Pediococcus acidilactici m4 for low-moisture fermented feed and application thereof

Abstract

The application provides a pediococcus acidilactici M4 for low-moisture fermented feed and an application thereof, and belongs to the field of microbial application and feed technology. The application uses the pediococcus acidilactici M4 to prepare fermented total mixed ration. The bacteria have the characteristics of high osmosis and fast acid production, and can reduce the pH value of the fermentation system in a short time; have strong broad-spectrum bacteriostatic effect on escherichia coli, chicken white dysentery salmonella and staphylococcus aureus; and perfectly adapt to the fermentation of low-moisture total mixed ration. The strain is used as the fermentation strain of low-moisture total mixed ration, can significantly reduce the content of acid detergent fiber and neutral detergent fiber, improve the crude protein level, and then improve the digestibility of the daily ration, increase the content of probiotics such as lactic acid bacteria and yeast in the daily ration, reduce the content of pathogenic bacteria such as escherichia coli in the daily ration, and also can improve the content of volatile fatty acid in the daily ration, improve the quality of the daily ration, and improve the feeding value of the daily ration.

Description

Technical Field

[0001] This invention belongs to the field of microbial application and feed technology, specifically relating to a strain of Pediococcus lactis M4 for use in low-moisture fermented feed and its application. Background Technology

[0002] my country has abundant crop straw resources, with an annual output of hundreds of millions of tons. Straw, as a potential source of roughage, has broad development prospects. Fermented total mixed rations (TMRs) are a form of feed made by mixing, wrapping, sealing, and fermenting complete diets. Under oxygen-free conditions, they achieve long-term storage through microbial action, offering advantages such as nutritional balance, high stability, and ease of transportation and commercialization. Therefore, in Xinjiang, where forage resources are unevenly distributed, developing fermented TMRs with straw as the main raw material is of great significance for alleviating local feed shortages and reducing livestock costs.

[0003] However, in actual production, the low moisture content, high crude fiber content, and strong buffering capacity of straw raw materials result in high residual oxygen and insufficient water activity within the fermented total mixed ration (TMR) wrappers. This severely affects the growth and metabolic activity of lactic acid bacteria, thereby inhibiting lactic acid production and feed acidification, ultimately impacting fermentation success rate and product quality. Therefore, providing a strain capable of efficient fermentation has become a key measure to improve the fermentation quality of straw-based TMRs. Existing single-inoculum agents have poor adaptability in low-moisture, high-crude-fiber straw-based TMR systems, making it difficult to achieve rapid, stable, and efficient fermentation, and failing to meet the actual production requirements for fermentation quality. Currently, there is no dedicated *Pediococcus lactis* strain capable of stable fermentation in straw-based TMR systems with 40%–50% low moisture and high osmotic pressure, making it difficult to meet the actual needs of low-moisture feed fermentation in arid regions of Xinjiang. Summary of the Invention

[0004] The purpose of this invention is to address the deficiencies in the prior art by providing a strain of *Pediococcus lactis* (…). Pediococcus acidilactici M4, the lactic acid cocci M4, can be used as a single inoculum agent for the efficient fermentation of low-moisture, high-coarse-fiber straw-based total mixed diets, improving the storage stability and feeding value of fermented total mixed diets, and improving the quality of fermented total mixed diets.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Pediococcus lactis M4, with accession number CCTCC NO: M 20253043.

[0006] This invention provides the application of the above-described Pediococcus lactis M4 in the preparation of lactic acid and / or the degradation of straw; the straw includes corn straw.

[0007] This invention provides the application of the above-described Pediococcus lactis M4 in inhibiting mold in feed fermentation.

[0008] This invention provides the application of the above-described Pyrococcus lactis M4 in feed preparation under low moisture conditions; the low moisture conditions include a fermentation system with a water content of 40% to 50%.

[0009] This invention provides a method for preparing feed, comprising the following steps: After mixing roughage and complete feed, inoculate with Pediococcus lactis M4, adjust the moisture content of the system to ≤50%, and carry out vacuum-sealed fermentation to obtain feed.

[0010] This invention provides a method for preparing fermented total mixed diets under low moisture conditions, comprising: After mixing roughage and complete feed, the moisture content of the mixture was adjusted to 40%~50%, and then inoculated with Pediococcus lactis M4 for fermentation to obtain fermented total mixed diet. The coarse feed includes corn stalks; the complete feed includes corn, sprayed corn husks, cottonseed meal, baking soda, expanded urea, bentonite, dicalcium phosphate, stone powder, and premixed feed. The mass ratio of the coarse feed to the complete feed is (19~20):(30~35).

[0011] This invention provides the application of fermentation of Pediococcus lactis M4 under low moisture conditions in improving feed quality; the low moisture conditions include a fermentation system with a moisture content of 40% to 50%.

[0012] Preferably, improving feed quality includes improving the content of volatile fatty acids in the feed and / or reducing the content of crude fiber in the feed; improving the content of volatile fatty acids in the feed includes increasing the content of acetic acid, propionic acid and butyric acid, and reducing the content of isobutyric acid, isovaleric acid and valeric acid; reducing the content of crude fiber in the feed includes reducing the content of neutral detergent fiber and acid detergent fiber in the feed.

[0013] The beneficial effects of this invention are: This invention provides a strain of *Pediococcus lactis* M4, with the preservation number CCTCC NO: M20253043. *Pediococcus lactis* M4 possesses three characteristics: high osmotic pressure, rapid acid production, and broad-spectrum antibacterial activity. *Pediococcus lactis* M4 exhibits highly efficient lactic acid production and can lower the pH of the fermentation system in a short time; it also has a strong broad-spectrum antibacterial effect against *Escherichia coli*, *Salmonella pullorum*, and *Staphylococcus aureus*; and it is perfectly suited for the fermentation of low-moisture total mixed rations (TMR). Using this strain as a fermentation starter for low-moisture TMR can significantly reduce the content of acid and neutral detergent fiber, promote bacterial protein synthesis, and increase crude protein levels, thereby improving the digestibility of the diet, enhancing the feed value of the fermented TMR, and improving the storage stability of the fermented TMR.

