A preparation method of microecological bacterial enzyme preparation and its application in feed additives

Abstract

The present invention discloses a method for preparing a microecological bacterial enzyme preparation and its application in feed additives, and belongs to the field of preparing fermented feed by using composite microbial strains and biological enzymes. The bacterial liquid cultured with acid-resistant fermentation bacteria is centrifuged and mixed to obtain a mixed bacterial precipitate, and the mixed bacterial precipitate is freeze-dried to obtain a mixed bacterial powder; the mixed enzyme liquid formed by mixing papain, bromelain and cellulase is frozen to obtain a mixed enzyme powder; the mixed bacterial powder and the mixed enzyme powder are mixed to form a microecological bacterial enzyme preparation. The microecological bacterial enzyme preparation, basic feed and water are mixed and stirred, and then fermented to obtain a fermentation mixture; the fermentation mixture is adjusted with hydrochloric acid to adjust the pH, and then mixed and stirred with aldehyded chitosan to obtain a cross-linked fermentation product, and the cross-linked fermentation product is dried with hot air to obtain a solid fermented feed.

Description

Technical Field

[0001] The invention belongs to the field of preparing fermented feed by combining composite microbial strains with biological enzymes, and specifically relates to a preparation method of a microecological bacterial enzyme preparation and its application in feed additives. Background Art

[0002] With the rapid development of the breeding industry, the quality and utilization efficiency of feed are receiving more and more attention. Traditional feed often has problems such as unbalanced nutrition, low digestibility and high content of anti-nutritional factors, which affect the growth and health of animals. As a new type of feed, fermented feed has gradually been favored by breeders due to its excellent nutritional content and prebiotic effects. In fermented feed, compound bacterial agents are often used to prepare fermented feed. Compound bacterial agents refer to active bacterial communities composed of a variety of beneficial microorganisms (such as lactic acid bacteria, yeast, Bacillus, etc.), which have a synergistic effect, can enhance the fermentation effect, and improve the nutritional and digestible characteristics of feed. In feed fermentation, compound bacterial agents can not only increase the fermentation rate, but also improve the flavor and nutritional content of the feed, providing animals with a better digestion and absorption environment. The benefits of compound bacterial fermentation include: (1) Improving feed digestibility: The probiotics in the compound bacterial fermentation can promote the degradation of cellulose in the feed and improve the digestibility of the feed; (2) Improving nutritional content: During the fermentation process, microorganisms can synthesize a variety of nutrients, such as vitamins, amino acids, etc., to enrich the nutritional value of the feed; (3) Inhibiting the growth of harmful bacteria: Lactic acid bacteria and other probiotics in the compound bacterial fermentation can inhibit the growth of harmful pathogens through competitive exclusion and production of antibacterial substances, thereby reducing the incidence of animal diseases; (4) Improving animal intestinal health: Probiotics can regulate the intestinal microecology, promote the growth of beneficial bacteria, and enhance the immunity of animals; (5) Improving the palatability of feed: Fermented feed usually has a better smell and taste, which can increase the feed intake of animals.

[0003] Patent CN116875483A discloses a composite bacterial agent, fermented feed and preparation method thereof, and the invention is synergistic by adding Clostridium butyricum, Enterococcus faecium, Lactobacillus bulgaricus and Bacillus cereus, so that the fermented feed made by the composite bacterial agent mixed therein has excellent antibacterial and growth-promoting effects. Clostridium butyricum and Enterococcus faecium are used to limit the survival of pathogenic bacteria and suppress their reproduction, and antibacterial effects are played. On this basis, Bacillus cereus and Lactobacillus bulgaricus are added to further strengthen the antibacterial effect, stimulate the appetite of animals, improve feed utilization, and promote animal growth. In addition, cellulolytic bacteria and acidophilic bacteria are also added to the added composite bacterial agent, and cellulose and complex carbohydrates in the fermented feed are effectively decomposed by cellulolytic bacteria, and organic acids such as lactic acid, acetic acid, and propionic acid are produced by rapid fermentation with acidophilic bacteria, which effectively improves the quality and fermentation efficiency of the fermented feed.

