CN114451486A - Fungus enzyme compound feed additive and preparation method and application thereof - Google Patents
Fungus enzyme compound feed additive and preparation method and application thereof Download PDFInfo
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- CN114451486A CN114451486A CN202210073769.0A CN202210073769A CN114451486A CN 114451486 A CN114451486 A CN 114451486A CN 202210073769 A CN202210073769 A CN 202210073769A CN 114451486 A CN114451486 A CN 114451486A
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- feed additive
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- enzyme compound
- bacterial
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/28—Silicates, e.g. perlites, zeolites or bentonites
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Birds (AREA)
- Microbiology (AREA)
- Inorganic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention provides a bacterial enzyme compound feed additive and a preparation method and application thereof, wherein the bacterial enzyme compound feed additive comprises the following components in percentage by weight: 3% -20% of glucose oxidase; coating 11% -25% of lactobacillus; 15% -35% of bacteriostatic bacillus subtilis; 1.0 to 6.0 percent of clostridium butyricum; 15 to 30 percent of bentonite and 10 to 20 percent of talcum powder. The bacterial enzyme compound feed additive has less dosage, for example, 200g/t of the bacterial enzyme compound feed additive can play a significant role when being added into a weaned pig compound feed, and the bacterial enzyme compound feed additive is specifically represented as follows: the daily gain of the weaned piglets is increased by 10 percent, the feed meat is 0.14 percent lower than that of a control group, and the diarrhea rate is reduced by 1/2; for example, 100g/t of compound feed for 50-week-old laying hens can slowly reduce the laying rate, and reduce the egg breaking rate and the death and culling rate by half.
Description
Technical Field
The invention relates to a composite functional feed additive, in particular to a bacterial enzyme composite feed additive taking various feeding live bacteria as main materials and a decomposition type enzyme preparation as an auxiliary material, and a preparation method and application thereof.
Background
Feed production enterprises stop producing commercial feeds containing growth-promoting drug feed additives from 7/1/7/2020, and the animal husbandry industry formally pulls open a 'feed nonreactive' curtain. According to research and analysis in the industry in recent years, the breeding industry faces huge challenges after resistance prohibition: the diarrhea complaints are increased, the production performance is reduced (the daily gain is reduced and the death and culling rate is increased), the feed cost is generally increased, the use amount of antibiotics for treatment in a culture site is increased, and the cost is increased. The pathogenic bacteria causing the poultry diarrhea are mainly clostridium perfringens (accounting for 54 percent), secondly pathogenic escherichia coli (accounting for 32 percent) and salmonella (accounting for 12 percent) through researching livestock and poultry intestinal pathogenic microorganisms, and staphylococcus aureus only accounts for 2 percent; the pathogenic bacteria causing the diarrhea of the swinery are mainly pathogenic escherichia coli (accounting for 83 percent), and secondly clostridium perfringens (accounting for 13 percent), and salmonella and staphylococcus aureus account for only 2 percent respectively; therefore, practitioners propose a core idea of 'micro-treatment', namely adding beneficial microorganisms into feed, reestablishing intestinal dominant flora, and inhibiting pathogenic microorganisms by utilizing trace metabolites (such as organic acids, bacteriocins, antibacterial peptides and the like) generated by rapid proliferation of the beneficial microorganisms, so that the balance of intestinal flora, the complete structure of intestinal mucosa and the complete digestion and absorption functions are finally realized, and the growth of cultured animals is promoted.
Single viable bacteria or compound viable bacteria preparations and compound additives with combined compatibility of viable bacteria, enzyme preparations, antibacterial peptides and Chinese herbal medicines exist in the market, but the products in the market have the problems of unstable use effect (weak bacteriostatic ability and high diarrhea rate), large addition amount, high use cost and the like, and the reasons for the problems are that the bacteriostatic effect of the used strains is undefined, the number and the types of viable bacteria entering the intestinal tracts of animals are small (the strains cannot tolerate the high-temperature high-humidity granulation of the feed and the gastric acid and the bile salt), and the compounding of the bacterial enzyme and other additives is not scientific, therefore, the coating lactobacillus, the bacteriostatic bacillus subtilis and the clostridium butyricum with definite bacteriostatic action are mainly selected, the series of problems can be well solved by taking the glucose oxidase which is a 'decomposition type enzyme preparation' for generating a large amount of antibacterial substances through oxidative decomposition as the auxiliary scientific proportion and exerting the synergistic effect of the bacterial enzymes.