[0014] Xinjiang's arid climate means that agricultural byproducts like corn stalks, used as roughage, have extremely low moisture content and a dense lignocellulose structure. Traditional high-moisture silage or fermentation processes typically require a moisture content of over 65%. In actual production in Xinjiang's arid regions, this not only consumes vast amounts of precious water resources but also makes the feed highly susceptible to freezing damage during the cold winter. Furthermore, excessively low moisture content during fermentation leads to loose compaction and high oxygen levels in the feed gaps, resulting in mycotoxin accumulation and fermentation failure. Therefore, developing low-moisture FTMR (Fluid-Free Regulated Mold) with a moisture content controlled at around 50% has become a core requirement for cost reduction and efficiency improvement in Xinjiang. However, the low moisture content and accompanying high osmotic pressure environment severely limit the fermentation efficiency of common lactic acid bacteria, resulting in slow or incomplete fermentation and difficulty in rapidly forming an acidic environment. To address this deficiency, this invention utilizes the homologous fermenting lactic acid spore strain M4. Example test results show that it exhibits high adaptability and excellent acid production efficiency in environments tolerant to high osmotic pressure and low moisture content.

[0015] This invention provides a method for preparing fermented total mixed ration (TMR) under low moisture conditions, comprising: mixing roughage and complete feed, adjusting the moisture content of the mixture to 40%–50%, inoculating with *Pediococcus lactis* M4, and fermenting to obtain the fermented TMR; wherein the roughage includes corn stalks; and the complete feed includes corn, sprayed corn husks, cottonseed meal, baking soda, expanded urea, bentonite, dicalcium phosphate, limestone powder, and premix; and the mass ratio of roughage to complete feed is (19–20):(30–35). The method provided by this invention uses *Pediococcus lactis* M4 as the fermentation inoculum, which can rapidly produce lactic acid, lower the pH of the fermentation system, efficiently promote the degradation of acid and neutral detergent fibers, increase crude protein content, and significantly improve the content of volatile fatty acids, increase the content of lactic acid bacteria and yeast, and reduce the content of *Escherichia coli*, thereby improving the quality of the diet. Simultaneously, *Pediococcus lactis* M4 can also inhibit mold growth during fermentation, ensuring the safety of the fermented feed for consumption.

[0016] Biological Preservation Instructions Pediococcus lactis M4, Latin scientific name: Pediococcus acidilactici It was deposited on December 29, 2025 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20253043. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a streak plate image of strain M4; Figure 2 Growth curves showing the lactic acid production capacity of the six strains obtained through screening. Figure 3 The growth curves for the six selected strains at pH are shown. Figure 4 OD of the 6 selected strains 600 Growth curve of the value; Figure 5 The graph shows the inhibitory effects of strain M4 on different pathogenic bacteria, where a, b, and c represent the inhibitory effects of strain M4 on Escherichia coli, Salmonella pullorum, and Staphylococcus aureus, respectively. Figure 6 This is a phylogenetic tree diagram of strain M4. Detailed Implementation

[0019] This invention provides a strain of Pediococcus lactis M4, with accession number CCTCC NO: M 20253043.

[0020] The *Pediococcus lactis* M4 strain provided by this invention is a probiotic with strong acid-producing ability, broad-spectrum antibacterial properties, and the ability to improve the nutritional value and digestibility of diets.

[0021] This invention isolates and screens lactic acid bacteria strains from naturally fermented corn stalks, exhibiting strong tolerance to high osmotic pressure, high acid production capacity, and broad-spectrum antibacterial activity. Through taxonomic identification, it was determined to be *Pediococcus lactis*, with the Latin scientific name (…). Pediococcus acidilactici The strain was numbered M4 and is called Pediococcus lactis M4.

[0022] In this invention, the lactic acid cocci M4 is screened from fermented straw. After being streaked and cultured on MRS agar solid plates containing calcium carbonate in a 20% PEG-6000 simulated hyperosmolar environment, a clear transparent ring is formed around the colony, indicating that it has outstanding acid production capacity and can be used for fermentation of low-moisture straw feed.

[0023] In this invention, the *Pediococcus lactis* M4 exhibits a significant antibacterial effect and can be used as an antibacterial agent. The antibacterial agent targets one or more of the following bacteria: *Escherichia coli*, *Salmonella pullorum*, *Staphylococcus aureus*, and molds. Through Oxford cup antibacterial tests, this invention demonstrates that *Pediococcus lactis* M4 has a strong broad-spectrum antibacterial effect against *Escherichia coli*, *Salmonella pullorum*, *Staphylococcus aureus*, and molds, with an inhibition zone of 28.47 mm against *Staphylococcus aureus*, 19.61 mm against *Salmonella pullorum*, and 27.03 mm against *Escherichia coli*.

[0024] The *Pediococcus lactis* M4 strain provided by this invention exhibits rapid lactic acid production and pH reduction during fermentation. When the *Pediococcus lactis* M4 strain was statically cultured at 37°C for 36 h, the lactic acid production reached 39.75 mmol / L, and the pH of the bacterial culture decreased to 3.48 after 48 h, significantly superior to conventional lactic acid bacteria strains. This demonstrates the ability to rapidly create an acidic fermentation environment and inhibit the growth of other microorganisms.

[0025] The *Pediococcus lactis* M4 provided by this invention, as a fermentation strain for low-moisture total mixed rations (TMR), can significantly reduce the content of acid detergent fiber and neutral detergent fiber, promote microbial protein synthesis, and increase crude protein levels, thereby improving the digestibility and feed value of the diet. When applied to the fermentation of low-moisture TMR, *Pediococcus lactis* M4 can improve the fermentation quality of the TMR, optimize the nutrient composition, and ensure the feed safety of the fermented TMR by inhibiting mold growth, thus possessing significant application value. In the preparation of low-moisture TMR, the *Pediococcus lactis* M4 provided by this invention significantly increased the crude protein content from 11.11% to 13.22%, significantly decreased the neutral detergent fiber content from 47.85% to 37.93%, and significantly decreased the acid detergent fiber content from 29.73% to 24.32%, greatly improving feed digestibility and optimizing nutrient composition.

[0026] The *Pediococcus lactis* M4 provided by this invention was applied to the preparation of low-moisture fermented total mixed diets (TMCs), and no aflatoxin B1, zearalenone, or vomitoxin was detected in the diets. *Pediococcus lactis* M4 can inhibit the growth of mold in low-moisture fermented TMCs, ensuring the safety of feeding low-moisture fermented TMCs.

