Antibiotic-free premix concentrate feed

Abstract

The application discloses a non-antibiotic premix concentrated feed, which comprises 16-25 parts by weight of an extract of Gynura bicolor DC, 60-80 parts by weight of concentrated dry powder of Morus alba L, 1-3 parts by weight of xylo-oligosaccharide and 6-12 parts by weight of a microbial compound preparation; the microbial compound preparation comprises cellulose-decomposing bacteria, bacillus, lactic acid bacteria, bifidobacterium and yeast. The premix core components (the extract of Gynura bicolor DC, Morus alba L, xylo-oligosaccharide and the compound microbial preparation) of the application all belong to natural plant-derived or microbial-derived substances and do not contain any antibiotic. Through synergistic effect of various active ingredients, the non-antibiotic premix concentrated feed can avoid drug residues in animals, generation of drug-resistant strains and public health risks of transmission to human beings through food chain caused by addition of antibiotics in feed.

Description

Technical Field

[0001] This invention belongs to the field of animal feed technology, and in particular relates to an antibiotic-free premixed concentrated feed. Background Technology

[0002] As livestock farming becomes increasingly large-scale and intensive, feed, as a crucial factor in ensuring the healthy growth of livestock and poultry, directly impacts animal growth rate, immune function, feed conversion rate, and the quality and safety of livestock products. Traditional feeds commonly contain antibiotic growth promoters to improve feed efficiency and reduce disease incidence. However, long-term use of antibiotics can lead to the development of drug-resistant and antibiotic-resistant strains in animals, which can then be transmitted to humans through the food chain, causing serious food safety and public health problems.

[0003] Currently, a series of antibiotic-free feed products claiming to have immunomodulatory functions have appeared on the market. These products mainly replace antibiotics to prevent diseases and promote growth by adding extracts of traditional Chinese medicine, probiotics, organic acids, and their compound preparations. For example, plant-based feed additives made from extracts of traditional Chinese medicines such as honeysuckle, isatis root, and astragalus, or microecological preparations such as Lactobacillus acidophilus and Bacillus subtilis are added to improve the intestinal flora structure and enhance immunity in animals. However, existing products or technologies still have many limitations in terms of stability, release of active ingredients, synergistic effects of components, and process controllability. Summary of the Invention

[0004] The purpose of this invention is to provide an antibiotic-free premixed concentrated feed to address the problems of antibiotic resistance and antibiotic-resistant strains in animals due to long-term use of antibiotics in feed, as well as the transmission of antibiotics to humans through the food chain.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: An antibiotic-free, immune-enhancing premixed concentrated feed, comprising: 16-25 parts by weight of *Chrysanthemum indicum* and *Panax notoginseng* extract, 60-80 parts by weight of concentrated mulberry protein powder, 1-3 parts by weight of xylooligosaccharides, and 6-12 parts by weight of a microbial compound preparation; the microbial compound preparation comprises cellulolytic bacteria, Bacillus, lactic acid bacteria, Bifidobacterium, and yeast; the method for preparing the compound microbial preparation is as follows: Step 1, raw material preparation: 30-60 parts by weight of rice bran or grass powder, 10-18 parts by weight of wheat bran, 10-18 parts by weight of corn, 10-18 parts by weight of oilseed cake, and molasses. Alternatively, use 1-3 parts by weight of brown sugar, 1-3 parts by weight of cellulose-decomposing bacteria, 1-3 parts by weight of Bacillus subtilis, 1-3 parts by weight of lactic acid bacteria, 1-3 parts by weight of Bifidobacterium, and 1-3 parts by weight of yeast; Step 2, dissolving the inoculum: dilute the evenly stirred inoculum with water to a ratio of 150:1; Step 3, mix the dissolved inoculum with 50 parts of rice bran or grass powder, 15 parts of wheat bran, 10 parts of corn, 10 parts of oilseed cake, and 2 parts of molasses or brown sugar evenly; Step 4, place the evenly mixed mixture into a container for fermentation, with the fermentation temperature not lower than 20 degrees Celsius; Step 5, ferment for 3 to 5 days until mature, dry and grind into powder to obtain a microbial compound preparation.

[0006] The present invention provides an antibiotic-free, immune-premixed, concentrated feed as described above. Preferably, the preparation process of the *Gynostemma pentaphyllum* extract is as follows: *Gynostemma pentaphyllum* leaves are removed, the stems are crushed, and the mixture is soaked in boiling water. The extract is then obtained through filtration, concentration, and paste collection.