Disclosure of Invention
In view of the above, the invention aims to provide a bacterium-enzyme composite feed additive, and a preparation method and an application thereof, wherein the bacterium-enzyme composite feed additive has high safety, no toxic or side effect, low addition amount and low use cost, has a remarkable inhibition effect on clostridium perfringens, pathogenic escherichia coli, salmonella and staphylococcus aureus, can be applied to livestock and poultry antibiotic-free breeding, and can remarkably reduce the diarrhea incidence and death rate of piglets, chicks, broilers and laying hens, promote the rapid growth of animals and improve the production performance.
In order to achieve the aim, the invention provides a bacterial enzyme compound feed additive which comprises the following components in percentage by weight: 3% -20% of glucose oxidase; coating 11% -25% of lactobacillus; 15% -35% of bacteriostatic bacillus subtilis; 1.0 to 6.0 percent of clostridium butyricum; 15 to 30 percent of bentonite and 10 to 20 percent of talcum powder.
Further, the coating lactic acid bacteria are granular lactic acid bacteria, wherein the granular lactic acid bacteria are prepared by screening and separating a strain Enterococcus faecium (Enterococcus faecium) HEW-A588 from rectal contents of healthy piglets, fermenting the strain in liquid to obtain active bacterial sludge, and then sequentially carrying out micro-capsule coating, granulation coating and enteric coating, and the granularity of the granular lactic acid bacteria is 30 meshes. The coated lactobacillus strain has clear source and strong stress resistance (high temperature resistance, high humidity resistance and granulation resistance) and strong tolerance (gastric acid resistance and cholate resistance) when entering all levels of digestive tracts of animals, thereby ensuring that enterococcus faecium with clear activity and enough quantity is fixedly planted and distributed in the whole intestinal tracts of the animals, quickly forming intestinal dominant flora and ensuring the health of the intestinal tracts of the animals. The Enterococcus faecium (Enterococcus faecium) HEW-A588 is preserved in China general microbiological culture Collection center (CGMCC for short), the address is No. 3 of West Luo No.1 of the North Chen West district, the China academy of sciences, the postal code is 100101, the preservation number is CGMCC No.10547, the preservation date is 2015, 2 months and 9 days, and the Enterococcus faecium (Enterococcus faecium) is classified and named.
Further, the bacterial enzyme compound feed additive comprises 3 beneficial bacteria with definite antibacterial performance, and the viable count of the beneficial bacteria is as follows: coating lactobacillus (enterococcus faecium) not less than 2.0 × 109CFU/g, bacterial inhibition type bacillus subtilis not less than 1.0×1010CFU/g, Clostridium butyricum not less than 1.0 x 108CFU/g; the glucose oxidase is solid powder with excellent antibacterial performance, and the activity of the glucose oxidase in the bacterial enzyme composite additive is not lower than 750U/g. For example, only 5% of the bacterial enzyme compound additive needs to be added to obtain 750U of activity.
In order to achieve the above purpose, the invention also provides a preparation method of the bacterial enzyme compound feed additive, which comprises the following steps:
respectively carrying out primary premixing on the glucose oxidase and the clostridium butyricum in the formula amount and one half of the bentonite in the formula amount to obtain a primary premix;
and then the primary premix is mixed with the coating lactic acid bacteria, the bacteriostatic bacillus subtilis and the talcum powder in formula amount and the bentonite in the rest half formula amount for the second level to obtain the bacterial enzyme compound feed additive.
Further, the rotation speed of the primary premixing is 20-40 rpm, and the mixing time is 200-300 seconds; the preferred speed of rotation is 30 rpm and the mixing time is 240 seconds.
Further, the rotation speed of the secondary mixing is 40-60 r/min, and the mixing time is 120-180 seconds; the preferred speed of rotation is 50 rpm and the mixing time is 150 seconds.
In order to achieve the purpose, the invention also provides application of the bacterial enzyme compound feed additive in livestock antibiotic-free feed.
Further wherein the applying comprises the steps of: the bacterial enzyme compound feed additive is directly added into the livestock feed.