[0004] Patent CN102499321A discloses a composite bacteria antibiotic-free fermented feed and a preparation method thereof. The Lactobacillus acidophilus and Bacillus subtilis in the composite bacteria of the invention can effectively inhibit the growth and reproduction of a large number of miscellaneous bacteria and harmful bacteria contained in the feeding system and feeding raw materials, ensure good fermentation of the feed, thereby improving the utilization rate of the feed and promoting the growth of animals.

[0005] Patent CN109234203A discloses a digestion-promoting composite bacterial agent for biological feed and its application. The invention mixes cellulosome-producing microbial agents, lactic acid bacteria agents and Bacillus subtilis agents to prepare a composite bacterial agent, and then ferments the basic feed to obtain a digestion-promoting enhanced biological feed.

[0006] Although the above-mentioned composite bacterial agent has a good promoting effect in the preparation of fermented feed, the feed obtained by the above fermentation cannot be dried at high temperature to prepare it into a solid feed form that is easy to store and transport due to the survival characteristics of active bacteria. Because high-temperature drying has a certain inactivation effect on active bacteria, the active bacteria in the feed cannot enter the animal body in a highly active form and be utilized.

[0007] Therefore, designing a microecological enzyme preparation so that the feed obtained after fermentation of the basic feed can withstand high-temperature drying and storage with less inactivation of active bacteria is of great significance for the storage and transportation of the feed. Summary of the invention

[0008] According to the deficiencies of the prior art, the present invention prepares acid-resistant fermentation bacteria, papain, bromelain and cellulase into solid powders and mixes them to form a microecological bacterial enzyme preparation, which is then mixed with a basic feed for fermentation to obtain a fermentation product, and the fermentation product is cross-linked with aldehyded chitosan after acid adjustment to obtain a cross-linked fermentation product with a high-temperature resistant dynamic imine bond structure, and the cross-linked fermentation product is then dried by hot air to obtain a solid fermented feed, thereby solving the technical problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents:

[0009] A method for preparing a microbial enzyme preparation, the method comprising the following steps:

[0010] The acid-resistant fermentation bacteria were cultured separately to a viable count of 1×10 7 CFU / mL~1×10 9 CFU / mL of bacterial liquid, then centrifugally separating and mixing the bacterial liquid to obtain a mixed bacterial precipitate, and freeze-drying the mixed bacterial precipitate at -50°C to -40°C for 15h to 20h to obtain a mixed bacterial powder;

[0011] The papain, bromelain and cellulase are mixed to form a mixed enzyme solution, and the mixed enzyme solution is freeze-dried at -50°C to -40°C for 15h to 20h to obtain a mixed enzyme powder;

[0012] The mixed bacterial powder and the mixed enzyme powder are mixed to form the microecological bacterial enzyme preparation.

[0013] Furthermore, the acid-resistant fermentation bacteria are Acetobacter pasteurianus, Lactobacillus paracasei, Streptococcus thermophilus and Saccharomyces cerevisiae.

[0014] Furthermore, the strain number of the Acetobacter pasteurianus is CICC 20761, the strain number of the Lactobacillus paracasei is CICC 20291, the strain number of the Streptococcus thermophilus is CICC 20367, and the strain number of the Saccharomyces cerevisiae is CICC 1405.

[0015] Furthermore, the centrifugal separation conditions include a centrifugal speed of 4000 r / min to 5000 r / min and a centrifugal time of 10 min to 15 min.

[0016] Furthermore, the mass ratio of papain:bromelain:cellulase is 1:1:1.

[0017] Furthermore, the mass ratio of the mixed bacterial powder to the mixed enzyme powder is 1:0.2-0.4.

[0018] A method for preparing a microecological bacterial enzyme preparation and application of the prepared microecological bacterial enzyme preparation in feed, the application comprising the following steps:

[0019] The microecological bacterial enzyme preparation, basic feed and water are mixed and stirred in a mass ratio of 0.005-0.02:1:5-10, and then fermented in a temperature environment of 30° C.-35° C. for 4-6 days to obtain a fermentation mixture;

[0020] The fermentation mixture is adjusted to pH 4.0-5.0 with hydrochloric acid, then mixed with aldehyded chitosan, stirred and heated to 30-35°C for reaction for 3-5 hours, and then naturally cooled to obtain a cross-linked fermentation product, and the cross-linked fermentation product is dried with hot air to obtain a solid fermented feed.