[0027] In summary, the *Pediococcus lactis* M4 provided by this invention possesses three characteristics: high osmotic pressure, rapid acid production, and a broad antibacterial spectrum. As a fermentation strain for low-moisture total mixed rations (TMR), *Pediococcus lactis* M4 can significantly reduce the content of acid and neutral detergent fiber, promote microbial protein synthesis, and increase crude protein levels, thereby improving the digestibility of the diet and enhancing its feed value. *Pediococcus lactis* M4 provides a theoretical basis and technical support for improving the utilization level of straw as feed in certain regions and promoting the sustainable development of animal husbandry.

[0028] This invention provides a microbial inoculant comprising *Pediococcus lactis* M4 as described in the above-mentioned technical solution. As an optional embodiment of this invention, the viable count of *Pediococcus lactis* M4 in the microbial inoculant is ≥1×10⁻⁶. 8 cfu / mL, or 1.08×10 8 cfu / mL ~1×10 9 cfu / mL.

[0029] This invention provides a method for preparing the microbial inoculant described in the above-mentioned technical solution, comprising: culturing the *Pediococcus lactis* M4 in a culture medium to obtain the microbial inoculant. This invention does not specifically limit the type of culture medium; any conventional culture medium in the art that allows *Pediococcus lactis* M4 to grow normally can be used. As an optional embodiment of this invention, the culture medium can be MRS broth medium. As an optional embodiment of the present invention, the culture temperature can be 15~37℃, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37℃; the culture time can be 6~48h, or 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48h; the culture is preferably static culture.

[0030] After cultivation, the present invention yields a culture medium, which can also be referred to as a fermentation broth. As an optional embodiment of the present invention, the fermentation broth can be directly used as a microbial inoculant. Alternatively, after obtaining the fermentation broth, the bacterial cells and culture supernatant can be separated, and the obtained bacterial cells or culture supernatant can be used as microbial inoculants, respectively.

[0031] This invention provides the application of the *Pediococcus lactis* M4 described in the above-mentioned technical solution, the microbial agent described in the above-mentioned technical solution, or the microbial agent prepared by the preparation method described in the above-mentioned technical solution in the preparation of antibacterial agents; the antibacterial agents target any one or more of the following bacteria: molds, *Escherichia coli*, *Salmonella pullorum*, and *Staphylococcus aureus*.

[0032] This invention provides the application of the above-described Pediococcus lactis M4, the above-described microbial agent, or the microbial agent prepared by the above-described preparation method in inhibiting mold in feed fermentation.

[0033] This invention provides the application of *Pediococcus lactis* M4, the microbial agent described in the above-mentioned technical solutions, or the microbial agent prepared by the preparation method described in the above-mentioned technical solutions in the preparation of feed under low-moisture conditions. The low-moisture conditions include a fermentation system with a moisture content of 40% to 50%. As an optional embodiment of this invention, the feed includes low-moisture fermented feed; the low-moisture fermented feed includes fermented total mixed ration (TMR). Conventional fermentation of TMR typically requires high moisture content, such as ≥70%, to make the fermentation process more efficient. However, the *Pediococcus lactis* M4 provided by this invention can be efficiently applied to the fermentation of TMR under conditions of ≤50% moisture content, such as 40% to 50%, significantly reducing cellulose content and increasing crude protein content, thereby improving the digestibility and feed value of the feed. As an optional embodiment of this invention, the temperature during feed preparation can be 20 to 30°C, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30°C.

[0034] This invention provides the application of *Pediococcus lactis* M4, the microbial agent described in the above-mentioned technical solutions, or the microbial agent prepared by the preparation method described in the above-mentioned technical solutions in the preparation of lactic acid and / or straw degradation. The *Pediococcus lactis* M4 provided by this invention can rapidly produce lactic acid during cultivation or fermentation and can be used for straw degradation. As an optional embodiment of this invention, the straw includes corn straw. The results of the embodiments of this invention show that *Pediococcus lactis* M4 can efficiently produce lactic acid after 8 hours of cultivation in the culture medium. When *Pediococcus lactis* M4 is applied to the degradation of corn straw and subsequently to the preparation of feed, the lactic acid yield of *Pediococcus lactis* M4 reaches as high as 26.75 mmol / g. This invention degrades corn straw using *Pediococcus lactis* M4 under low moisture content conditions of 40%~50%, which can significantly reduce the content of neutral detergent fiber and acid detergent fiber in corn straw and increase the crude protein content.

[0035] This invention provides a method for preparing feed, comprising the following steps: Roughage and complete feed are mixed and inoculated with Pediococcus lactis M4. The moisture content of the system is adjusted to ≤50%, and vacuum fermentation is carried out to obtain feed. As an optional embodiment of the present invention, the feed includes a fermented total mixed ration (TMR). The following description of the preparation method of the fermented TMR specifically describes the feed preparation method.

[0036] This invention provides a method for preparing fermented total mixed diets under low moisture conditions, comprising: After mixing roughage and complete feed, the moisture content of the mixture was adjusted to 40%~50%, and then inoculated with Pediococcus lactis M4 for fermentation to obtain fermented total mixed diet. The coarse feed includes corn stalks; the complete feed includes corn, sprayed corn husks, cottonseed meal, baking soda, expanded urea, bentonite, dicalcium phosphate, stone powder, and premixed feed. The mass ratio of the coarse feed to the complete feed is (19~20):(30~35).

[0037] This invention mixes coarse feed and complete feed to obtain a fermentation substrate. As an optional embodiment of this invention, the coarse feed includes straw; the straw includes corn straw; the straw is preferably dried straw. This invention does not specifically limit the composition, preparation method, or source of the complete feed; conventional compositions, preparation methods, and products in the art are all acceptable. As an optional embodiment of this invention, the complete feed may consist of corn, sprayed corn husks, cottonseed meal, baking soda, expanded urea, bentonite, dicalcium phosphate, limestone powder, and premix. As an optional embodiment of this invention, by weight, the complete feed may consist of: 16.76 parts corn, 6.85 parts sprayed corn husks, 3.20 parts cottonseed meal, 0.55 parts baking soda, 0.91 parts expanded urea, 0.52 parts bentonite, 0.21 parts dicalcium phosphate, 0.55 parts limestone powder, and 0.92 parts premix. As an optional embodiment of this invention, the complete feed may be purchased from Fuhai County Big Tail Sheep Co., Ltd.

