A composite probiotic leavening agent, a preparation method thereof, and a pre-fermented bacterial grass feed made therefrom

Abstract

The application discloses a composite probiotic fermenting agent, a preparation method thereof and a pre-fermented forage grass feed made of the composite probiotic fermenting agent. The preparation method of the composite probiotic fermenting agent comprises the following steps: mixing Pichia membranifaciens C200-1 and Brevibacillus reus RF10-5 in a proportion to obtain aerobic mixed bacteria, mixing Pediococcus acidilactici, Lactobacillus plantarum, Lactobacillus buchneri and Bifidobacterium in a proportion to obtain anaerobic mixed bacteria, and then mixing the two kinds of bacteria in a proportion of 1: (45-55) to obtain seed liquid. The seed liquid is diluted and expanded by molasses, and then sprayed on crushed forage grass (such as giant reed) to ferment under a closed condition, so that the pre-fermented forage grass feed is obtained. The pre-fermented forage grass feed is used for pig feeding, can significantly improve feed utilization, regulate intestinal flora, enhance immunity, reduce the feed-meat ratio and improve the breeding environment, and provides an effective technical scheme for realizing grass instead of grain and saving grain.

Description

Technical Field

[0001] This invention relates to the field of microbial feed additive technology, and in particular to a compound probiotic fermentation agent and its application in the preparation of pre-fermented fungal and grass feed. Background Technology

[0002] The efficient utilization of feed raw material resources is one of the key directions for the current development of the livestock industry. Juncao (a type of grass used for animal husbandry) is a high-yield and highly adaptable feed raw material with good development potential.

[0003] Juncao can be made into feed through silage fermentation, but traditional silage mainly relies on the natural environment microorganisms attached to the raw materials for fermentation. The microbial community is complex and uncontrollable, and it is prone to spoilage and mold due to slow fermentation start-up and insufficient acid production, resulting in nutrient loss. Ultimately, the feed quality is unstable, the safety is poor, and the feed value is limited.

[0004] Furthermore, since the ban on antibiotics in 2020, the livestock industry urgently needs green alternatives to ensure animal health. Although there are attempts to use single or a few types of probiotics (such as Lactobacillus plantarum) for fermentation in existing technologies, they generally suffer from problems such as limited strain function, arbitrary compatibility, and unclear synergistic effects. In particular, for Juncao raw materials with high fiber content and complex structure, technical bottlenecks often occur, such as incomplete fermentation, weak probiotic function, and limited grain substitution ratio. Summary of the Invention

[0005] The purpose of this invention is to provide a compound probiotic fermentation agent with clearly defined strain combinations that targets the characteristics of Juncao raw materials, can simultaneously improve the nutritional value of feed and its antibiotic alternative function, and its efficient application method.

[0006] To achieve the above objectives, the solution of the present invention is: a method for preparing a compound probiotic fermentation agent, comprising the following steps:

[0007] S1. Prepare Pichia pastoris C200-1, Bacillus reuteri RF10-5, Pediococcus lactis, Lactobacillus plantarum, Lactobacillus brunelli and Bifidobacterium;

[0008] Each strain was activated, cultured in a large scale, and cultured in a fermenter to obtain individual bacterial cultures.

[0009] S2. Mix Pichia pastoris C200-1 bacterial suspension and Bacillus reuteri RF10-5 bacterial suspension at a volume ratio of (0.8~1.2):1 to obtain an aerobic mixed bacterial suspension;

[0010] S3. Mix the *Pediococcus lactis* culture, *Lactobacillus plantarum* culture, *Lactobacillus brucelli* culture and *Bifidobacterium* culture obtained in step S1 at a volume ratio of 1:1:1:1 to obtain an anaerobic mixed culture.

[0011] S4. The aerobic mixed bacterial solution obtained in step S2 and the anaerobic mixed bacterial solution obtained in step S3 are mixed at a volume ratio of 1:(45~55) to prepare a compound probiotic fermentation agent seed liquid; the compound probiotic fermentation agent seed liquid is then activated and cultured to obtain a compound probiotic fermentation agent.