[0021] Furthermore, the basic feed includes soybean meal, peanut meal, wheat bran and rice straw.

[0022] Furthermore, the preparation of the aldehyde-modified chitosan is to dissolve chitosan in acetate buffer and oxidize it with sodium periodate.

[0023] Furthermore, the hot air drying conditions include a drying temperature of 70° C. to 90° C. and a drying time of 8 h to 12 h.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses acid-resistant fermentation bacteria with acid resistance as fermentation bacteria, and freeze-dries the bacteria to prepare mixed bacteria powder in solid form. Then, cellulase with the ability to break cell walls to release intracellular proteins and papain and bromelain with the ability to decompose proteins to form small molecule peptides or amino acids are mixed and freeze-dried to prepare mixed enzyme powder in solid form, and then the mixed bacteria powder and the mixed enzyme powder are mixed to form a microecological bacteria enzyme preparation, and then the microecological bacteria enzyme preparation is mixed with high-protein basic feed and water for fermentation to obtain a fermentation mixture containing amino acids and small molecule peptides, and then the fermentation mixture is adjusted to acidity, and then aldehyde-modified chitosan with aldehyde functional groups after oxidation is added to mix with the acidic fermentation mixture, and the amino functional groups on the amino acids and small molecule peptides can react with the aldehyde groups on the aldehyde-modified chitosan under certain temperature and acidic conditions to form a dynamic imine bond cross-linked network structure with high temperature resistance, so as to achieve the encapsulation of the fermentation bacteria, thereby playing a role in high temperature protection of the fermentation bacteria, and the dynamic imine bond structure can be hydrolyzed and broken when entering the stomach and intestines of animals, thereby releasing the fermentation bacteria, thereby improving the utilization efficiency of the fermentation bacteria in the fermented feed.

[0026] (2) The Lactobacillus paracasei and Streptococcus thermophilus used in the present invention are mainly used to produce lactic acid, which helps to improve the digestibility of feed. Yeast fermentation can produce fermentation products with high nutritional value such as amino acids, but it also produces more alcohol. Excessive accumulation of alcohol has a damaging effect on other fermentation bacteria. Therefore, Acetobacter pasteurianus, which can convert ethanol into acetic acid, is used to improve the acidity of feed and help inhibit the growth of harmful microorganisms. At the same time, acetic acid can promote digestion and absorption of animals. Through the synergistic mechanism of Acetobacter pasteurianus, Lactobacillus paracasei, Streptococcus thermophilus and Saccharomyces cerevisiae, the fermentation efficiency of the fermentation bacteria is improved. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below through 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 those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0029] Acetobacter pasteurianus with strain number CICC 20761, Lactobacillus paracasei with strain number CICC 20291, Streptococcus thermophilus with strain number CICC 20367, Saccharomyces cerevisiae with strain number CICC 1405, Bacillus licheniformis with strain number CICC10266 and Enterococcus faecalis with strain number CICC 10266 were all purchased from China Industrial Microbiological Culture Collection Center.

[0030] Papain, bromelain, and cellulase were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0031] Preparation Example 1:

[0032] Preparation of Acetobacter pasteurianus liquid:

[0033] Use a sterilized inoculation loop to take a strain of CICC 20761 Acetobacter pasteurianus and inoculate it into an acetic acid bacteria solid culture medium (8g glucose, 10g yeast powder, 5g calcium carbonate, 20g agar and deionized water to 1L, adjusted to pH 5.5) and culture for 24h. After the culture is completed, use an inoculation loop to take a loop from the surface of the acetic acid bacteria solid culture medium and inoculate it into an acetic acid bacteria liquid culture medium (10g glucose, 10g yeast powder and water to 1L, adjusted to pH 5.5) and culture it in a shaker at 30℃ until the number of viable bacteria in the bacterial solution is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0034] Preparation Example 2:

[0035] Preparation of Lactobacillus casei liquid:

[0036] Use a sterilized inoculation loop to take a Lactobacillus paracasei strain numbered CICC 20291 and inoculate it into MRS solid medium for 24 hours. After the culture is completed, use an inoculation loop to take a loop from the surface of the MRS solid medium and inoculate it into MRS liquid medium. Cultivate in a shaker at 35°C until the number of viable bacteria in the bacterial liquid is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0037] Preparation Example 3:

[0038] Preparation of thermophilic Streptococcus bacterial solution:

[0039] Use a sterilized inoculation loop to take a thermophilic Streptococcus strain numbered CICC 20367 and inoculate it into M17 solid medium for 24 hours. After the culture is completed, use an inoculation loop to take a loop from the surface of the M17 solid medium and inoculate it into M17 liquid medium and culture it in a shaker at 35°C until the number of viable bacteria in the bacterial liquid is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0040] Preparation Example 4:

[0041] Preparation of brewer's yeast culture liquid:

[0042] Use a sterilized inoculation loop to take a loop of Saccharomyces cerevisiae strain numbered CICC 1405 and inoculate it into YPD solid medium for 24 hours. After the culture is completed, use an inoculation loop to take a loop from the surface of the YPD solid medium and inoculate it into YPD liquid medium. Cultivate in a shaker at 28°C until the number of viable bacteria in the bacterial solution is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0043] Preparation Example 5:

[0044] Preparation of Bacillus licheniformis bacterial solution:

[0045] Use a sterilized inoculation loop to take Bacillus licheniformis with strain number CICC 10266 and inoculate it into LB solid medium for 24 hours. After the culture is completed, use an inoculation loop to take a loop from the surface of the LB solid medium and inoculate it into LB liquid medium. Cultivate in a shaker at 28°C until the number of viable bacteria in the bacterial liquid is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0046] Preparation Example 6:

[0047] Preparation of Enterococcus faecium culture solution:

[0048] Use a sterilized inoculation loop to take a piece of Enterococcus faecium with strain number CICC 10266 and inoculate it into MRS solid medium for 24 hours. After the culture is completed, use an inoculation loop to take a loop from the surface of the MRS solid medium and inoculate it into MRS liquid medium. Incubate in a shaker at 28°C until the number of viable bacteria in the bacterial liquid is measured by a bacterial counter at 1×10 7 CFU / mL~1×10 9 CFU / mL, ready for use.

[0049] Preparation Example 7:

[0050] Preparation of aldehyde-modified chitosan:

[0051] Chitosan was dissolved in an acetate buffer solution with a pH of 6.0 and stirred evenly, and then sodium periodate of 1 times the weight of chitosan was added for mixing and protected from light, and reacted in a water bath at 25°C for 1 hour. After the reaction, ethanol with a sodium periodate of 3 times the weight of sodium periodate was added to quench the sodium periodate to obtain a mixed solution, and then the mixed solution was dialyzed with a dialysis bag with a molecular weight cutoff of 500Da to obtain aldehyde-modified chitosan.

[0052] Preparation Example 8:

[0053] Preparation of aldehyde-modified gelatin:

[0054] Gelatin was dissolved in an acetate buffer solution with a pH of 6.0 and stirred evenly, and then sodium periodate with a weight of 1 times the weight of gelatin was added to mix and protect from light, and reacted in a water bath at 25°C for 1 hour. After the reaction, ethanol with a weight of 3 times the weight of sodium periodate was added to quench the sodium periodate to obtain a mixed solution, and then the mixed solution was dialyzed with a dialysis bag with a molecular weight cutoff of 500Da to obtain aldehyde-modified gelatin.

[0055] Embodiment 1:

[0056] A method for preparing a microecological bacterial enzyme preparation specifically comprises the following steps:

[0057] The Acetobacter pasteurianus liquid, Lactobacillus paracasei liquid, Streptococcus thermophilus liquid and Saccharomyces cerevisiae liquid obtained in Preparation Examples 1 to 4 were centrifuged in a centrifuge at a rotation speed of 4000 r / min to remove the culture medium to obtain bacterial precipitates, the bacterial precipitates were rinsed with sterile water three times and then mixed to form a mixed bacterial precipitate, the mixed bacterial precipitate was dispersed in glycerol, and then treated in a freeze dryer at -50°C for 15 h to obtain a solid block, which was processed into a mixed bacterial powder using a grinder.

