Fermented feed for animal husbandry and preparation method thereof

Abstract

The invention provides a fermented feed for animal husbandry and a preparation method of the fermented feed, belongs to the technical field of biological fermented feeds, and particularly relates to the technical field of the biological fermented feeds. According to the preparation method, bacillus subtilis, lactobacillus plantarum and saccharomyces cerevisiae are used as complex microbial inoculants to ferment the feed, and by means of use of a Saccharomyces cerevisiae extract in a fermentation substrate, the fermentation efficiency is improved; the feed additive can promote colonization of probiotics in intestinal tracts, prevent growth of pathogenic microorganisms, maintain reasonable intestinal flora structure of livestock and intestinal development of the livestock, reduce diarrhea rate, improve digestion and absorption functions of the intestinal tracts and improve growth performance. The fermented feed for animal husbandry and the preparation method thereof have the advantages of being low in cost, short in production cycle, environmentally friendly, capable of improving the intestinal function of livestock and improving the growth performance and high in economic benefit.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-fermented feed, and particularly relates to a fermented feed for livestock and its preparation method. Background Art

[0002] With the enhancement of consumers' food safety awareness and the emergence of pathogenic microorganism drug resistance, antibiotic-free farming has become an irreversible trend. Therefore, finding alternatives to antibiotics and developing alternative technologies to ensure the healthy development of livestock production is a major scientific issue faced by animal nutritionists. The microbial fermentation feed technology is a technology developed in recent years and has great application prospects. The feed reduces the anti-nutritional factors in the feed through the fermentation process, improves the content of nutritional components, and plays functions such as regulating the microecological balance of the animal intestine, promoting animal growth, and enhancing the body immunity, and plays an important role in healthy farming.

[0003] Intestinal health encompasses multiple positive aspects of the gastrointestinal tract, such as the effective digestion and absorption of food, protection from gastrointestinal diseases, the normal and stable intestinal microbiota, and an effective immune state. In addition to playing the physiological functions of nutritional digestion and absorption, the intestine is also the largest immune organ of animals because, essentially, the intestine is the first line of defense for animals to resist microbial stress from the environment, especially pathogens invading from the gastrointestinal tract.

[0004] The prior art, such as the Chinese invention patent with the authorization announcement number of CN 119184201 B, discloses a fermented feed for improving intestinal health and its preparation method, which relates to the technical field of feed processing; the fermented feed for improving intestinal health is composed of ZIF-8@clove basil oil nanoparticles, porphyra polysaccharide active traditional Chinese medicine composite gel, corn flour, soybean meal powder, grass powder, Bacillus subtilis, yeast, and papain; the porphyra polysaccharide active traditional Chinese medicine composite gel is composed of porphyra polysaccharide, chitosan, compound traditional Chinese medicine ultrafine powder, and citric acid; by combining the ZIF-8@clove basil oil nanoparticles with the porphyra polysaccharide active traditional Chinese medicine composite gel and then coating the fermented feed, it is effectively isolated from the external environment, maintaining the activity of the microorganisms in the fermented feed, increasing the stability of the feed, and can also adsorb harmful substances in the feed, reducing the adverse effects on the digestive tract, significantly improving intestinal health, thereby promoting digestion and absorption and helping to improve the growth performance of animals. Summary of the Invention

[0005] The purpose of the present invention is to provide a fermented feed for livestock and its preparation method. The fermented feed has a low cost, a short production cycle, is environmentally friendly, can improve the intestinal function of livestock, improve the growth performance, and has a high economic benefit.

[0006] The technical solution adopted by the present invention to achieve the above purpose is as follows: The present invention discloses a preparation method of fermented feed for livestock and poultry, comprising the following steps: A. Mix crushed corn, soybean meal, wheat bran, wheat flour, corn germ meal, and vitex agnus-castus extract evenly according to a mass ratio of 80-120:100-150:30-50:300-500:20-40:8-15 to obtain a fermentation substrate; B. Mix the compound bacterial liquid evenly with the fermentation substrate, adjust the water content to 45wt%-55wt% with water, and ferment it sealed at 30-35°C for 2-4d to obtain a product; The above compound bacterial liquid is obtained by mixing Bacillus subtilis bacterial liquid, Lactobacillus plantarum bacterial liquid, and Saccharomyces cerevisiae bacterial liquid. Adding a certain amount of vitex agnus-castus extract to the fermentation substrate can improve the adhesion ability of probiotics, facilitate the colonization of probiotics in the intestine, prevent the growth of pathogenic microorganisms, maintain a reasonable intestinal flora structure, reduce the diarrhea rate, improve the intestinal digestion and absorption function, increase the feed-to-weight ratio of Hu sheep lambs, and increase economic benefits.