[0038] As an optional embodiment of the present invention, when the coarse feed and the complete feed are mixed, the mass ratio of the coarse feed and the complete feed can be (19~20):(30~35), or it can be 19.53:30.47. In this invention, after mixing the coarse feed and the complete feed, *Pediococcus lactis* M4 is inoculated. In this invention, the *Pediococcus lactis* M4 can be inoculated in the form of *Pediococcus lactis* M4 fermentation broth. As an optional embodiment of the present invention, the viable count of the *Pediococcus lactis* M4 fermentation broth is preferably ≥1×10⁻⁶. 8 cfu / mL, or 1.08×10 8 cfu / mL ~1×10 9cfu / mL. As an optional embodiment of the present invention, the inoculum amount of the *Pediococcus lactis* M4 fermentation broth can be 0.36 mL / g fermentation substrate; the fermentation substrate is a mixture of crude material and complete material.

[0039] After inoculation, the present invention preferably adjusts the water content of the system. As an optional embodiment of the present invention, the water content can be adjusted to ≤50%, or to 40%~50%, or to 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0040] After adjusting the moisture content, the present invention performs vacuum-sealed fermentation to obtain feed. In this invention, the temperature of the vacuum-sealed fermentation can be 15~37℃, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37℃; the time of the vacuum-sealed fermentation is 20~30 days, or 20 days. After the vacuum-sealed fermentation is completed, the present invention obtains a fermented total mixed ration (TMR).

[0041] The fermentation process described in the above technical solution of this invention is carried out under low moisture conditions. The *Pediococcus lactis* M4, as the fermentation strain, can rapidly produce lactic acid, lower the pH value of the fermentation system, efficiently promote the degradation of acidic and neutral detergent fibers, increase crude protein content, and significantly improve the content of volatile fatty acids, increase the content of lactic acid bacteria and yeast, and reduce the content of *Escherichia coli*, thereby improving the quality of the diet. Simultaneously, *Pediococcus lactis* M4 can also inhibit mold growth during fermentation, ensuring the safety of the fermented feed for consumption.

[0042] This invention provides a fermented total mixed diet (TMD), prepared by the method described in the above technical solution. The fermented TMD has a high lactic acid content, significantly reduced acid detergent fiber and neutral detergent fiber content, and significantly increased crude protein content. No aflatoxin B1, zearalenone, or vomitoxin was detected. Simultaneously, the fermented TMD shows a significant increase in the content of lactic acid bacteria and yeast, a significant decrease in the content of Escherichia coli, a significant increase in the content of acetic acid, propionic acid, and butyric acid, and a significant decrease in the content of isobutyric acid, isovaleric acid, and valeric acid.

[0043] The results of the embodiments of this invention show that the crude protein content of the fermented total mixed diet prepared by the described method is significantly increased from 11.11% to 13.22%, the neutral detergent fiber content is significantly decreased from 47.85% to 37.93%, and the acid detergent fiber content is significantly decreased from 29.73% to 24.32%, which greatly improves feed digestibility and optimizes nutrient composition. Furthermore, no aflatoxin B1, zearalenone, or vomitoxin were detected in the diet. Further, after fermentation, the pH value of the resulting fermented total mixed diet decreased to 4.36, and the lactic acid content reached 26.75 mmol / g. It also significantly increased the content of lactic acid bacteria and yeast, significantly decreased the content of Escherichia coli, and significantly increased the content of monoacyl, propionic, and butyric acids, while significantly decreasing the content of isobutyric, isovaleric, and valerate, thus improving the quality of the diet.

[0044] This invention provides the application of fermentation of Pediococcus lactis M4 under low moisture conditions in improving feed quality; the low moisture conditions include a fermentation system with a water content of 40% to 50%. In this invention, the improvement of feed quality includes at least one of the following: (1) increasing the crude protein content in the feed; (2) decreasing the crude fiber content in the feed; (3) eliminating any one or more of vomitoxin, zearalenone, and aflatoxin B1 in the feed; (4) increasing the lactic acid bacteria content in the feed; (5) increasing the yeast content in the feed; (6) decreasing the Escherichia coli content in the feed; (7) improving the volatile fatty acid content in the feed; (8) decreasing the pH value of the feed; and (9) increasing the lactic acid content in the feed.

[0045] As an optional embodiment of the present invention, improving the volatile fatty acid content in feed includes increasing the content of acetic acid, propionic acid, and butyric acid, and decreasing the content of isobutyric acid, isovaleric acid, and valeric acid; reducing the crude fiber content in feed includes decreasing the content of neutral detergent fiber and acid detergent fiber in feed. In the present invention, the feed may also be referred to as a diet.

[0046] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0047] Corn stalks are corn stalks that have been harvested at full maturity and then naturally dried.

[0048] Example 1 Isolation, screening and identification of Pediococcus lactis M4 (1) Test materials Corn stalks were provided by Sanping Ranch of Xinjiang Agricultural University. Harvested at full maturity, the stalks were naturally sun-dried and pulverized to 2-3 cm for later use. PEG-6000 (polyethylene glycol-6000 analytical grade) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd. MRS broth and MRS agar were purchased from Haibo Biotechnology (Qingdao) Co., Ltd., and NB medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. Pathogenic bacteria: Escherichia coli (CVCC1382, Escherichia coli Salmonella pullorum (CVCC525), Salmonella pullorum Staphylococcus aureus (CVCC2257), Staphylococcus aureus All samples were purchased from the China Veterinary Microbial Culture Collection Center (Isolation and Identification of Ferulic Acid Esterase-Producing Lactic Acid Bacteria and Their Effects on Production Performance, Rumen Fermentation and Meat Quality of Bashibai Sheep, Qiu Mingxin, Xinjiang Agricultural University, 2025).