[0007] The antibiotic-free, immune-premixed, concentrated feed of the present invention, as described above, preferably includes the following process for preparing the extract of *Gynostemma pentaphyllum*: *Gynostemma pentaphyllum* is crushed by removing the leaves and leaving the stems, then soaked in boiling water. After filtration, concentration, and other processes, a primary extract of *Gynostemma pentaphyllum* is obtained. The primary extract of *Gynostemma pentaphyllum* is then fermented with *Lactobacillus plantarum* in an anaerobic environment to obtain a secondary extract of *Gynostemma pentaphyllum* after fermentation and transformation.

[0008] The present invention provides an antibiotic-free, immune-premixed, concentrated feed as described above. Preferably, the method for preparing concentrated mulberry protein powder is as follows: the mulberry protein plant is harvested before the stems form woody plants, the mulberry protein plant is crushed and dried, and then the dried mulberry protein plant is ground into powder to obtain concentrated mulberry protein powder.

[0009] In the antibiotic-free premixed concentrated feed of the present invention as described above, preferably, the molecular weight of the xylooligosaccharide is 300 Da to 2000 Da. More preferably, the molecular weight of the xylooligosaccharide is 700 Da to 1500 Da.

[0010] The antibiotic-free, immune-enhancing premixed concentrated feed of the present invention, as described above, preferably includes, in the method for preparing the compound microbial preparation, 2 parts by weight of cellulose-decomposing bacteria, 3 parts by weight of Bacillus, 1 part by weight of lactic acid bacteria, 2 parts by weight of Bifidobacterium, and 3 parts by weight of yeast.

[0011] The antibiotic-free, immunomodulatory premixed concentrated feed of the present invention, as described above, preferably includes the following steps in the method for preparing the compound microbial preparation: Step 1: At 35-40°C and with moderate aeration, the growth of cellulose-decomposing bacteria and Bacillus is preferentially promoted to decompose cellulose raw materials and generate heat and enzymes; Step 2: At 30-32°C with reduced or stopped aeration, the growth of lactic acid bacteria and Bifidobacteria is promoted to produce acid, inhibiting other bacteria, while yeast functions in a microaerobic environment. More preferably, Step 4 is further divided into the following steps: Step 1: At 38°C with moderate aeration, the growth of cellulose-decomposing bacteria and Bacillus is preferentially promoted to decompose cellulose raw materials and generate heat and enzymes; Step 2: At 31°C with reduced or stopped aeration, the growth of lactic acid bacteria and Bifidobacteria is promoted to produce acid, inhibiting other bacteria, while yeast functions in a microaerobic environment.

[0012] The beneficial effects of this invention are as follows: the core components of the premix (Gynostemma pentaphyllum extract, mulberry protein, xylooligosaccharides, and compound microbial preparations) are all natural plant-derived or microbial-derived substances, completely free of any antibiotics. Through the synergistic effect of multiple active ingredients, it is possible to avoid drug residues in animals, the development of drug-resistant strains, and public health risks transmitted to humans through the food chain caused by the addition of antibiotics to feed. Detailed Implementation

[0013] The embodiments described herein are specific implementations of the present invention, used to illustrate the concept of the invention, and are illustrative and exemplary, and should not be construed as limiting the implementation or scope of the invention. In addition to the embodiments described herein, those skilled in the art can employ other obvious technical solutions based on the content disclosed in the claims and specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein.

[0014] The preparation process of *Gynostemma pentaphyllum* extract 1 is as follows: 1. Fresh *Gynostemma pentaphyllum* medicinal materials were collected from the planting base in Tiantai County, Zhejiang Province. After identification, the leaves and fibrous roots were removed, and the main stem and stolons were retained. After rinsing with purified water, the herbs were drained and cut into small sections of about 1-2 cm. 2. 1000g of the treated *Gynostemma pentaphyllum* stem sections were weighed and placed in a multi-functional extraction tank. Eight times the volume (w / v, i.e., 8000 mL) of purified water was added, and the mixture was heated to boiling and refluxed for 2 hours at a gentle boil. After extraction, the mixture was filtered while hot through a 200-mesh double-layer filter cloth, and the filtrate was collected. The residue was then added to six times the volume of purified water, and the extraction was repeated for 1 hour, followed by filtration. The two filtrates were combined. 3. The combined filtrate was transferred to a vacuum concentration tank and concentrated under conditions of temperature ≤60°C and vacuum degree ~0.08 MPa until a thick paste was obtained, which is *Gynostemma pentaphyllum* water extract 1.