[0007] Preferably, in the above step B, the mass ratio of the compound bacterial liquid to the fermentation substrate is 5-10:100.

[0008] Preferably, the viable bacteria number ratio of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae in the above compound bacterial liquid is 2:2-3:1-1.5.

[0009] Preferably, the total viable bacteria number of the compound bacteria in the above compound bacterial liquid ≥ 2×10 9 CFU / mL.

[0010] Preferably, the product in the above step B is subjected to a drying treatment, and the water content is controlled at 10wt%-15wt%.

[0011] Preferably, the temperature of the above drying treatment is 20-40°C.

[0012] Preferably, the preparation steps of the above compound bacterial liquid include: a. Strain activation: Take Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, and inoculate them into a liquid medium for activation culture respectively; b. Bacterial liquid culture: Transfer the three kinds of bacterial liquids after activation culture in step a to the corresponding liquid media respectively for enlarged culture; c. Compound: Mix the bacterial liquids after enlarged culture and compound them in proportion to obtain a compound bacterial liquid.

[0013] Preferably, the liquid medium of the above Bacillus subtilis is LB liquid medium.

[0014] Preferably, the liquid medium of the above Lactobacillus plantarum is MRS liquid medium.

[0015] Preferably, the liquid medium of the above-mentioned Saccharomyces cerevisiae is YPD liquid medium.

[0016] Preferably, the above-mentioned fermentation substrate further includes butterfly pea pollen and honeysuckle extract.

[0017] Preferably, the total content of butterfly pea pollen and honeysuckle extract in the above-mentioned fermentation substrate is 5wt%-9wt%.

[0018] Preferably, the mass ratio of the above-mentioned butterfly pea pollen to honeysuckle extract is 1-2:2.5-4. Adding butterfly pea pollen and honeysuckle extract with a mass ratio of 1-2:2.5-4 to the fermentation substrate can promote the secretion of ileal mucin MUC-2 in Hu sheep lambs, prevent pathogenic bacteria from entering the intestinal mucus, enhance the gastrointestinal barrier function, improve intestinal health, reduce the diarrhea rate, increase the feed-to-weight ratio, and increase economic benefits.

[0019] The present invention discloses a fermented feed for livestock, which is prepared by using the above-mentioned preparation method.

[0020] The present invention also discloses a breeding method for Hu sheep lambs, which feeds the Hu sheep lambs with a mixed feed obtained by mixing a basal diet and the above-mentioned fermented feed for livestock, so as to achieve the effects of improving the intestinal function of Hu sheep lambs and enhancing the growth performance.

[0021] Preferably, the mass ratio of the above-mentioned basal diet to the fermented feed for livestock is 8-9:1-2.

[0022] Since a certain amount of vitex agnus-castus extract is included in the fermentation substrate when preparing the fermented feed in the present invention, the following beneficial effects are achieved: it can improve the adhesion ability of probiotics, thereby promoting the colonization of probiotics in the intestine, preventing the growth of pathogenic microorganisms, promoting the intestinal development of livestock, reducing the diarrhea rate, improving the intestinal digestion and absorption function, and improving the growth performance.

[0023] Since a specific proportion of butterfly pea pollen and honeysuckle extract is added to the fermentation substrate when preparing the fermented feed in the present invention, the following beneficial effects are achieved: it can promote the secretion of intestinal mucin, prevent pathogenic bacteria from entering the intestinal mucus, enhance the gastrointestinal barrier function, improve intestinal health, reduce the diarrhea rate, and improve the growth performance.

[0024] The present invention provides a preparation method for a fermented feed for livestock. The preparation method has low cost, short production cycle, and environmental protection. The prepared feed can improve the intestinal function of livestock, enhance the growth performance, and has high economic benefits. Description of the Drawings

[0025] Figure 1 It is the measurement result of the number of adherent bacteria in Test Example 1 of the present invention.