[0049] (2) Screening of high-osmotic-tolerant strains Under aseptic conditions, 5 g of corn stalk sample was weighed and added to 45 mL of sterile deionized water. The mixture was shaken at room temperature for 30 min and thoroughly mixed. The residue was removed by filtration through sterile filter paper to obtain the bacterial suspension. 4 mL of this suspension was pipetted into MRS broth containing 20% ​​PEG-6000 and incubated at 37°C for 48 h. Then, the suspension was streaked onto MRS agar solid medium containing 0.75 wt.% calcium carbonate and incubated at 37°C for 48 h. Strains forming clear spots around the colonies were selected as candidate strains, i.e., the initial screening strains. These initial screening strains were then inoculated into MRS broth containing 20% ​​PEG-6000 and cultured at 37°C for 48 h. The OD of the bacterial suspension was measured every 4 h using a UV-Vis spectrophotometer. 600 Value, lasting 48 hours, based on OD 600 The growth curve of the strain was plotted by measuring the changes in pH value; at the same time, the pH value of the bacterial solution was measured with a pH meter, and the lactic acid content of the bacterial solution was measured with a portable lactic acid meter.

[0050] Six strains with large, clear, and well-formed colonies were selected from the initial screening petri dishes and labeled M1, M2, M3, M4, M5, and M6 for subsequent rescreening experiments. The streak plate image of strain M4 is shown below. Figure 1 As shown.

[0051] Growth curves of lactic acid production capacity, pH growth curves, and OD values ​​of the six selected strains. 600 The growth curves of the values ​​are shown in Tables 1-3 and Figures 2-4 As shown.

[0052] Table 1. Results of lactic acid production capacity of the six selected strains within a 48-hour culture period.

[0053] Table 2. pH detection results of the six selected strains during the 48-hour culture period.

[0054] Table 3. OD values ​​of the six selected strains during a 48-hour culture period. 600 Value detection results

[0055] From Tables 1-3 and Figures 2-4 It can be seen that, based on lactic acid production capacity, pH, OD 600 The values ​​show that strains M4 and M5 have superior performance. Strain M4 reached a peak lactic acid content of 39.75 mmol / L at 40 h, and the pH dropped to a minimum of 3.48 at 48 h. OD... 600 The value peaked at 0.82 at 24 h. Strain M5 reached a peak lactic acid content of 38.12 mmol / L at 44 h, and the pH dropped to a minimum of 3.46 at 48 h. OD 600 The pH value reached a peak of 0.76 at 36–40 h. Strains M4 and M5 were superior to other strains in terms of rapid pH reduction and high acid production capacity. Antibacterial tests were conducted using strains M4 and M5.

[0056] (3) Antibacterial test The screened lactic acid bacteria strains were incubated statically in MRS broth at 37°C for 48 h. After incubation, the bacterial cells were resuspended to obtain candidate bacterial suspensions. Simultaneously, *Escherichia coli*, *Salmonella pullorum*, and *Staphylococcus aureus* were cultured with shaking at 37°C and 170 r / min, and the OD values ​​were measured. 600 A pathogenic bacterial suspension with an OD value of 0.8 was used as an indicator solution. 0.1 mL of the bacterial suspension (indicator solution) was rapidly mixed with 15 mL of sterile solid culture medium at approximately 60°C, and quickly poured into sterile petri dishes. After the petri dishes solidified, sterile Oxford cups were gently placed evenly on the culture medium, and 0.2 mL of OD value was taken from each cup. 600 A candidate bacterial suspension at 0.8 g was poured into Oxford cups and allowed to diffuse on the culture medium at 4°C for 12 h. The plates were then incubated at 37°C for 24 h. The diameter of the inhibition zone was measured using calipers via the cross-hatching method. The results are shown in Table 4. Figure 5 As shown. Figure 5 The graph shows the inhibitory effects of strain M4 on different pathogenic bacteria, where a, b, and c represent the inhibitory effects of strain M4 on Escherichia coli, Salmonella pullorum, and Staphylococcus aureus, respectively.

[0057] Table 4. Diameter of inhibition zones (mm) of strains M4 and M5 against different pathogens determined by the Oxford cup method.

[0058] Note: Different capital letters in the superscript of data in the same row indicate extremely significant differences between groups (P < 0.01), while the same capital letter indicates no significant differences (P > 0.05); differences between groups were analyzed using independent samples t-tests, and the same applies below.

[0059] Antibacterial tests on strains M4 and M5 revealed that the inhibition zone diameter of strain M4 against *Escherichia coli*, *Salmonella pullorum*, and *Staphylococcus aureus* was significantly larger than that of strain M5 (P < 0.01). Considering the overall performance of strain rescreening and antibacterial activity, strain M4 demonstrated superior acid production, pH reduction, and fermentation performance compared to strain M5. Ultimately, one dominant strain, M4, was selected for subsequent strain identification.

[0060] (4) Species identification The dominant bacterial strains selected in step (3) were activated for three generations or more, and bacterial DNA was extracted. The 16S rDNA was amplified using primers 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO.1) and 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2). The amplified products were sequenced by Shanghai Sangon Biotech Co., Ltd., and the sequencing results were compared using BLAST in the NCBI database. A phylogenetic tree was constructed using MEGA4.0 software for homology analysis.

[0061]

[0062] Using Blast to perform a homology search in the GenBank gene database and through similarity comparison, strain M4 was found to be related to *Pediococcus lactis* (…). Pediococcus acidilactici The similarity between strain M4 and DSM 20284 (GenBank accession number: NR 042057.1) is over 99%, and they are at the same node in the phylogenetic tree with a bootstrap value of 95. This further confirms that strain M4 is *Pediococcus lactis* M4. The phylogenetic tree of strain M4 is as follows: Figure 6 As shown. Currently, strain M4 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20253043.

[0063] Example 2 Fermented diets were prepared by fermenting total mixed rations (TMCs) with Pediococcus lactis M4. The resulting fermented diet can also be called a fermented total mixed ration. The specific method is as follows: 1. The preparation method of Pediococcus lactis M4 fermentation broth is as follows: Pediococcus lactis M4 is inoculated into MRS broth medium at an inoculation rate of 2%–3%, and fermented statically at 37℃ for 12–24 h to obtain Pediococcus lactis M4 fermentation broth. The viable count of Pediococcus lactis M4 fermentation broth is adjusted to 1.08 × 10⁻⁶ cells / mL. 8 cfu / mL.