[0015] The production process of *Gynostemma pentaphyllum* extract 2 is as follows: 1. Preparation of primary extract: Prepare a water-extracted thick paste of *Gynostemma pentaphyllum* according to the method described in extract 1, and use it as the primary extract for later use. 2. Preparation of *Lactobacillus plantarum* fermentation broth: Inoculate *Lactobacillus plantarum* (CICC 20261) freeze-dried bacterial powder into MRS liquid medium and activate it under anaerobic (nitrogen-filled) conditions at 37°C for 18 hours to obtain activated seed liquid. 3. Fermentation conversion: Dilute the above primary extract with sterile purified water to a solid content of 10% (w / v) and dispense it into fermenters. Inoculate the seed liquid into the diluted extract at an inoculation rate of 5% (v / v) and stir evenly. Adjust the initial pH to 6.0, maintain the anaerobic environment, and ferment at a constant temperature of 37°C for 48 hours. 4. Post-treatment: After fermentation, heat the fermentation broth to 90°C and maintain it for 30 minutes to terminate fermentation and inactivate the bacterial cells. After cooling, centrifuge at 4°C and 8000 rpm for 15 minutes and collect the supernatant. Concentrate the supernatant again under reduced pressure at ≤60°C until it becomes a thick paste to obtain the fermented extract of Panax notoginseng 2.

[0016] Detection of flavonoid aglycones: Weigh 0.2g of the secondary extract of Panax notoginseng into a 25mL volumetric flask, add 10mL of 80% ethanol solution, shake well, and extract by sonication at 50℃ for 30min. Centrifuge at 3000r / min for 15min, take 1mL of the supernatant, dilute 5 times with 80% methanol, filter through a 0.45μm filter membrane, and then inject. Detection of flavonoid glycosides and flavonoid aglycones in the sample by high performance liquid chromatography: Column: SB-C18 column (4.6mm×250mm, 5μm); Column temperature: 35℃; Flow rate: 1.0mL / min; Injection volume: 20μL; UV detector wavelength: 260nm; Mobile phase: 0.2% glacial acetic acid + methanol; Gradient elution program: 0-5min, methanol from 20% to 25%; 5-25min, methanol from 25% to 80%; 25-30min, methanol from 80% to 20%, elution ends. Flavonoid aglycone conversion rate (%) = Flavonoid aglycone content / (Flavonoid glycoside content + Flavonoid aglycone content) × 100%. Test results: The flavonoid aglycone conversion rate in *Chrysanthemum indicum* aqueous extract 1 was 15%, and the conversion rate in *Chrysanthemum indicum* aqueous extract 2 was 63%. *Chrysanthemum indicum* aqueous extract 2 contained a higher content of flavonoid aglycones.

[0017] Method for making concentrated mulberry protein powder: 1. Raw material harvesting and pretreatment: Harvest mulberry protein in spring when the plant height reaches 80-100cm and the stem is not yet lignified. Harvest all the tender stems and leaves above ground. Rinse thoroughly with clean water to remove mud and impurities, then place in a ventilated area to drain surface moisture. 2. Crushing and drying: Use a crusher to cut the drained mulberry protein raw material into small pieces of about 1-2cm in length to increase the drying surface area. Spread the crushed mulberry protein evenly on a tray, with a thickness not exceeding 2cm, and place it in an electric heating forced-air drying oven. Dry at 65±5°C for 5 hours until the material feels crisp and the moisture content drops below 8%. 3. Grinding and pulverizing: Grind the dried mulberry protein using a universal pulverizer, then pass it through an 80-mesh sieve. Collect the sieve-passing material to obtain concentrated mulberry protein powder.