[0026] Figure 2This is the measurement result of the material weight ratio in Test Example 2 of the present invention.

[0027] Figure 3 It is the measurement result of diarrhea rate in Test Example 2 of the present invention.

[0028] Figure 4 The ileum of the Hu sheep lamb in the test example 2 of the present invention Muc-2 mRNA expression level. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be pointed out that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other without conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are purchased from the market.

[0031] The Bacillus subtilis involved in the embodiments provided by the present invention was purchased from the China Industrial Microorganism Culture Collection Administration Center with a number of CICC24534; Lactobacillus plantarum was purchased from the China Industrial Microorganism Culture Collection Administration Center with a number of CICC20265; Saccharomyces cerevisiae was purchased from the China Industrial Microorganism Culture Collection Administration Center with a number of CICC1005. Butterfly pea pollen was purchased from Shaanxi Senyuan Biotechnology Co., Ltd. Chasteberry extract, product specification 10:1, was purchased from Shaanxi Bohong Health Industry Co., Ltd. Honeysuckle extract, product specification 10:1, was purchased from Fufeng Ciyuan Biotechnology Co., Ltd.

[0032] Embodiment 1: 1. A method for preparing fermented feed for animal husbandry, comprising the following steps: 1.1 Crush corn, soybean meal, bran, and corn germ meal and pass them through a 60-mesh sieve. Then, mix the crushed corn, soybean meal, bran, corn germ meal, wheat flour, and chasteberry extract in a mass ratio of 100:120:40:30:400:12 to obtain a fermentation substrate.

[0033] 1.2 Preparation of the compound bacterial liquid, including: 1.2.1 Activation of the bacterial strains: YPD medium: 20 g of tryptone, 20 g of glucose, 10 g of yeast extract, made up to 1000 mL with distilled water, pH value is natural, and 18 g of agar is added for solid medium.

[0034] LB liquid medium: 10 g of peptone, 10 g of sodium chloride, 5.0 g of yeast powder, made up to 1000 mL with distilled water, and the pH is adjusted to 7.4.

[0035] MRS medium: 10 g of peptone, 5 g of glucose, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium hydrogen phosphate, 0.25 g of manganese sulfate, 2 g of ammonium citrate, 2 g of sodium acetate trihydrate, 1.0 mL of Tween-80, 0.5 g of magnesium sulfate, made up to 1000 mL with distilled water, and the pH is adjusted to 6.5. 18 g of agar is added for solid medium.

[0036] Streak plate activation of the strains Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae preserved in glycerol. Select single colonies and inoculate them into 50 mL of liquid medium for subculture. Saccharomyces cerevisiae is cultured with YPD liquid medium at 30 °C and 180 r / min for 36 h by shaking. Bacillus subtilis is cultured with LB liquid medium at 37 °C and 150 r / min for 24 h by shaking. Lactobacillus plantarum is cultured with MRS liquid medium at 37 °C for 24 h by static culture. The above three kinds of cultured bacterial liquids are used as seed liquids.

[0037] 1.2.2 Inoculate the seed liquid into 250 mL of liquid medium according to an inoculation amount of 1.2% (v / v). Saccharomyces cerevisiae is inoculated into YPD liquid medium and cultured statically at 30 °C for 30 h. Bacillus subtilis is inoculated into LB liquid medium and cultured statically at 37 °C for 24 h. Lactobacillus plantarum is inoculated into MRS liquid medium and cultured statically at 37 °C for 24 h. Measure the OD 600nm value and perform dilution coating on the plate to calculate the cell density. The viable count of the Bacillus subtilis bacterial liquid is calculated to be 7.6×10 9 CFU / mL, the viable count of the Lactobacillus plantarum bacterial liquid is 6.8×10 9 CFU / mL, and the viable count of the Saccharomyces cerevisiae bacterial liquid is 3.6×10 9 CFU / mL. Mix the three kinds of bacterial liquids evenly according to the volume ratio of 1:1.5:1.5 to obtain the compound bacterial liquid.