[0064] 2. The diet formula, by weight parts, consists of: 30.47 parts complete feed and 19.53 parts corn stalks. Based on the weight parts of corn stalks, the complete feed composition is: 16.76 parts corn, 6.85 parts sprayed corn husks, 3.20 parts cottonseed meal, 0.55 parts baking soda, 0.91 parts expanded urea, 0.52 parts bentonite, 0.21 parts dicalcium phosphate, 0.55 parts limestone powder, and 0.92 parts premix. The premix provides the following per kilogram of diet: Vitamin A 8.0-12.0 KIU / kg; Vitamin D3 2000-3000 IU / kg; Vitamin E 30-50 IU / kg; Ferrous sulfate 80-120 mg; Copper sulfate 10-15 mg; Zinc sulfate 60-80 mg; Manganese sulfate 40-60 mg; Sodium selenite 0.3-0.5 mg; Potassium iodide 0.5-1.0 mg.

[0065] The complete feed was provided by Fuhai County Big Tail Sheep Co., Ltd., and the product name is Jinshengkang, sheep feed concentrate supplement 611.

[0066] 3. Crush the nearly dry corn stalks into 2-3 cm pieces using a shredder. Then mix them evenly with the complete feed (the complete feed is dried complete feed) to obtain the diet substrate (also known as the fermentation substrate).

[0067] The fermentation broth of *Pediococcus lactis* M4 was inoculated into the mixed system at a rate of 0.36 mL / g of dietary substrate. The overall moisture content was adjusted to 50%, and the mixture was thoroughly mixed. The amount of dietary substrate for each experimental group was 200 g, and three parallel experiments were set up. Fermentation was carried out under vacuum conditions at a temperature of 20℃ for 30 days.

[0068] After fermentation is complete, fermented total mixed ration is obtained.

[0069] Comparative Example 1 A total mixed diet, with the same composition as the fermented total mixed diet in Example 2, was prepared by mixing complete feed and corn stalks and adjusting the moisture content to 50% before subsequent testing. It was not fermented for 30 days.

[0070] Application Example 1 The fermented total mixed diet of Example 2 and the diet obtained in Comparative Example 1 were tested. (1) Measurement indicators and methods Take feed samples and dry them at 65℃ to constant weight. After grinding them with a pulverizer, pass them through a 40-mesh sieve for later use. Determine the routine nutrients in the feed: crude protein, crude ash, neutral detergent fiber, and acid detergent fiber according to the methods in "Feed Analysis and Feed Quality Testing Technology".

[0071] Take 10 g of sample and mix it with 90 mL of deionized water. Shake well with a stirrer for 10 min, let it stand for 24 h, and measure the pH of the feed using a pH meter. The lactic acid content in the fermented feed was determined using a lactic acid analyzer. The contents of vomitoxin, zearalenone, and aflatoxin B1 in the sample were determined using a quantitative real-time mycotoxin detector.

[0072] After fermentation in Example 2, feed samples were taken, and samples from Comparative Example 1 were also taken for testing of lactic acid bacteria, yeast, and Escherichia coli.

[0073] After fermentation in Example 2, feed samples were taken, along with samples from Comparative Example 1, and the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid were tested.

[0074] Methods for determining volatile fatty acids Referring to T / NAIA 005-2020 "Determination of Volatile Fatty Acids in Rumen Fluid by Gas Chromatography", the contents of volatile fatty acids acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid were determined using a gas chromatograph.

[0075] Determination of viable bacteria count in feed Referring to GB / T 13093-2023 "Determination of Total Bacterial Count in Feed", the dilution plating method was used to count viable bacteria in the samples.

[0076] ①Gradual dilution of bacterial culture samples First, mix 10.0g of feed sample with 90ml of deionized water in a magnetic stirrer for 2 minutes, then filter through 4 layers of nylon gauze and qualitative filter paper to obtain feed extract. Take 1ml of extract, add 9ml of sterile water and mix well to prepare a 10⁻¹ dilution; and so on, to prepare 10⁻² and other graded dilutions.

[0077] ②Prepare culture medium plates and spread them for inoculation. Prepare MRS, PDA, and LB broth sterile culture media. In a laminar flow hood, use a pipette to draw 100 μL of the corresponding dilution of the sample bacterial solution and drop it onto the center of the sterile plate. Then, use a glass spreader (which should be sterilized with alcohol and cooled beforehand to avoid killing the bacteria with high temperature) to gently spread the bacterial solution from the center of the plate outwards, so that the bacterial solution is evenly distributed on the surface of the culture medium.

[0078] ③ Culture counting After inoculation, the plates were inverted and placed in a 37°C incubator for 48 hours. The colony counts were then performed at dilutions of 30-300 CFU / plate (National Standard 8.2.1).

[0079] Result calculation: Total bacterial count (CFU / g or CFU / mL) = (Average colony count on plate × Dilution factor) / 0.1 mL (2) Data statistical analysis Data were statistically analyzed using Excel software. SPSS 24.0 was used for statistical analysis. All data conformed to a normal distribution and satisfied Levene's test for homogeneity of variance. The differences between the total mixed diet control group and the fermented total mixed diet treatment group were analyzed using independent samples t-tests. Results are expressed as mean ± standard deviation. This experiment only involved two independent sample comparisons; therefore, uppercase letters were used to indicate extremely significant differences between groups (P < 0.01), and the same letter indicated no significant differences (P > 0.05).

[0080] (3) Detection results of diet-related indicators in Example 2 and Comparative Example 1 The pH value and lactic acid content of the diets of Example 2 and Comparative Example 1 are shown in Table 5.

[0081] Table 5. Results of pH and lactic acid content detection in the diets of Example 2 and Comparative Example 1

[0082] As shown in Table 5, compared with the control group, the pH of the M4 fermentation group was significantly lower (P<0.01), and the lactic acid content increased by 20.58 times (P<0.01), indicating that the M4 fermentation strain can significantly reduce the pH and increase the lactic acid content.

[0083] The results of the determination of crude protein, crude ash, neutral detergent fiber and acid detergent fiber in the diets of Example 2 and Comparative Example 1 are shown in Table 6.

[0084] Table 6. Results of determination of crude protein, crude ash, neutral detergent fiber, and acid detergent fiber in the diets of Example 2 and Comparative Example 1.

[0085] As shown in Table 6, compared with the control group, the crude protein content in the diet fermented with Pediococcus lactis M4 increased by 18.99% (P < 0.01). Neutral detergent fiber decreased by 20.74% and acid detergent fiber decreased by 18.97% (P < 0.01).