[0018] Preparation method of microbial compound preparation 1: The microbial compound preparation includes cellulolytic bacteria, Bacillus, lactic acid bacteria, Bifidobacterium, and yeast; the preparation method of the compound microbial preparation is as follows: Step 1, raw material preparation: 50 parts by weight of rice bran, 15 parts by weight of wheat bran, 10 parts by weight of corn, 10 parts by weight of oilseed cake, 2 parts by weight of molasses or brown sugar; 2 parts by weight of cellulolytic bacteria, 3 parts by weight of Bacillus, 1 part by weight of lactic acid bacteria, 2 parts by weight of Bifidobacterium, and 3 parts by weight of yeast; Step 2, dissolution of the inoculum: add water and mix well under stirring. Step 3: Dilute the inoculum with water to 150 times; Step 4: Mix the dissolved inoculum with rice bran or grass powder, wheat bran, corn, oilseed cake, molasses or brown sugar evenly; Step 5: Place the evenly mixed mixture in a container for fermentation. The fermentation process is divided into two stages: Stage 1: At 38℃ and with ventilation, the growth of cellulose-decomposing bacteria and Bacillus is preferentially promoted, decomposing fibrous raw materials and generating heat and enzymes; Stage 6: At 31℃ and with reduced or stopped ventilation, the growth of lactic acid bacteria and Bifidobacteria is promoted and acid is produced, while yeast plays a role in a microaerobic environment. Step 7: Fermentation matures in 3 to 5 days, then dry and grind to obtain the microbial compound preparation. Preparation method of microbial compound preparation 2: Step 4: Place the evenly mixed mixture in a container for fermentation at 35℃; the remaining conditions are the same as the preparation method of microbial compound preparation 1. The method for detecting the number of viable bacteria refers to the method specified in the national standard GB4789.34-2016 "National Food Safety Standard for Microbiological Examination of Food - Bifidobacterium Examination". The number of Bifidobacterium in microbial compound preparation 1 is 18*10. 9 CFU / ml, Bifidobacterium 13*10 in microbial compound preparation 2 9 CFU / ml. By modifying the original single fermentation to a two-stage fermentation, this process can optimize the proportion of microbial community structure and increase the number of viable bacteria (especially strictly anaerobic Bifidobacteria).

[0019] The preparation of antibiotic-free immunomodulatory premixed concentrated feed includes: 16-25 parts by weight of *Gynostemma pentaphyllum* extract, 60-80 parts by weight of concentrated mulberry protein powder, 1-3 parts by weight of xylooligosaccharides, and 6-12 parts by weight of a microbial compound preparation. The preparation method is as follows: Core premixing: According to the formula ratio, take all the xylooligosaccharides and a portion (approximately 10%-20%) of the concentrated mulberry protein powder as a "carrier" or "diluent" and put them into a small horizontal mixer. Mixing: Mix for 5-10 minutes to ensure thorough homogenization. This step involves small-scale premixing of the smaller but highly active components (xylooligosaccharides, microbial compound preparation) to prevent uneven distribution in the final product. Main mixing: a. Put all the remaining concentrated mulberry protein powder into a large twin-shaft paddle mixer. b. Then add the pretreated *Gynostemma pentaphyllum* extract powder. c. Finally, slowly add the "core premix" prepared in the previous step. Mixing parameters: Close the mixer inlet. Mix each batch for 5-8 minutes, and do not mix for too long. The entire mixing process is best carried out at room temperature and low humidity (ambient humidity <60%) to protect microbial activity. Prepare different antibiotic-free immune premixed concentrated feeds according to the raw material ratios in Table 1 for later use.

[0020] Table 1

[0021] Note: In the "Panax notoginseng extract" column of the table, the first number indicates either "Panax notoginseng extract 1" or "Panax notoginseng extract 2," and the second number is the weight parts of the extract. In the "microbial compound preparations" column of the table, the first number indicates either "microbial compound preparation 1" or "microbial compound preparation 2," and the second number is the weight parts of the compound preparation.

[0022] Experimental group feed formula: Corn: 60 parts by weight; Soybean meal: 18 parts by weight; Fish meal: 5 parts by weight; Rapeseed meal: 8 parts by weight; Antibiotic-free premixed concentrate feed: 5-8 parts by weight (according to Table 1). Control group feed formula: Corn: 60 parts by weight; Soybean meal: 30 parts by weight; Fish meal: 5 parts by weight; Rapeseed meal: 8 parts by weight; Antibiotic-free premixed concentrate feed: 5-8 parts by weight. The experimental animals were fed with these feeds.