[0012] Furthermore, in step S1, the activation and expansion culture process for each strain is as follows:

[0013] Pichia pastoris C200-1, which was frozen with glycerol, was inoculated into PDB medium and cultured at 26-30℃ until OD600=0.45; Bacillus reuteri RF10-5, which was frozen with glycerol, was inoculated into LB medium and cultured at 32-37℃ until OD600=0.7.

[0014] Take glycerol-frozen strains of Pediococcus lactis, Lactobacillus plantarum, Lactobacillus brunelli and Bifidobacterium, and inoculate them separately on MRS medium for culture until OD600 = 0.6~1.5, and control the temperature at 35~38℃.

[0015] Furthermore, before mixing, the viable cell concentration of each individual bacterial suspension is controlled within (0.5~1.5) × 10⁻⁶. 8 Within the range of CFU / mL.

[0016] Furthermore, the viable cell concentration of each single bacterial culture was controlled to be 1×10⁻⁶. 8 CFU / mL; and the Pichia pastoris C200-1 bacterial suspension and Bacillus reuteri RF10-5 bacterial suspension are mixed at a volume ratio of 1:1, and the bacterial suspensions of Pediococcus lactis, Lactobacillus plantarum, Lactobacillus brunelli and Bifidobacterium are mixed at a volume ratio of 1:1:1:1.

[0017] Furthermore, in step S4, the specific methods for activating and culturing the compound probiotic fermentation agent seed liquid are as follows:

[0018] Take the seed liquid of compound probiotic fermentation agent, dilute it with 3-5% sugarcane molasses at a ratio of 1:200 (v / v), and then place it in a 30℃ environment for static culture for 8-12 hours to obtain the activated compound probiotic fermentation agent.

[0019] A compound probiotic starter is prepared by the above method.

[0020] A pre-fermented mushroom and grass feed, the preparation method of which is as follows: the above-mentioned compound probiotic fermentation agent is evenly sprayed onto giant reed grass raw material, with 50-150 L of compound probiotic fermentation agent sprayed per ton of giant reed grass; then the mixture is fermented in a sealed environment at 20-40℃ for 10-15 days to obtain pre-fermented mushroom and grass feed.

[0021] Furthermore, before spraying the compound probiotic fermentation agent, the giant reed grass is pretreated. The pretreatment method is as follows: harvest the giant reed grass with a plant height of about 2.0 meters on a sunny day, leave a stubble of 5-7 cm, and after preliminary chopping, transport it to the processing workshop and crush it to a particle size of 1-3 mm using a shredder.

[0022] Furthermore, the pre-fermented fungal feed is used as pig feed.

[0023] After adopting the above solution, the beneficial effects of the present invention are as follows:

[0024] 1. The Pichia pastoris C200-1 and Bacillus reuteri RF10-5 strains used in this invention were isolated from Yongchun aged vinegar. They have excellent acid resistance, can tolerate the low pH ring produced by fermentation, and have highly efficient aerobic growth capabilities.

[0025] Combining these two methods in the fermentation of Juncao (a type of feed) can rapidly and synergistically deplete the oxygen in the sealed environment in the early stages, creating an anaerobic environment for anaerobic fermentation. Even if oxygen re-enters due to damage to the fermentation bag or opening the package to remove materials, the strains in this combination can preferentially utilize oxygen to quickly resume growth. Through competitive site occupation and metabolic products, they continuously inhibit the growth of other aerobic harmful bacteria (especially molds), thereby significantly improving the stability and safety of the fermented feed.

[0026] 2. This invention combines strictly anaerobic bacteria (Bifidobacterium, Lactobacillus brunelli) with facultative anaerobic acid-producing bacteria (Lactobacillus plantarum, Pediococcus lactis) to form an anaerobic bacterial community. In the anaerobic environment created by the lead aerobic bacterial community, this anaerobic bacterial community can efficiently initiate and rapidly produce acid, utilizing substrates (including readily available components produced by aerobic bacteria degradation) to generate large amounts of organic acids such as lactic acid and acetic acid, causing the pH value of the system to drop rapidly. This strongly inhibits various miscellaneous bacteria, including putrefactive bacteria and pathogenic bacteria, ensuring the biological safety of fermented feed. In addition, this fermentation process not only accumulates active probiotics but also produces abundant microbial metabolites (postbiotics). These components can exert stable immune regulation and intestinal health promotion functions that exceed those of the live bacteria themselves when fed to animals.