[0058] Papain, bromelain and cellulase were mixed together in equal amounts to form a mixed enzyme solution, and then the mixed enzyme solution was mixed with glycerol and placed in a -50°C freeze dryer for 15 hours to obtain a solid block, which was then processed into mixed enzyme powder using a grinder.

[0059] The obtained mixed bacterial powder and mixed enzyme powder are mixed and dispersed in a mass ratio of 1:0.2 to form a microecological bacterial enzyme preparation.

[0060] Embodiment 2:

[0061] A method for preparing a microecological bacterial enzyme preparation specifically comprises the following steps:

[0062] The Acetobacter pasteurianus liquid, Lactobacillus paracasei liquid, Streptococcus thermophilus liquid and Saccharomyces cerevisiae liquid obtained in Preparation Examples 1 to 4 were centrifuged in a centrifuge at a rotation speed of 4500 r / min to remove the culture medium to obtain bacterial precipitates, the bacterial precipitates were rinsed three times with sterile water and then mixed to form a mixed bacterial precipitate, the mixed bacterial precipitate was dispersed in glycerol, and then treated in a freeze dryer at -45°C for 17 h to obtain a solid block, which was processed into a mixed bacterial powder using a grinder.

[0063] Papain, bromelain and cellulase were mixed together in equal amounts to form a mixed enzyme solution, and then the mixed enzyme solution was mixed with glycerol and placed in a -45°C freeze dryer for 17 hours to obtain a solid block, which was then processed into mixed enzyme powder using a grinder.

[0064] The obtained mixed bacterial powder and mixed enzyme powder are mixed and dispersed in a mass ratio of 1:0.3 to form a microecological bacterial enzyme preparation.

[0065] Embodiment 3:

[0066] A method for preparing a microecological bacterial enzyme preparation specifically comprises the following steps:

[0067] The Acetobacter pasteurianus liquid, Lactobacillus paracasei liquid, Streptococcus thermophilus liquid and Saccharomyces cerevisiae liquid obtained in Preparation Examples 1 to 4 were centrifuged in a centrifuge at a rotation speed of 5000 r / min to remove the culture medium to obtain bacterial precipitates, the bacterial precipitates were rinsed with sterile water three times and then mixed to form a mixed bacterial precipitate, the mixed bacterial precipitate was dispersed in glycerol, and then treated in a freeze dryer at -40°C for 20 h to obtain a solid block, which was processed into a mixed bacterial powder using a grinder.

[0068] Papain, bromelain and cellulase were mixed together in equal amounts to form a mixed enzyme solution, and then the mixed enzyme solution was mixed with glycerol and placed in a -50°C freeze dryer for 20 hours to obtain a solid block, which was then processed into mixed enzyme powder using a grinder.

[0069] The obtained mixed bacterial powder and mixed enzyme powder are mixed and dispersed in a mass ratio of 1:0.4 to form a microecological bacterial enzyme preparation.

[0070] Embodiment 4:

[0071] A method for preparing a microecological bacterial enzyme preparation specifically comprises the following steps:

[0072] The Acetobacter pasteurianus solution and the Lactobacillus paracasei solution in Example 3 were replaced by the Bacillus licheniformis solution in Preparation Example 5 and the Enterococcus faecium solution in Preparation Example 6, respectively, and the other conditions remained the same as in Example 3.

[0073] Embodiment 5:

[0074] A method for preparing a microecological bacterial enzyme preparation specifically comprises the following steps:

[0075] The Acetobacter pasteurianus liquid in Example 3 was removed and not added, and the other conditions remained the same as in Example 3.

[0076] Application Example 1:

[0077] Preparation of solid fermented feed:

[0078] The microecological enzyme preparation obtained in Example 1, the basic feed (mixed with soybean meal, peanut meal, wheat bran and straw in the same mass) and water were mixed and stirred evenly in a mass ratio of 0.005:1:5, and then fermented in an environment of 30°C for 4 days (the oxygen content was controlled at 5%). After the fermentation, the pH of the fermentation mixture was adjusted to 4.0 with hydrochloric acid, and then the aldehyde-modified chitosan obtained in Preparation Example 7 was added to the fermentation mixture at this pH value for mixing and stirring, and then heated to 30°C for timed reaction for 3 hours. After the reaction was completed, it was naturally cooled to room temperature to obtain a cross-linked fermentation product, and the cross-linked fermentation product was placed in a hot air dryer at 70°C and vacuum dried for 8 hours to obtain a solid fermented feed.