[0038] 1.3 Inoculate the above compound bacterial liquid into the fermentation substrate according to a ratio of 8 wt%, mix evenly, ferment in a sealed state at 35 °C for 3 d, and air-dry at 25 °C until the water content is 12 wt% after fermentation to obtain the fermented feed.

[0039] Example 2: Compared with Example 1, the differences are as follows: 1.1 The corn, soybean meal, wheat bran, and corn germ meal are pulverized and then passed through a 60-mesh sieve. Then, the pulverized corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract are mixed evenly according to the mass ratio of 100:120:40:30:400:3 to obtain the fermentation substrate.

[0040] Example 3 Compared with Example 1, the differences are as follows: 1.1 The corn, soybean meal, wheat bran, and corn germ meal are pulverized and then passed through a 60-mesh sieve. Then, the pulverized corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract are mixed evenly according to the mass ratio of 100:120:40:30:400:18 to obtain the fermentation substrate.

[0041] Example 4: Compared with Example 1, the differences are as follows: 1.1 The corn, soybean meal, wheat bran, and corn germ meal are pulverized and then passed through a 60-mesh sieve. Then, the pulverized corn, soybean meal, wheat bran, corn germ meal, and wheat flour are mixed evenly according to the mass ratio of 100:120:40:30:400 to obtain the fermentation substrate.

[0042] Example 5: Compared with Example 1, the differences are as follows: 1.1 The corn, soybean meal, wheat bran, and corn germ meal are pulverized and then passed through a 60-mesh sieve. Then, the pulverized corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract are mixed evenly according to the mass ratio of 100:120:40:30:400:12, and then a mixture of 6 wt% of butterfly pea pollen and honeysuckle extract is added, where the mass ratio of butterfly pea pollen to honeysuckle extract is 1:2, and the mixture is mixed evenly to obtain the fermentation substrate.

[0043] Example 6: Compared with Example 1, the differences are as follows: 1.1 The corn, soybean meal, wheat bran, and corn germ meal are pulverized and then passed through a 60-mesh sieve. Then, the pulverized corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract are mixed evenly according to the mass ratio of 100:120:40:30:400:12, and then a mixture of 6 wt% of butterfly pea pollen and honeysuckle extract is added, where the mass ratio of butterfly pea pollen to honeysuckle extract is 1:3.5, and the mixture is mixed evenly to obtain the fermentation substrate.

[0044] Example 7: Compared with Example 1, the differences are as follows: 1.1 Corn, soybean meal, wheat bran, and corn germ meal were crushed and passed through a 60-mesh sieve. Then, the crushed corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract were mixed evenly according to a mass ratio of 100:120:40:30:400:12. Next, a mixture of 6 wt% butterfly pea pollen and honeysuckle extract was added, where the mass ratio of butterfly pea pollen to honeysuckle extract was 1:0.8, and the mixture was mixed evenly to obtain a fermentation substrate.

[0045] Example 8: Compared with Example 1, the differences are as follows: 1.1 Corn, soybean meal, wheat bran, and corn germ meal were crushed and passed through a 60-mesh sieve. Then, the crushed corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract were mixed evenly according to a mass ratio of 100:120:40:30:400:12. Next, a mixture of 6 wt% butterfly pea pollen and honeysuckle extract was added, where the mass ratio of butterfly pea pollen to honeysuckle extract was 1:5, and the mixture was mixed evenly to obtain a fermentation substrate.

[0046] Example 9: Compared with Example 1, the differences are as follows: 1.1 Corn, soybean meal, wheat bran, and corn germ meal were crushed and passed through a 60-mesh sieve. Then, the crushed corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract were mixed evenly according to a mass ratio of 100:120:40:30:400:12. Next, 6 wt% butterfly pea pollen was added and mixed evenly to obtain a fermentation substrate.

[0047] Example 10: Compared with Example 1, the differences are as follows: 1.1 Corn, soybean meal, wheat bran, and corn germ meal were crushed and passed through a 60-mesh sieve. Then, the crushed corn, soybean meal, wheat bran, corn germ meal, wheat flour, and vitex agnus-castus extract were mixed evenly according to a mass ratio of 100:120:40:30:400:12. Next, 6 wt% honeysuckle extract was added and mixed evenly to obtain a fermentation substrate.