Furthermore, the addition proportion of the livestock feed is 0.15-0.3 per mill, and the addition proportion of the poultry feed is 0.1-0.2 per mill. For example, each ton of pig feed is added with 300g of 150-.
The bacterial enzyme compound feed additive provided by the invention comprises the following components in part by weight:
the coated lactic acid bacteria are microcapsule particles prepared by carrying out liquid fermentation on enterococcus faecium in lactic acid bacteria with good probiotic effect and then adopting a stabilizing coating process, have the characteristics of storage resistance, feed processing resistance and gastric juice and intestinal juice resistance, can be rapidly disintegrated after reaching intestinal tracts, release a large amount of live enterococcus faecium to rapidly occupy intestinal tract mucous membrane sites, rapidly proliferate into intestinal tract dominant flora and protect the health of the intestinal tracts. Enterococcus faecium belongs to facultative anaerobe, can adapt to aerobic and anaerobic environments, plays a role in the whole intestinal tract through mechanisms such as adhesion, sterilization, repair, anti-inflammation and the like, is a normal dominant flora in the intestinal tract of human and animals, and has strong intestinal adhesion and colonization capacity. On one hand, enterococcus faecium constructs the dominant flora of the intestinal probiotics by means of competing host intestinal mucosa structural sites and competing nutrients required by microorganisms, so that pathogenic bacteria adhesion and infection are excluded, and flora balance and intestinal health are maintained; on the other hand, enterococcus faecium can generate organic acids such as lactic acid and acetic acid, reduce the pH value of intestinal tracts, enhance the activity of digestive enzymes, promote the digestion and absorption of nutrient substances, generate antibacterial substances such as bacteriocin and antibacterial peptide, inhibit the reproduction and intestinal infection of intestinal putrefying bacteria and pathogenic bacteria (such as clostridium perfringens, staphylococcus aureus, escherichia coli, salmonella and the like), reduce the content of ammonia and endotoxin in blood, reduce the diarrhea rate, maintain the structural functional integrity of the intestinal tracts, promote the health of animals, and is one of the probiotic strains which are preferably used in animal husbandry production. The stabilized coated lactic acid bacteria are added to supplement the deficiency of endogenous lactic acid bacteria in animal intestinal tracts, help the endogenous lactic acid bacteria to proliferate into intestinal dominant flora, maintain the balance of intestinal flora, have no promotion effect when being added too little, and inhibit the activity of the endogenous lactic acid bacteria when being added excessively.
The bacteriostatic bacillus subtilis is an aerobic bacterial strain, can form high-temperature-resistant, acid-base-resistant and high-osmotic-pressure-resistant dormant spores, quickly adheres and occupies the front section of an animal intestinal tract, competitively excludes pathogenic bacteria for field planting, quickly proliferates by capturing oxygen, forms a local anaerobic environment, and inhibits the growth of aerobic bacteria; meanwhile, active substances such as subtilin, polymyxin, gramicidin and the like generated in the growth process of the antibacterial bacillus subtilis have obvious inhibition effect on pathogenic bacteria or conditional pathogenic bacteria of endogenous infection, can also generate important digestive enzymes such as bacteriocin, amylase, protease and the like, promotes the digestion and absorption of intestinal chyme, enhances the immunity of animals, and is a spore strain commonly used for livestock breeding. The bacteriostatic bacillus subtilis is added to strengthen the effect of inhibiting harmful bacteria proliferation, synergistically promote lactobacillus to become intestinal dominant flora and regulate the balance of intestinal flora, cannot strengthen the bacteriostatic action if the addition amount is too small, and can cause overlarge local anaerobic environment of the intestinal tract and inhibit the activity and proliferation of endogenous lactobacillus if the addition amount is excessive.