[0079] Application Example 2:

[0080] Preparation of solid fermented feed:

[0081] The microecological enzyme preparation obtained in Example 2, the basic feed (mixed with soybean meal, peanut meal, wheat bran and straw in the same mass) and water were mixed and stirred evenly in a mass ratio of 0.01:1:7, and then fermented in an environment of 32°C for 5 days (the oxygen content was controlled at 5%). After the fermentation, the pH of the fermentation mixture was adjusted to 4.5 with hydrochloric acid, and then the aldehyde-modified chitosan obtained in Preparation Example 7 was added to the fermentation mixture at this pH value for mixing and stirring, and then heated to 32°C for a timed reaction of 4 hours. After the reaction was completed, it was naturally cooled to room temperature to obtain a cross-linked fermentation product, and the cross-linked fermentation product was placed in a hot air dryer at 80°C and vacuum dried for 10 hours to obtain a solid fermented feed.

[0082] Application Example 3:

[0083] Preparation of solid fermented feed:

[0084] The microecological enzyme preparation obtained in Example 3, the basic feed (mixed with soybean meal, peanut meal, wheat bran and straw in the same mass) and water were mixed and stirred evenly in a mass ratio of 0.02:1:10, and then fermented in an environment of 35°C for 6 days (the oxygen content was controlled at 5%). After the fermentation, the pH of the fermentation mixture was adjusted to 5.0 with hydrochloric acid, and then the aldehyde-modified chitosan obtained in Preparation Example 7 was added to the fermentation mixture at this pH value for mixing and stirring, and then heated to 35°C for timed reaction for 5 hours. After the reaction was completed, it was naturally cooled to room temperature to obtain a cross-linked fermentation product, and the cross-linked fermentation product was placed in a hot air dryer at 90°C and vacuum dried for 12 hours to obtain a solid fermented feed.

[0085] Application example 4:

[0086] Preparation of solid fermented feed:

[0087] The microecological bacterial enzyme preparation in Application Example 3 was replaced by the microecological bacterial enzyme preparation obtained in Example 4, and the other conditions remained the same as those in Application Example 3.

[0088] Application Example 5:

[0089] The microecological bacterial enzyme preparation in Application Example 3 was replaced by the microecological bacterial enzyme preparation obtained in Example 5, and the other conditions remained the same as those in Application Example 3.

[0090] Application Example 6:

[0091] Preparation of solid fermented feed:

[0092] The aldehyde-modified chitosan in Application Example 3 was removed and not added, and the other conditions remained the same as those in Application Example 3.

[0093] Application Example 7:

[0094] Preparation of solid fermented feed:

[0095] The aldehyde-modified chitosan in Application Example 3 was replaced by the aldehyde-modified gelatin obtained in Preparation Example 8, and the other conditions were kept the same as those in Application Example 3.

[0096] The solid fermented feed obtained in Examples 1 to 7 was tested for total viable bacteria count, and then placed at room temperature (25°C ± 2°C) for one month and the growth of mold on the surface was observed. The results are as follows:

[0097]

[0098] From the above results, we can know that:

[0099] (1) It can be seen from Application Examples 1 to 3 that the cross-linked fermentation product formed by the reaction of the fermentation mixture obtained by the microecological bacterial enzyme preparation constructed by the present invention after fermenting feed with aldehyde-modified chitosan has a good promoting effect on the preparation of solid-state fermented feed that is easy to store and transport.

[0100] (2) It can be seen from Application Example 4 that since Bacillus licheniformis and Enterococcus faecium are suitable for survival in a neutral environment, their growth is greatly inhibited in acidic conditions, resulting in a low number of live bacteria and the production of some mold after storage for a period of time.