[0048] Test Example 1: 1. Probiotic adhesion ability test 1.1 Reagents used: DMEM culture medium was purchased from Invitrigen, USA.

[0049] DMEM complete culture medium: 78% (v / v) DMEM culture medium, 20% (v / v) fetal bovine serum, 1% (v / v) streptomycin (100 μg / mL), 1% (v / v) penicillin (100 U / mL).

[0050] PBS solution: Weigh 8 g NaCl, 0.2 g KCl, 1.42 g Na2 HPO 4 ·12H 2 O, 0.24 g KH 2 PO 4 Dissolve in 800 mL of ultrapure water, adjust the pH to 7.4, and make up to 1 L with ultrapure water.

[0051] Trypsin cell digestive solution: Weigh 0.25 g of trypsin and 0.02 g of EDTA-Na, add 90 mL of deionized sterilized water, mix well and make up to about 100 mL, filter through a 0.22-μm MILLEX GP disposable filter and dispense.

[0052] Cell lysate: Pipette 0.5 mL of Triton X-100 into 99.5 mL of PBS solution, mix well and store in a 4°C refrigerator for later use.

[0053] 1.2 Cell culture: After thawing the Caco-2 cells stored in a liquid nitrogen tank, place them in a culture flask. Add DMEM complete culture medium and incubate in a 37°C, 5% CO 2 2 incubator. When the cells grow well (70% confluence), passage them; discard the culture medium, rinse twice with PBS solution to wash away the DMEM complete medium. Then add 1 mL of trypsin digestive solution and digest in a 37°C, 5% CO 2 2 incubator for 3 min, and observe the cell digestion under a microscope. When the cells are dispersed into single cells and become round, gently tap the flask wall. After the cells completely detach from the flask wall, add 1 mL of DMEM complete culture medium to terminate the digestion. Transfer the liquid to a 15-mL centrifuge tube, centrifuge at 500 r / min, 37°C for 5 min, and discard the supernatant. Then add 1 mL of DMEM complete culture medium, gently pipette to mix evenly to make a cell suspension. Divide the cell suspension into new medium at a ratio of 1:5, that is, pipette 200 μL and add it to 4 mL of new DMEM complete culture medium. Replace the culture medium every 2 days and passage every 4 days. After 3 passages, perform the adhesion experiment. After trypsin digestion, inoculate at a density of 2×10 5 cells / mL into a 48-well plate. Observe the cell adhesion situation and use it for experiments after 4 days.

[0054] 1.3 Preparation of probiotic suspension: Take the supernatant of the feed after fermentation and before air-drying, centrifuge at 6000 r / min, 4°C for 5 min, collect the bacterial cells, wash twice with PBS solution, and resuspend the bacterial cells in DMEM culture medium, adjusting the bacterial cell concentration to 4×10 8 CFU / mL.

[0055] 1.4 Adhesion counting: Digest the cultured Caco-2 cells, dilute the cells with DMEM complete culture medium to make the concentration reach about 5×10 5cells / mL, take 1 mL of cells and drop them into a cell culture dish, and incubate in a 5% CO₂ incubator at 37 °C. When the cells grow to a monolayer, wash them twice with PBS solution. Add 1 mL of probiotic suspension to each culture dish. After incubating for 2 h, wash 5 times with PBS solution to remove the non - adhered probiotics. Add 1 mL of cell lysate to the culture dish, mix well and let it stand for 10 min to lyse the cells. Transfer the mixture to a test tube, after serial dilution, perform plate colony counting to calculate the total viable count of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae in the mixture. Select another culture dish that has grown to a monolayer, use a hemocytometer to count the cells, and calculate the number of cells in the culture dish. Each treatment is done in 3 replicates, and calculate the average number of bacteria adhered to each cell according to the following formula. 2 The number of adhered bacteria (per cell) = total viable count / number of cells in the culture dish.

[0056] The results of the determination of the number of adhered bacteria are shown in

[0057] Figure 1

[0058] Test Example 2: 1. Animal experiment Select 300 weaned Hu sheep lambs with similar body weights, randomly divide them into 10 groups, and feed them 15 wt% of the fermented feed prepared in the above - mentioned example + 85 wt% of the basal diet. Each group has 3 replicates, and each replicate has 10 Hu sheep lambs. The test period is 42 d. The composition and nutritional level of the basal diet are shown in Table 1. The lambs in each group are raised in the same feeding environment and management conditions. The sheep are allowed to eat and drink freely, and the sheep house is disinfected and immunized and dewormed according to the conventional method.