[0027] 3. The special compound probiotic composition provided by this invention combines specific aerobic bacteria with anaerobic bacteria at a ratio of 1:45-55. This combination can rapidly consume oxygen, degrade macromolecules, and quickly produce acid to lower pH, effectively inhibiting the growth of harmful bacteria, improving feed palatability, and overcoming the high failure risk caused by traditional silage relying on natural microbial communities.

[0028] 4. Feeding trials have shown that adding 30%-50% of the pre-fermented mushroom and grass feed of this invention to the diet of finishing pigs can significantly increase daily weight gain and reduce feed conversion ratio while ensuring the same feed intake, directly saving the amount of concentrate feed used. At the same time, the highly active compound probiotics in the feed can colonize the animal's intestines, optimize the intestinal flora balance, enhance immunity, and significantly reduce the diarrhea rate of piglets. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the embodiments.

[0030] Example 1: Preparation of Aerobic Strains

[0031] 1.1 Pichia membranifaciens C200-1

[0032] This strain is disclosed for the first time in this application. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, China, on August 3, 2022, with accession number CCTCC NO: M20221232. This application provides proof of the strain's viability at the collection. The metabolic processes of *Pichia pastoris* C200-1 can produce volatile esters, acids, and other aromatic substances. This unique odor can induce and attract pigs to compete for feed, increasing their feed intake.

[0033] Glycerol tube activation: Take 500 μL of frozen Pichia pastoris C200-1 in glycerol and inoculate it into 50 mL of PDB liquid medium (formulation g / L: potato extract 200, glucose 20; pH 5.6±0.2, sterilized at 121 ℃ for 20 min), and culture at 28℃ and 220 rpm with shaking until OD600≈0.45.

[0034] Expanded culture: The above culture was transferred to 200 mL of YPD medium and cultured at 28°C with shaking at 220 rpm (under the same conditions) until OD600≈0.45.

[0035] Fermentation in a 5L tank: YPD medium, 70% filling volume, 5% inoculum (v / v), culture conditions: 28℃, stirring speed 400 rpm, aeration rate 1 vvm, cultured until OD600≈0.45, then diluted to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL available for use.

[0036] 1.2 Lacticaseibacillus paracasei RF10-5

[0037] This strain is not disclosed for the first time in this application. It is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, Hubei Province, on December 13, 2022, with accession number CCTCC NO: M20221968. Existing research has confirmed that this strain can efficiently degrade zearalenone toxins, achieving a degradation rate of up to 99.8%. It can be used to degrade zearalenone toxins in feed and other products, significantly reducing the harm of zearalenone to animal reproductive performance and improving animal production performance (see patent CN202310648355.0, "A strain of *Bacillus reuteri* degrading zearalenone and its application"). Therefore, its application in mushroom and grass fermentation can degrade mycotoxins that may be present in the raw materials before and after fermentation.

[0038] Glycerol tube activation: Take 500 μL of frozen Bacillus reuteri RF10-5 in glycerol and inoculate it into 50 mL of LB liquid medium (g / L peptone 10, yeast extract 5, NaCl 10, pH 7.0~7.2, sterilized at 121 ℃ for 20 min), and culture at 35 ℃ and 220 rpm with shaking until OD600≈0.7.

[0039] Expanded culture: The above culture was transferred to 200 mL LB medium and cultured at 35°C and 220 rpm shaking (under the same conditions) until OD600≈0.7.

[0040] Fermentation in a 5L tank: LB medium, 70% filling volume, 5% inoculum (v / v), culture conditions: 35℃, stirring speed 400 rpm, aeration rate 0.5 vvm, cultured until OD600≈0.7, then diluted to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL available for use.

[0041] The *Pichia pastoris* C200-1 and *Bacillus reuteri* RF10-5 used in this invention were isolated from fermentation samples of Yongchun aged vinegar. They exhibit excellent acid resistance, can tolerate the low pH environment generated during fermentation, and are aerobic bacteria with high oxygen utilization efficiency. Combining these two bacteria in the fermentation of *juncao* (a type of fermented plant), they can rapidly and synergistically consume residual oxygen in the system during the initial fermentation stage, creating an anaerobic environment for subsequent anaerobic lactic acid bacteria.

