO RAD) Column temperature: 40℃ Detector: Differential refractive index detector (RID-20A, manufactured by Shimadzu Corporation) Mobile phase: 0.01 N sulfuric acid Fluid delivery speed: 0.6 mL / min

[0041] [Table 8]

[0042] As shown in Table 8, general feed rice silage prepared using a mixed preparation of Pediococcus inopinatus IWT685 and Lactococcus lactis SBS-0001-S, which is a specific example of a mixed lactic acid bacteria preparation for feed preparation of the present invention, had a higher lactic acid content, lower acetic acid and butyric acid contents, and tended to have a lower VBN, even in full-scale silage, compared to the commercially available product, Chikusa No. 1 Plus, and resulted in a higher V2 score. These results confirm that the mixed lactic acid bacteria preparation for feed preparation of the present invention can achieve a significant decrease in pH, an increase in lactic acid production, and an inhibition of acetic acid production and butyric acid production earlier than known techniques, and has excellent fermentation properties even at room temperature, making it extremely useful in practical situations. [Industrial Applicability]

[0043] The mixed lactic acid bacteria preparation for feed preparation of the present invention maintains the characteristics of the lactic acid bacteria Pediococcus inopinatus IWT685 strain (NITE P-03829), such as its excellent low-temperature growth ability and the ability to promote fermentation by increasing the production of organic acids such as lactic acid even in low-temperature environments, enabling the production of high-quality livestock feed.However, when used in combination with Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099), the growth ability is significantly improved, resulting in a significant decrease in pH, an increase in lactic acid production, and an inhibition of butyric acid production, making it useful for producing high-quality livestock feed. Furthermore, the mixed lactic acid bacteria preparation for preparing feed of the present invention has excellent fermentation properties even at room temperature, and therefore has the new effect of significantly contributing to livestock feeding, making it extremely useful industrially.

Claims

1. Pediococcus inopinatus IWT685 strain (NITE P-03829) and Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099), A mixed lactic acid bacteria preparation for feed preparation containing

2. A feed containing Pediococcus inopinatus IWT685 strain (NITE P-03829) and Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099).

3. Pediococcus inopinatus IWT685 strain (NITE P-03829) and Lactococcus lactis SBS-0001 strain (NITE BP-1107) and / or Lactococcus lactis SBS-0001-S strain (NITE BP-04099), Contains fermented feed.

4. A method for preparing fermented feed, comprising using the mixed lactic acid bacteria preparation for preparing feed according to claim 1 and a fermented feed material.

5. The method for preparing fermented feed according to claim 4, wherein the fermented feed material is feed rice.

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