[0059] Table 1 Composition and nutritional level of the basal diet for Hu sheep lambs (air - dried basis)

[0060] Note: ① The premix is purchased from Zhangzhou Dabeinong Agro - animal Husbandry Technology Co., Ltd. ② The crude protein in the nutritional level is the measured value, and the rest are calculated values.

[0061] 2. Test indicators and methods 2.1 Growth performance During the test, accurately record the feed consumption of each replicate lamb. Weigh the lambs on an empty stomach for 12 h at the beginning and end of the test, accurately record the number of diarrhea cases of each group of lambs, and calculate the growth performance data such as average daily feed intake, average daily gain, diarrhea rate, and feed - to - gain ratio. The results of the determination of the feed - to - gain ratio are shown in Figure 2 . The results of the determination of the diarrhea rate are shown in Figure 3

[0062] Average daily gain = (final weight - initial weight) / number of test days ​​​Average daily feed intake = Total feed consumption / Number of experimental days Feed conversion ratio = Average daily feed intake / Average daily weight gain Diarrhea rate = [ (Number of diarrhea cases × Diarrhea days) / (Total number of animals × Number of experimental days) ] × 100% 2.2 In the ileum tissue samples of the intestine Muc-2 Determination of gene expression level On the 42nd day of the experiment, 15 Hu sheep lambs were randomly selected from each group for slaughter. The middle part of the ileum was retrieved with a sterile scalpel, rinsed thoroughly with physiological saline, blotted dry with filter paper, and mucosal samples were scraped onto a glass slide and placed in a cryotube, wrapped with tin foil, quickly frozen in liquid nitrogen, and stored in a -80 °C refrigerator.

[0063] Extraction of total RNA: Weigh about 0.1 g of the sample, and then follow the operating instructions of the kit (Trizol Reagent, TaKaRa, Japan).

[0064] RNA concentration determination: Use a nucleic acid protein detector (Beckman DU-800, CA, USA) for detection. The ratio of A260 / A280 represents the purity of RNA, and this ratio is preferably 2.0.

[0065] Synthesis of cDNA: Follow the instructions of the reverse transcription kit (Prime ScriptTM Regent Kit, TaKaRa, Japan).

[0066] Primer design: The primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd. For the gene Muc-2 the forward primer is: 5’-CACCACCCTTCCAGGCTC-3’, and the reverse primer is: 5’-GAGGTAGGGCTTGTGCTGAG-3’; for the reference gene β-actin the forward primer is: 5’-CGCAAGTACTCCGTGTGGAT-3’, and the reverse primer is: 5’-TAACGCAGCTAACAGTCCGC-3’.

[0067] Real-time fluorescence quantitative PCR: The reaction system is: 0.4 µL of 10 µmol / L forward primer, 0.4 µL of 10 µmol / L forward primer, 1.0 µL of cDNA template, 0.2 µL of ROX reference dye(50×), 3.0 µL of RNase Free H 2 O, 5.0 µL SYBR Premix Ex Taq TM The reaction program is: 95 °C for 30 s, 95 °C for 10 s, 60 °C for 25 s, for 40 cycles. The relative expression level of the target gene is based on β-actin the gene as the internal reference gene, using 2−ΔΔCt Calculated by the method. The ileum of Hu sheep lambs Muc-2 The mRNA expression levels are shown in Figure 4 .

[0068] It can be seen from Figure 1 that the number of probiotics adhering to cells in the fermented feed prepared in Example 1 is significantly more than that in Example 2, Example 3, and Example 4, and the adhesion ability of probiotics is stronger. It can be seen from Figure 2 , Figure 3 that after feeding Hu sheep lambs with the fermented feeds prepared in Examples 1-4 respectively, compared with Example 2, Example 3, and Example 4, the diarrhea rate and feed-to-weight ratio of the Hu sheep lambs fed with Example 1 are significantly reduced. However, there is no significant difference in the above indexes between Example 2, Example 3, and Example 4. This shows that adding a certain amount of vitex agnus-castus extract to the fermentation substrate can improve the adhesion ability of probiotics, which is beneficial to promoting the colonization of probiotics in the intestines of Hu sheep lambs, preventing the growth of pathogenic microorganisms, maintaining a reasonable intestinal flora structure in Hu sheep lambs, reducing the diarrhea rate, improving the intestinal digestion and absorption function, increasing the feed-to-weight ratio of Hu sheep lambs, and increasing economic benefits.