[0042] The *Pediococcus lactis*, *Lactobacillus plantarum*, *Lactobacillus brunelli*, and *Bifidobacterium* used in this invention refer to strains of these species with corresponding functions and clearly defined taxonomically. Their sources include, but are not limited to, specific strains from public microbial culture collections (such as ATCC, CCTCC, etc.), or strains of the same species isolated from the natural environment and identified as possessing equivalent or superior performance. The following examples use recognized standard strains as examples, but the scope of protection of this invention is not limited thereto.

[0043] Example 2: Preparation of Anaerobic Strains

[0044] 2.1 Pediococcus acidilactici

[0045] In this embodiment, Pediococcus lactis (ATCC 8043) was selected.

[0046] Glycerol tube activation: Take 500 μL of frozen Pediococcus lactis in glycerol, inoculate it into 50 mL of MRS medium, and incubate at 37 ℃ (or 50 rpm) until OD600≈1.2~1.5.

[0047] Expanded culture: Transfer the above culture to 200 mL MRS medium and incubate at 37 ℃ (or with micro-oscillation at 50 rpm) until OD600≈ 1.2~1.5.

[0048] Fermentation in a 5 L tank: MRS medium, 70% fill factor; inoculum 5% (v / v); culture conditions: 37℃, static or low-speed stirring at 50 rpm; pH not controlled: culture to OD600 ≈ 1.2~1.5, then dilute to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL is used for subsequent fermentation agent formulation.

[0049] The formulation of the above MRS medium (based on 1L): 10.0 g peptone, 10.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate (K₂HPO₄), 2.0 g triammonium citrate, 5.0 g sodium acetate (NaAc·3H₂O), 0.58 g magnesium sulfate (MgSO₄·7H₂O), 0.25 g manganese sulfate (MnSO₄·H₂O), and 1.0 ml Tween-80. Dilute to 1L with distilled water, adjust the pH to 6.2-6.4, and autoclave at 121℃ for 20 minutes before use.

[0050] 2.2 Lactiplantibacillus plantarum

[0051] In this embodiment, Lactobacillus plantarum (ATCC 14917) was selected.

[0052] Glycerol tube activation: Take 500 μL of Lactobacillus plantarum frozen in glycerol, inoculate it into 50 mL of MRS medium, and incubate at 37 ℃ (or with micro-shaking at 50 rpm) until OD600 ≈ 0.8~1.0.

[0053] Expanded culture: Transfer the above culture to 200 mL MRS medium and incubate at 37 ℃ (or with micro-oscillation at 50 rpm) until OD600≈0.8~1.0.

[0054] Fermentation in a 5 L tank: MRS medium, 70% fill factor; inoculum 5% (v / v); temperature 37 ℃, static or low-speed stirring at 50 rpm; pH not controlled; culture to OD600≈0.8~1.0, then dilute to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL is used for subsequent fermentation agent formulation.

[0055] 2.3 Lactobacillus buchneri

[0056] In this embodiment, Lactobacillus blight (ATCC 4005) was selected.

[0057] Glycerin tube activation: Take 500 μL of frozen Lactobacillus brunelli in glycerol, inoculate it into 50 mL of MRS medium, and incubate at 37 ℃ (or with micro-shaking at 50 rpm) until OD600 ≈ 0.6~0.8.

[0058] Expanded culture: Transfer the above culture to 200 mL MRS medium and incubate at 37 ℃ (or with micro-oscillation at 50 rpm) until OD600≈0.6~0.8.

[0059] Fermentation in a 5 L tank: MRS medium, 70% fill factor; inoculum 5% (v / v); temperature 37 ℃, incubation at rest or with low stirring at 50 rpm; pH not controlled; culture to OD600≈0.6~0.8; then dilute to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL is used for subsequent fermentation agent formulation.

[0060] 2.4 Bifidobacterium

[0061] Bifidobacterium (ATCC 27673) was used in this embodiment.