[0086] The results of detecting vomitoxin, zearalenone, and aflatoxin B1 in the diets of Example 2 and Comparative Example 1 after fermentation are shown in Table 7.

[0087] Table 7. Detection results of vomitoxin, zearalenone, and aflatoxin B1 content in the diets of Example 2 and Comparative Example 1.

[0088] As shown in Table 7, no mycotoxins were detected in either the control group or the M4 fermentation group. After fermentation with M4, the fermented total mixed diet was safe and reliable.

[0089] The control group consisted of samples that had not undergone fermentation, while Example 2 was a low-moisture fermentation group with the addition of *Pediococcus lactis* M4. Under low-moisture fermentation conditions, the material has high water activity and is rich in nutrients, making it highly susceptible to the growth of harmful molds such as *Aspergillus flavus* and *Fusarium*, which can then produce various mycotoxins such as aflatoxin B1, zearalenone, and vomitoxin, posing a serious risk of mold contamination and safety. The test results showed that the control group samples were negative for aflatoxin B1, zearalenone, and vomitoxin; the samples from Example 2, which underwent low-moisture fermentation, were also negative for these three mycotoxins, indicating no detectable mycotoxin contamination. The results demonstrate that by adding *Pediococcus lactis* M4 during low-moisture fermentation, this invention effectively inhibits the growth and reproduction of harmful molds in the fermentation system, prevents the production of mycotoxins, and significantly reduces the risk of mold contamination during low-moisture fermentation. This fully demonstrates that *Pediococcus lactis* has excellent antibacterial and antifungal effects, ensuring the safety and stability of low-moisture fermented products.

[0090] Xinjiang has a consistently dry climate, and agricultural byproducts such as corn stalks, when used as roughage, have extremely low moisture content and a dense lignocellulose structure. When corn stalks are mixed with complete feed to adjust the moisture content to 40-50%, mycotoxins often accumulate during fermentation, leading to fermentation failure. The *Pediococcus lactis* M4 described in this invention not only efficiently ferments corn stalks under these conditions, but also produces a fermentation product in which no vomitoxin, zearalenone, or aflatoxin B1 was detected. This fully demonstrates the effectiveness of *Pediococcus lactis* M4 in inhibiting mold growth.

[0091] The detection results of lactic acid bacteria, yeast and Escherichia coli in the diets of Example 2 and Comparative Example 1 are shown in Table 8.

[0092] Table 8. Detection results of lactic acid bacteria, yeast, and Escherichia coli in the diets of Example 2 and Comparative Example 1.

[0093] Table 8 shows that the lactic acid bacteria concentration increased from 3.65±0.09 to 4.59±0.33 log10 cfu / g (P<0.001), which was beneficial for rapidly lowering the pH and inhibiting harmful bacteria. The yeast concentration increased from 2.45±1.20 to 4.25±0.26 log10 cfu / g (P=0.005), which helped improve the fermentation environment. The Escherichia coli concentration decreased from 3.72 to 1.61 log10 cfu / g, achieving significant inhibition of harmful bacteria.

[0094] The results of the detection of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in the diets of Example 2 and Comparative Example 1 are shown in Table 9.

[0095] Table 9. Detection results of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid in the diets of Example 2 and Comparative Example 1.

[0096] Volatile fatty acids (VFAs), including acetic acid, propionic acid, and butyric acid, are the main metabolites produced by microorganisms in FTMR (fermented total mixed diet) when utilizing substrate nutrients (nitrogen and carbon sources). The yield of acetic acid and propionic acid can indicate the fermentation type; heterofermentation by lactic acid bacteria produces acetic acid and carbon dioxide. Isovaleric acid and isobutyric acid have distinctive odors, and reducing their content can improve feed odor or reduce adverse effects. Acetic acid is the only fermentation product that can stably improve the aerobic stability of silage. Propionic acid, as a short-chain fatty acid, has an inhibitory effect on fungi, significantly reducing the risk of secondary fermentation caused by fungal proliferation. It also has good antibacterial properties, promotes the uptake and metabolism of sugars by lactic acid bacteria, thereby accelerating lactic acid production, optimizing fermentation quality, and increasing feed intake in livestock and poultry. Therefore, the composition and content of VFAs have become important indicators for evaluating the fermentation quality of FTMR.

[0097] Anaerobic fermentation significantly increased the number of lactic acid bacteria and yeast in FTMR (P < 0.01), while also significantly increasing the content of lactic acid and beneficial volatile fatty acids such as acetic acid, propionic acid, and butyric acid (P < 0.01), accompanied by a significant decrease in the content of undesirable flavor compounds such as isobutyric acid and isovaleric acid (P < 0.05). This study confirms that anaerobic fermentation of TMR can significantly improve the fermentation quality of FTMR, thereby helping to improve the conversion efficiency of FTMR in livestock and poultry, enhance the intestinal mucosal barrier function, and improve digestive health. This provides important scientific evidence for the efficient utilization of FTMR in modern livestock and poultry farming and the sustainable development of the feed industry.

[0098] Example 3 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that the overall moisture content is adjusted to 40% after inoculation with the inoculum.

[0099] Example 4 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that the overall moisture content is adjusted to 45% after inoculation with the inoculum.

[0100] Example 5 The method for preparing fermented diets using the total mixed diet fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that after inoculation with the inoculum, the overall moisture content is adjusted to 50%.

[0101] Example 6 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that after inoculation with the inoculum, the overall moisture content is adjusted to 40%, and the fermentation temperature is 25°C.

[0102] Example 7 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that after inoculation with the inoculum, the overall moisture content is adjusted to 45% and the fermentation temperature is 25°C.

[0103] Example 8 The method for preparing fermented diets using total mixed diets fermented with Pediococcus lactis M4 is the same as in Example 2, except that the fermentation temperature is 25℃.

[0104] Example 9 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that after inoculation with the inoculum, the overall moisture content is adjusted to 40%, and the fermentation temperature is 30°C.

[0105] Example 10 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that after inoculation with the inoculum, the overall moisture content is adjusted to 45% and the fermentation temperature is 30℃.

[0106] Example 11 The method for preparing fermented diets using total mixed diets fermented with Pleurotus ostreatus M4 is the same as in Example 2, except that the fermentation temperature is 30℃.