[0023] 1. Experimental animals and grouping Animal selection: Select piglets of similar breed, parity, age (e.g., weaned piglets, 25-30 days old), weight, and good health, who are negative for swine fever antigen / antibody. Feeding and management: All pigs should be housed in the same independent isolation unit within the same farm, with completely identical environmental conditions (temperature, humidity, ventilation), using the same basal diet and feeding management procedures. Strict biosecurity measures should be implemented to prevent accidental infection.

[0024] 2. Experimental Procedure Adaptation period (7 days): All pigs enter the farm and are fed a basal diet to adapt to the environment. Treatment period (14 days): The experimental group begins to be fed a diet containing additives. Virus strain used: A standard virulent classical swine fever strain (such as virulent strain C) is used. Route and dosage of challenge: A certain dose of virus (such as 1000 ID50) is administered via intramuscular injection or intranasal inoculation.

[0025] 3. Data recording and indicator statistics Morbidity: The percentage of pigs exhibiting typical clinical symptoms of classical swine fever (high fever (>40.5℃), depression, anorexia, constipation followed by diarrhea, cyanosis of the skin, petechiae, neurological symptoms, etc.) out of the total number of pigs.

[0026] Calculation formula: Incidence rate (%) = (Number of infected animals in the group / Total number of animals in the group) × 100%.

[0027] Mortality rate: The percentage of pigs that died (including those near death and culled) between the time of viral challenge and the end of the observation period.

[0028] Calculation formula: Mortality rate (%) = (Number of deaths within the group / Total number of deaths in the group) × 100%.

[0029] Short-chain fatty acids, as important metabolites of gut microbiota, play a vital role in inhibiting the growth of pathogenic bacteria in the gut, maintaining the intestinal barrier, managing oxidative stress, inflammation, and enhancing immunity.

[0030] The experiment used GC-MS to determine the content of short-chain fatty acids (total acetic acid, propionic acid, and butyric acid) in digested extracts from animal intestines. The experiment was conducted in triplicate. Experimental method: 1.2 g of digested extract was centrifuged at 10,000 rpm for 10 min at 4℃. 500 μL of the supernatant was collected and mixed with an equal proportion of diethyl ether. The mixture was vortexed for 2 min to ensure sufficient reaction between the supernatant and the organic solvent. The vortexed mixture was then placed on a flotation plate and sonicated at 400 W for 3 min. After standing for 10 min, the mixture was filtered through a 0.22 μm inorganic filter membrane and ready for analysis. The content of short-chain fatty acids in different digested extracts was determined using gas chromatography-mass spectrometry (GC-MS).

[0031] GC-MS analysis conditions: Column: HPFF-AP; Injector temperature: 250℃; Detector temperature: 250℃; Temperature program: Initial temperature 80℃, increase to 140℃ at 7.5℃ / min and hold for 1 min, increase to 200℃ at 15℃ / min and hold for 3 min; Split ratio: 10:1; Injection volume: 1.0 μL.

[0032] Table 2

[0033] 1. Effectively replaces antibiotics, fundamentally solving the problems of drug residues and drug resistance. The core components of the premix of this invention (Gynostemma pentaphyllum extract, mulberry protein, xylooligosaccharides, and compound microbial preparations) are all natural plant-derived or microbial-derived substances and do not contain antibiotics. Through the synergistic effect of multiple active ingredients, it replaces the growth-promoting and disease-preventing effects of antibiotics, thereby completely avoiding the drug residues in animals, the emergence of drug-resistant strains, and the public health risks transmitted to humans through the food chain caused by the addition of antibiotics to feed.

[0034] 2. Significantly enhances animal immunity and effectively reduces morbidity and mortality after infection with specific viruses. As shown in the efficacy test in the specific implementation method, animals fed with the premix of this invention (experimental group) are expected to have significantly lower morbidity and mortality rates than the control group (CK group) after challenge with a virulent strain of classical swine fever. This is mainly due to the synergistic effect of multiple immune-enhancing components in the formula: *Panax notoginseng extract: rich in flavonoids (such as secondary extracts after fermentation, with a flavonoid aglycone conversion rate as high as 63%). Flavonoid aglycones have better biological activity and can effectively fight viruses, resist oxidation, and regulate the body's immune function. * Xylooligosaccharides and microbial complex preparation: together constitute a "prebiotic-probiotic" synbiotic system, which indirectly and persistently enhances the systemic immune response by optimizing the intestinal flora.