[0062] Glycerin tube activation: Take 50 μL of frozen Bifidobacterium glycerol, inoculate it into 50 mL of MRS medium, and incubate at 37 ℃ (or with micro-shaking at 50 rpm) until OD600 ≈ 0.8~1.0.

[0063] Expanded culture: Add the above culture to 200 mL of MRS medium and incubate at 37 ℃ (or with micro-oscillation at 50 rpm) until OD600≈0.8~1.0.

[0064] Fermentation in a 5 L tank: MRS medium, 70% fill factor; inoculum 5% (v / v); temperature 37 ℃, incubation at rest or low stirring at 50 rpm; pH not controlled; culture to OD600≈0.8~1.0, then dilute to a viable cell concentration of 1×10⁻⁶. 8 CFU / mL is used for subsequent fermentation agent formulation.

[0065] Of the four anaerobic bacteria used in this invention, Bifidobacterium and Lactobacillus bryonicus are strict anaerobes, while Lactobacillus plantarum and Pediococcus lactis are facultative anaerobes. Under anaerobic conditions, they exhibit excellent growth and acid production performance, rapidly reducing the pH of the system and thus strongly inhibiting various miscellaneous bacteria, including putrefactive bacteria and pathogens, ensuring the biological safety of fermented feed. In addition, this fermentation process not only accumulates active probiotics but also produces abundant microbial metabolites (postbiotics). These components can exert stable immune regulation and intestinal health promotion functions that surpass those of the live bacteria themselves when fed to animals.

[0066] Example 3: Preparation of compound probiotic fermentation agent seed liquid

[0067] 3.1 Preparation of aerobic mixed bacteria:

[0068] The Pichia pastoris C200-1 bacterial culture and Bacillus reuteri RF10-5 bacterial culture prepared in Example 1 were mixed at a volume ratio of 1:1 to form an aerobic mixed bacterial culture.

[0069] 3.2 Preparation of anaerobic mixed bacteria:

[0070] The *Pediococcus lactis* solution, *Lactobacillus plantarum* solution, *Lactobacillus brucelli* solution, and *Bifidobacterium* solution prepared in Example 2 were mixed in a volume ratio of 1:1:1:1 to form an anaerobic mixed bacteria.

[0071] 3.3 Preparation of seed liquids of compound probiotic fermentation agents with different proportions:

[0072] Take the above-mentioned aerobic mixed bacterial solution and anaerobic mixed bacterial solution, and mix them separately according to the following three volume ratios, and mix thoroughly to obtain three portions of compound probiotic fermentation agent seed liquid with different proportions:

[0073] Seed culture A: aerobic mixed bacterial culture: anaerobic mixed bacterial culture = 1:45 (v / v).

[0074] Seed culture B: aerobic mixed bacterial culture: anaerobic mixed bacterial culture = 1:50 (v / v).

[0075] Seed culture C: aerobic mixed bacterial culture: anaerobic mixed bacterial culture = 1:55 (v / v).

[0076] Example 4: Preparation of pre-fermented fungal-straw feed using seed liquid of different proportions

[0077] 4.1 Preparation of Juncao (a type of medicinal herb):

[0078] Harvest giant reeds that are about 2.0 meters tall on a sunny day, leaving a stubble of 5-7 cm. After preliminary chopping, transport them to the processing workshop and use a shredder to grind them to a particle size of 1-3 mm. The natural moisture content is sufficient and no additional adjustment is needed.

[0079] 4.2 Seed liquid activation:

[0080] One portion each of seed solutions A, B, and C prepared in Example 3 was taken and diluted with a 4% (w / v) sugarcane molasses aqueous solution that had been sterilized at 121°C for 20 minutes, at a volume ratio of 1:200 (seed solution: molasses solution). The diluted bacterial solutions were placed in a 30°C environment and incubated for 10 hours to obtain activated compound probiotic fermentation agent spray solutions A, B, and C.

[0081] 4.3 Inoculation and Fermentation:

[0082] Take three portions of crushed giant reed grass raw materials. Spray the spraying solutions A, B, and C evenly onto the corresponding three groups of giant reed grass raw materials, with an inoculation amount of 100 L of spraying solution per ton of raw materials.