[0069] It can be seen from Figure 2 , Figure 3 , Figure 4 that after feeding Hu sheep lambs with the fermented feeds prepared in Examples 1, 5-10 respectively, compared with Example 1, Example 7-10, the ileum MUC-2 mRNA expression levels of the Hu sheep lambs fed with Example 5 and Example 6 are significantly higher, and the diarrhea rate and feed-to-weight ratio of the Hu sheep lambs are significantly reduced. And there is no significant difference in the above indexes between Example 7-10 and Example 1. This shows that adding butterfly pea pollen and honeysuckle extract with a mass ratio of 1-2:2.5-4 to the fermentation substrate can promote the secretion of mucin MUC-2 in the ileum of Hu sheep lambs, prevent pathogenic bacteria from entering the intestinal mucus, enhance the gastrointestinal barrier function, improve intestinal health, reduce the diarrhea rate, increase the feed-to-weight ratio of Hu sheep lambs, and increase economic benefits.

[0070] The above-mentioned embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any form of limitation on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but it should still be regarded as the same technology or embodiment as the present invention in essence.

[0071] In this text, specific examples are used to elaborate on the principles and implementation manners of the present application. The descriptions of the above embodiments are only used to help understand the method of the present application and its core idea. The above are only the preferred implementation manners of the present application. It should be noted that due to the limited nature of literal expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for preparing fermented feed for animal husbandry, characterized in that: The steps include: A. Evenly mix the crushed corn, soybean meal, bran, wheat flour, corn germ meal and chasteberry extract in a mass ratio of 80-120:100-150:30-50:300-500:20-40:8-15 to obtain a fermentation substrate; B. Mix the composite bacterial liquid and the fermentation substrate evenly, adjust the water content to 45wt%-55wt% with water, and ferment at 30-35°C for 2-4 days to obtain the product; The composite bacterial liquid is obtained by uniformly mixing a bacillus subtilis bacterial liquid, a lactobacillus plantarum bacterial liquid and a saccharomyces cerevisiae bacterial liquid.

2. The method for preparing a fermented feed for animal husbandry according to claim 1, characterized in that: In step B, the mass ratio of the composite bacterial liquid to the fermentation substrate is 5-10:

100.

3. The method for preparing a fermented feed for livestock according to claim 1, characterized in that: The ratio of the number of live bacteria of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae in the composite bacterial liquid is 2:2-3:1-1.

5.

4. The method for preparing a fermented feed for animal husbandry according to claim 3, characterized in that: The total number of viable bacteria in the composite bacterial solution is ≥ 2×10 9 CFU / mL.

5. The method for preparing a fermented feed for animal husbandry according to claim 1, characterized in that: The product in step B is dried to control the moisture content to be between 10wt% and 15wt%.

6. The method for preparing a fermented feed for animal husbandry according to claim 5, characterized in that: The temperature of the drying process is 20-40°C.

7. The method for preparing fermented feed for livestock according to claim 1, characterized in that: The steps of preparing the composite bacterial solution include: a. Activation of bacterial strains: Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae are inoculated into liquid culture medium for activation culture respectively; b. Bacterial liquid culture: transfer the three bacterial liquids after activation culture in step a to corresponding liquid culture medium respectively, and expand the culture; c. Compounding: Mix the bacterial solutions after expanded culture and compound them according to the proportion to obtain a composite bacterial solution.

8. A fermented feed for animal husbandry, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 7.

9. A method for breeding Huyang lambs, characterized in that: The mixed feed prepared by mixing the basic daily diet with the fermented feed for animal husbandry prepared by the preparation method according to claim 1 is fed to the Hu sheep lambs.

10. The method for breeding Huyang lambs according to claim 9, characterized in that: The mass ratio of the basic diet to the fermented feed for animal husbandry is 8-9:1-2.

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