[0107] The moisture and temperature conditions for fermentation in Examples 3-11 are detailed in Table 10.

[0108] Table 10. Moisture and temperature conditions for fermentation in Examples 3-11

[0109] Application Example 2 The crude protein content, neutral detergent fiber and acid detergent fiber degradation rate of the fermented total mixed diets of Examples 3-11 were determined, and the results are shown in Tables 11-13.

[0110] Table 11 Crude protein content in fermented total mixed diets of Examples 3-11

[0111] Table 12 Effect of fermentation on the degradation rate of FTMR neutral detergent fiber in Examples 3-11

[0112] Table 13 Effect of fermentation on the degradation rate of FTMR acid detergent fibers in Examples 3-11

[0113] As shown in Tables 11-13, when the diet substrate was fermented with Pediococcus lactis M4, it was still able to efficiently increase the crude protein content in the fermented diet under the conditions of 20℃ and 40% moisture, and maintain a high degradation rate for neutral detergent fiber and acid detergent fiber.

[0114] The *Pediococcus lactis* M4 strain described in this invention maintains excellent fermentation performance in a corn stalk and concentrate mixture system, under low moisture conditions of 40%–50% and a low temperature of 20°C, demonstrating significant advantages over conventional corn stalk fermentation systems. Conventional corn stalk fermentation typically requires higher moisture content (e.g., above 65%) and higher temperatures to proceed smoothly. High moisture content leads to high transportation costs and poor storage stability, while higher temperatures not only increase energy consumption but also easily cause spoilage, nutrient loss, and off-odors.

[0115] This invention can initiate and complete stable fermentation at a low temperature of 20℃ without additional heating, resulting in lower energy consumption, safer operation, and wider applicability. Furthermore, even under low moisture conditions of 40%~50%, *Pediococcus lactis* can still rapidly colonize and efficiently produce acid, ensuring the stability of the fermentation system and inhibiting the growth of harmful microorganisms.

[0116] The test results show that the crude protein content in the fermentation product of this invention is significantly increased, and the degradation rate of acid detergent fiber (ADF) and neutral detergent fiber (NDF) is significantly improved. This indicates that under the dual conditions of low temperature and low moisture, the lactic acid cocci can still effectively degrade cellulose, hemicellulose and other difficult-to-use fiber components, significantly improving the nutritional value and digestibility of fermented feed.

[0117] In summary, this invention achieves efficient fermentation of a corn stalk concentrate mixture system under low temperature (20℃) and low moisture content (40%–50%). It solves the problems of high energy consumption and inconvenient storage and transportation associated with conventional high-moisture and high-temperature fermentation, and significantly improves the nutritional quality and fiber degradation effect of the fermentation products. It has obvious technical advantages and practical value.

[0118] Xinjiang's arid climate means that agricultural byproducts like corn stalks, used as roughage, have extremely low moisture content and a dense lignocellulose structure. Traditional high-moisture silage or fermentation processes typically require a moisture content of over 65%. In actual production in Xinjiang's arid regions, this not only consumes vast amounts of precious water resources but also makes the feed highly susceptible to freezing damage during the cold winter. Furthermore, excessively low moisture content during fermentation leads to loose compaction and high oxygen levels in the feed gaps, resulting in mycotoxin accumulation and fermentation failure. Therefore, developing low-moisture FTMR with a moisture content controlled at around 50% has become a core requirement for cost reduction and efficiency improvement in Xinjiang. However, the low moisture content and accompanying high osmotic pressure environment severely limit the fermentation efficiency of common lactic acid bacteria, resulting in slow or incomplete fermentation and difficulty in rapidly forming an acidic environment. To address this deficiency, the first phase of this invention simulated a low-moisture, high-osmotic-pressure environment using 20% ​​PEG-6000, screening out the homologous fermenting *Pediococcus lactis* M4. Experimental results showed that it exhibited high adaptability to drought-tolerant environments and excellent acid production efficiency.

[0119] Application Example 3 Inhibitory effect of Pietrococcus lactis M4 against mold The Oxford cup method and mycelial growth rate method showed that Pediococcus lactis M4 had a strong antifungal effect on molds. It is speculated that the low levels of vomitoxin, zearalenone, and aflatoxin B1 in the fermented diet are related to the antifungal effect of Pediococcus lactis M4 on molds.

[0120] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Pediococcus lactis* M4, characterized in that, The accession number is CCTCC NO: M 20253043.

2. The use of the *Pediococcus lactis* M4 of claim 1 in the preparation of lactic acid and / or the degradation of straw; wherein the straw includes corn straw.

3. The application of the *Pediococcus lactis* M4 as described in claim 1 in inhibiting mold growth during feed fermentation.

4. The application of the *Pediococcus lactis* M4 of claim 1 in the preparation of feed under low moisture conditions; the low moisture conditions include a fermentation system with a moisture content of 40% to 50%.

5. A method for preparing feed, characterized in that, Includes the following steps: After mixing roughage and complete feed, inoculate with Pediococcus lactis M4, adjust the moisture content of the system to ≤50%, and carry out vacuum-sealed fermentation to obtain feed.

6. A method for preparing fermented total mixed rations under low moisture conditions, characterized in that, include: After mixing roughage and complete feed, the moisture content of the mixture was adjusted to 40%~50%, and then inoculated with Pediococcus lactis M4 for fermentation to obtain fermented total mixed diet. The coarse feed includes corn stalks; the complete feed includes corn, sprayed corn husks, cottonseed meal, baking soda, expanded urea, bentonite, dicalcium phosphate, stone powder, and premixed feed. The mass ratio of the coarse feed to the complete feed is (19~20):(30~35).

7. Application of fermentation of Pediococcus lactis M4 under low moisture conditions in improving feed quality; the low moisture conditions include fermentation system with a moisture content of 40%~50%.

8. The application according to claim 7, characterized in that, Improving feed quality includes improving the content of volatile fatty acids in feed and / or reducing the content of crude fiber in feed; improving the content of volatile fatty acids in feed includes increasing the content of acetic acid, propionic acid and butyric acid, and reducing the content of isobutyric acid, isovaleric acid and valeric acid; reducing the content of crude fiber in feed includes reducing the content of neutral detergent fiber and acid detergent fiber in feed.

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