[0083] The giant reeds sprayed with bacterial solution were divided into 40 kg fermentation bags, and the bags were compacted layer by layer to remove the air inside. The bags were then sealed.

[0084] All three fermentation bags were placed at room temperature (25~30℃) and fermented for 14 days. After fermentation, they were opened and tested, and pre-fermented mushroom and grass feed A, feed B, and feed C were obtained respectively.

[0085] Example 5: Application of pre-fermented mushroom and straw feed with different proportions in pig farming

[0086] The pre-fermented mushroom grass of this invention contains a large number of probiotics, which can regulate the balance of intestinal flora in pigs, improve feed utilization, enhance their immunity, and improve the breeding environment.

[0087] 5.1 Application in piglets:

[0088] 120 healthy piglets with similar weights (7.0±0.5 kg) were randomly divided into 4 groups of 30 piglets each.

[0089] Control group: fed 100% commercial starter feed.

[0090] Experimental Group A: Feeded with a diet consisting of 90% commercial creep feed + 10% pre-fermented mushroom and grass feed A

[0091] Experimental Group B: Feeded with a diet consisting of 90% commercial creep feed + 10% pre-fermented mushroom and grass feed B.

[0092] Experimental group C: fed a diet consisting of 90% commercial creep feed + 10% pre-fermented mushroom and grass feed C.

[0093] The preliminary trial period was 3 days, and the formal trial period was 14 days. During the trial, the pigs had free access to feed and water, and their health status and diarrhea were observed and recorded daily.

[0094]

[0095] Note: Diarrhea incidence (%) = (Total number of diarrheal episodes / (Number of experimental pigs × Number of experimental days)) × 100%.

[0096] As shown in Table 1, compared with the control group, the incidence of diarrhea in piglets in each experimental group with 10% of the pre-fermented mushroom and grass feed of the present invention was significantly reduced, proving that the fermented feed of the present invention has a good intestinal health regulation function.

[0097] 5.2 Application in fattening pigs:

[0098] In fattening pigs, feeding them with 30%–50% pre-fermented mushroom and grass feed B plus 50%–70% complete feed resulted in better weight gain and health than feeding them with only complete feed. The experimental methods and results are as follows:

[0099] 120 healthy fattening pigs of similar weight were selected and randomly divided into 4 groups of 30 pigs each, and kept in 4 pens.

[0100] Control group: fed 100% complete compound feed.

[0101] Experimental Group I: fed a mixed diet consisting of 70% complete compound feed + 30% pre-fermented mushroom and grass feed B.

[0102] Experimental Group II: Feeded with a mixed diet consisting of 60% complete compound feed + 40% pre-fermented mushroom and grass feed B.

[0103] Experimental Group III: Feeded with a mixed diet consisting of 50% complete compound feed + 50% pre-fermented mushroom and grass feed B.

[0104] The preliminary trial period was 3 days, and the formal trial period was 20 days. During the trial, participants had free access to food and water. The total feed intake of each group was accurately recorded daily. At the beginning and end of the trial, each animal was weighed on an empty stomach, and the feed conversion ratio was recorded. The results are shown in Table 2.

[0105]

[0106] Note: Unit: jin (500g)

[0107] Feed conversion ratio (%) = (total feed intake / average weight gain per head) × 100%.

[0108] Total feed intake = Average daily feed intake × Number of experimental days

[0109] Average head weight gain = Final average weight gain - Initial average weight gain.

[0110] As shown in Table 2, with the same average daily feed intake, the daily weight gain of all experimental groups was higher than that of the control group, and the feed conversion ratio was lower in all experimental groups than in the control group. This indicates that the pre-fermented mushroom and grass feed prepared in this invention can effectively improve feed conversion efficiency, promote the growth of fattening pigs, and reduce feed costs.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the design of this case. All equivalent changes made based on the key design features of this case shall fall within the protection scope of this case.