[0035] The technical features disclosed above are not limited to the combinations of the disclosed features with other features. Those skilled in the art can also make other combinations of the technical features according to the purpose of the invention to achieve the purpose of the invention.

Claims

1. An antibiotic-free premixed concentrated feed, characterized in that, include: The mixture comprises 16-25 parts by weight of Panax notoginseng extract, 60-80 parts by weight of concentrated mulberry protein powder, 1-3 parts by weight of xylooligosaccharides, and 6-12 parts by weight of a microbial compound preparation; the microbial compound preparation includes cellulose-decomposing bacteria, Bacillus, lactic acid bacteria, Bifidobacterium, and yeast; the preparation method of the compound microbial preparation is as follows: Step 1, raw material preparation: 30-60 parts by weight of rice bran or grass powder, 10-18 parts by weight of wheat bran, 10-18 parts by weight of corn, 10-18 parts by weight of oilseed cake, 1-3 parts by weight of molasses or brown sugar, and fiber... Step 1: Dissolve the microbial inoculum (1-3 parts by weight), Bacillus subtilis (1-3 parts by weight), Lactobacillus (1-3 parts by weight), Bifidobacterium (1-3 parts by weight), and yeast (1-3 parts by weight); Step 2: Dissolve the microbial inoculum by adding water while stirring and diluting it to 150 times; Step 3: Mix the dissolved microbial inoculum with rice bran or grass powder, wheat bran, corn, oilseed cake, molasses or brown sugar; Step 4: Place the well-mixed mixture in a container for fermentation at a temperature not lower than 20 degrees Celsius; Step 5: Ferment for 3 to 5 days until mature, then dry and grind into powder to obtain a microbial compound preparation.

2. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, The production process of the *Gynostemma pentaphyllum* extract is as follows: remove the leaves from *Gynostemma pentaphyllum*, keep the stems, crush them, soak them in boiling water, and then obtain the extract through filtration, concentration, and paste-forming processes.

3. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, The production process of the *Gynostemma pentaphyllum* extract is as follows: the leaves of *Gynostemma pentaphyllum* are removed, the stems are crushed and soaked in boiling water, and then processed through filtration, concentration and paste-making processes to obtain the primary extract of *Gynostemma pentaphyllum*; the primary extract of *Gynostemma pentaphyllum* is fermented with *Lactobacillus plantarum* in an anaerobic environment to obtain the secondary extract of *Gynostemma pentaphyllum* after fermentation and transformation.

4. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, Method for making concentrated mulberry protein powder: Harvest mulberry before the stems form woody plants, crush the mulberry and dry it, then grind the dried mulberry into powder to obtain concentrated mulberry protein powder.

5. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, The molecular weight of the xylooligosaccharide is 700 Da to 1500 Da.

6. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, In the method for preparing compound microbial preparations, there are 50 parts by weight of rice bran or grass powder, 15 parts by weight of wheat bran, 10 parts by weight of corn, 10 parts by weight of oilseed cake, 2 parts by weight of molasses or brown sugar, 2 parts by weight of cellulose-decomposing bacteria, 3 parts by weight of Bacillus, 1 part by weight of lactic acid bacteria, 2 parts by weight of Bifidobacterium, and 3 parts by weight of yeast.

7. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, In the method for preparing compound microbial preparations, the specific process of step 4 is as follows: Stage 1: At 35-40℃ and under ventilation, the growth of cellulose-decomposing bacteria and Bacillus is preferentially promoted to decompose cellulose raw materials and generate heat and enzymes; Stage 2: At 30-32℃ and with reduced or stopped ventilation, the growth of lactic acid bacteria and bifidobacteria is promoted to produce acid, while yeast plays a role in a micro-aerobic environment.

8. The antibiotic-free premixed concentrated feed according to claim 1, characterized in that, In the method for preparing compound microbial preparations, the specific process of step 4 is as follows: Stage 1: At 38°C and with moderate ventilation, the growth of cellulose-decomposing bacteria and Bacillus is preferentially promoted to decompose cellulose raw materials and generate heat and enzymes; Stage 2: At 31°C and with reduced or stopped ventilation, the growth of lactic acid bacteria and bifidobacteria is promoted to produce acid, while yeast plays a role in a micro-aerobic environment.

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