[0101] (3) It can be seen from Application Example 5 that due to the lack of Acetobacter Pasteurellii that consumes alcohol, the alcohol production of brewer's yeast is relatively high, while the other bacterial species are unable to utilize and consume alcohol, resulting in excessive accumulation of alcohol, causing the remaining bacteria to be damaged by alcohol, resulting in a low number of live bacteria, and some mold was produced after storage for a period of time.

[0102] (4) It can be seen from Application Example 6 that due to the lack of dynamic imine bond cross-linking structure of formaldehyded chitosan, a large number of live bacteria are lost during high-temperature hot air drying, and a large amount of mold is generated after storage for a period of time, which is not conducive to the preparation of solid fermented feed that is easy to store and transport.

[0103] (5) It can be seen from Application Example 7 that although formaldehyde-modified gelatin aldehyde can also form a cross-linked structure with dynamic imine bonds, since gelatin does not have antibacterial properties, relying solely on the acidic substances produced by live bacteria to inhibit the growth of harmful microorganisms cannot meet storage requirements.

[0104] The solid fermented feed obtained in Application Examples 1 to 7 was fed to beef cattle weighing 400±25kg at a feeding rate of 1.0kg per day. The weight of the beef cattle was weighed after the 90th day. The results are as follows:

[0105]

[0106] It can be seen from the above results that the solid-state fermented feed obtained by applying the microecological bacterial enzyme preparation of the present invention to feed fermentation has the effect of promoting the growth of beef cattle.

[0107] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a microecological bacterial enzyme preparation, characterized in that: The preparation method comprises the following steps: The acid-resistant fermentation bacteria were cultured separately to a viable count of 1×10 7 CFU / mL~1×10 9 CFU / mL of bacterial liquid, then centrifugally separating and mixing the bacterial liquid to obtain a mixed bacterial precipitate, and freeze-drying the mixed bacterial precipitate at -50°C to -40°C for 15h to 20h to obtain a mixed bacterial powder; The papain, bromelain and cellulase are mixed to form a mixed enzyme solution, and the mixed enzyme solution is freeze-dried at -50°C to -40°C for 15h to 20h to obtain a mixed enzyme powder; The mixed bacterial powder and the mixed enzyme powder are mixed to form the microecological bacterial enzyme preparation; The acid-resistant fermentation bacteria are Acetobacter pasteurianus, Lactobacillus paracasei, Streptococcus thermophilus and Saccharomyces cerevisiae; The strain number of the Acetobacter pasteurianus is CICC 20761, the strain number of the Lactobacillus paracasei is CICC 20291, the strain number of the Streptococcus thermophilus is CICC 20367, and the strain number of the Saccharomyces cerevisiae is CICC 1405; The mass ratio of papain:bromelain:cellulase is 1:1:1; The mass ratio of the mixed bacterial powder to the mixed enzyme powder is 1:0.2-0.

4.

2. The method for preparing a microecological bacterial enzyme preparation according to claim 1, characterized in that: The centrifugal separation conditions include a centrifugal speed of 4000 r / min to 5000 r / min and a centrifugal time of 10 min to 15 min.

3. An application of a microecological bacterial enzyme preparation prepared by the preparation method of any one of claims 1 to 2 in feed, characterized in that: The application comprises the following steps: The microecological bacterial enzyme preparation, basic feed and water are mixed and stirred in a mass percentage of 0.5% to 2%: 1: 5 to 10, and then fermented in a temperature environment of 30° C. to 35° C. for 4 to 6 days to obtain a fermentation mixture; The fermentation mixture is adjusted to pH 4.0-5.0 with hydrochloric acid, then mixed with aldehyded chitosan, stirred and heated to 30-35°C for reaction for 3-5 hours, and then naturally cooled to obtain a cross-linked fermentation product, and the cross-linked fermentation product is dried with hot air to obtain a solid fermented feed.

4. The application of the microecological bacterial enzyme preparation prepared by the preparation method of a microecological bacterial enzyme preparation according to claim 3 in feed is characterized in that, The basic feed comprises soybean meal, peanut meal, wheat bran and rice straw.

5. The application of the microecological bacterial enzyme preparation prepared by the preparation method of a microecological bacterial enzyme preparation according to claim 3 in feed is characterized in that, The preparation of the aldehyde-modified chitosan is that chitosan is dissolved in an acetate buffer and oxidized with sodium periodate.

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