Claims

1. A method for preparing a compound probiotic starter, characterized in that, Includes the following steps: S1. Prepare Pichia pastoris membrane ( Pichia membranifaciens C200-1, Bacillus reuteri ( Brevibacillus reuszeri ) RF10-5, Pediococcus acidilactici ( Pediococcus acidilactici Lactobacillus plantarum ( ‌Lactiplantibacillus plantarum Lactobacillus bruneri ( Lactobacillus buchneri ) and Bifidobacteria ( Bifidobacterium ATCC 27673; The preservation number of the Pichia pastoris C200-1 is CCTCC NO: M20221232; The preservation number of Bacillus reuteri RF10-5 is CCTCC NO: M20221968; Each strain was activated, cultured in a large-scale manner, and cultured in a fermenter to obtain individual bacterial suspensions. The viable cell concentration of each bacterial suspension was (0.5~1.5) × 10⁻⁶. 8 Within the range of CFU / mL; S2. Mix Pichia pastoris C200-1 bacterial suspension and Bacillus reuteri RF10-5 bacterial suspension at a volume ratio of (0.8~1.2):1 to obtain an aerobic mixed bacterial suspension; S3. Mix the *Pediococcus lactis* culture, *Lactobacillus plantarum* culture, *Lactobacillus brucelli* culture and *Bifidobacterium* culture obtained in step S1 at a volume ratio of 1:1:1:1 to obtain an anaerobic mixed culture. S4. The aerobic mixed bacterial solution obtained in step S2 and the anaerobic mixed bacterial solution obtained in step S3 are mixed at a volume ratio of 1:(45~55) to prepare a compound probiotic fermentation agent seed liquid; the compound probiotic fermentation agent seed liquid is then activated and cultured to obtain a compound probiotic fermentation agent. The specific methods for activating and culturing the seed liquid of the compound probiotic fermentation agent are as follows: Take the seed liquid of compound probiotic fermentation agent, dilute it with 3-5% sugarcane molasses at a ratio of 1:200 v / v, and then place it in a 30℃ environment for static culture for 8-12 hours to obtain the activated compound probiotic fermentation agent.

2. The preparation method of the compound probiotic starter as described in claim 1, characterized in that: In step S1, the activation and expansion culture process of each strain is as follows: Pichia pastoris C200-1, stored in glycerol, was inoculated into PDB medium and cultured at 26-30°C until OD500. 600 =0.45; Glycerol-frozen Bacillus reuteri RF10-5 was inoculated onto LB medium and cultured at 32-37℃ until OD... 600 =0.7; Frozen strains of *Pediococcus lactis*, *Lactobacillus plantarum*, *Lactobacillus bruneri*, and *Bifidobacterium* were taken from glycerol and inoculated separately onto MRS medium for culture until OD500. 600 =0.6~1.5, temperature controlled at 35~38℃.

3. The preparation method of the compound probiotic starter as described in claim 1, characterized in that: The viable cell concentration of each single bacterial culture was controlled to be 1×10⁻⁶. 8 CFU / mL; and the Pichia pastoris C200-1 bacterial suspension and Bacillus reuteri RF10-5 bacterial suspension are mixed at a volume ratio of 1:1, and the bacterial suspensions of Pediococcus lactis, Lactobacillus plantarum, Lactobacillus brunelli and Bifidobacterium are mixed at a volume ratio of 1:1:1:

1.

4. A compound probiotic starter, characterized in that: It is prepared by the method according to any one of claims 1 to 3.

5. A pre-fermented mushroom and grass feed, characterized in that, The preparation method of the pre-fermented mushroom and grass feed is as follows: the compound probiotic fermentation agent described in claim 4 is evenly sprayed onto the giant reed grass raw material, with 50-150 L of compound probiotic fermentation agent sprayed per ton of giant reed grass; then the mixture is fermented in a sealed environment at 20-40℃ for 10-15 days to obtain the pre-fermented mushroom and grass feed.

6. The pre-fermented mushroom and grass feed as described in claim 5, characterized in that: Before spraying the compound probiotic fermentation agent, the giant reed grass is pretreated. The pretreatment method is as follows: harvest the giant reed grass with a plant height of about 2.0 meters on a sunny day, leave a stubble of 5-7 cm, and after preliminary chopping, transport it to the processing workshop and crush it to a particle size of 1-3 mm using a shredder.

7. The pre-fermented fungal feed as described in claim 5, characterized in that: The pre-fermented mushroom and grass feed is used as pig feed.

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