The clostridium butyricum is strictly anaerobic clostridium, has certain high temperature resistance and animal gastrointestinal tract environment resistance, and mainly plays a role in the posterior segment of the intestinal tract. The clostridium butyricum can maintain or recover dominant flora of host intestinal tract in vivo as probiotics, inhibit the growth of common intestinal pathogenic bacteria such as escherichia coli, salmonella, clostridium perfringens and the like by secreting short-chain fatty acid, antibacterial peptide, clostridium butyricum and the like, can enhance the activity of antioxidase by means of metabolic production of butyrate, hydrogen and the like, reduce the oxidative damage of intestinal tract epithelial tissues, can rapidly provide energy for colon epithelial cell metabolic utilization, promote the growth of intestinal villi on intestinal mucosa, rapidly repair mucosal damage caused by diarrhea, promote the development of intestinal tract and improve the digestive absorption capacity of animal intestinal tract. A large number of research results show that the clostridium butyricum can regulate the intestinal microecological balance, inhibit the growth of harmful bacteria and promote the proliferation of beneficial bacteria, and the feed digestibility is increased and the growth performance of animals is obviously improved after the clostridium butyricum is fed. The clostridium butyricum is added in the livestock production, because the butyrate generated by the metabolism of the clostridium butyricum in vivo has the functions of repairing mucosal injury and promoting intestinal development, the addition of too little clostridium butyricum has the function of not repairing mucosal injury, and the addition of excessive clostridium butyricum can generate too much butyrate to cause animal intestinal dysfunction and other anaphylactic reactions.
The glucose oxidase is a third-generation decomposition type enzyme preparation, is an aerobic dehydrogenase, is a biological reaction catalyst, can specifically oxidize and decompose beta-D-glucose into gluconic acid and hydrogen peroxide, the gluconic acid is an organic acid (the pH value is 3-6) with stronger acidity, can reduce the pH value of the animal gastrointestinal tract environment, simultaneously provides better reproduction and multiplication conditions for beneficial bacteria acidophilic in the digestive tract, simultaneously consumes a large amount of oxygen in the intestinal tract, prevents the growth and the reproduction of the aerobic bacteria, and is beneficial to the reproduction of anaerobic probiotics represented by bifidobacterium; the hydrogen peroxide generated by the metabolism of the glucose oxidase has a broad-spectrum bactericidal effect, and the gluconic acid has the property similar to prebiotics, is rarely absorbed in small intestine, can be utilized by inhabitant flora to generate butyric acid when reaching the rear section of the intestinal tract (mainly large intestine), provides energy for the epithelial cells of the large intestine, and has good effects of stimulating the growth of the epithelial cells of the intestine and promoting the reabsorption efficiency of sodium and water; the glucose oxidase has an anti-oxidation effect, can remove free radicals and protect the integrity of intestinal epithelial cells; therefore, glucose oxidase is easy to show its effect under poor cultivation conditions (especially under the condition that a large amount of pathogenic microorganisms exist), and is widely popularized and used in livestock cultivation in recent years. The glucose oxidase is added in the invention, because the product generated after the glucose oxidase is decomposed has the effects of reducing the pH value of the intestinal tract and inhibiting the proliferation of harmful bacteria, the product cannot play a role of strengthening bacteriostasis if the product is added too little, and the product can greatly reduce the acid environment of the gastrointestinal tract and cause the adverse effects of reduced food consumption of animals, accelerated intestinal tract peristalsis, digestive tract mucosa shedding and the like if the product is added too much.
Bentonite is clay which takes montmorillonite as a main component, has good water absorption and fluidity, is commonly used as a carrier of a feed additive, and plays roles of diluting effective components, adsorbing harmful substances, absorbing moisture, lubricating and the like; in addition, the bentonite contains macroelements and microelements (such as magnesium, calcium, ferrum, phosphorus, potassium, sodium, chromium, manganese and the like) which are necessary for the growth and development of animals, and can supplement the mineral requirements of livestock and poultry and promote the growth and development of animals. According to the invention, the bentonite is used as a carrier (also called as a diluent), and due to the large volume weight (the weight of a unit volume is large), the addition of too little bentonite can not effectively dilute and disperse the viable bacteria component and the glucose oxidase, and the addition of too much bentonite can excessively dilute the viable bacteria component and the glucose oxidase, so that the growth effect of the microbial enzyme compound feed on livestock and poultry is weakened.
Talcum powder is an aluminosilicate mineral, contains more than 20 major and trace elements such as calcium, phosphorus, potassium, sodium, magnesium, zinc, copper, manganese, etc., and can promote animal metabolism, improve disease resistance of organism, and promote animal growth while being used as a feed carrier. The addition proportion of the talcum powder in the livestock and poultry compound feed is generally not more than 5 wt%, and excessive addition can cause the inner wall of the stomach of an animal to be corroded, and the feed intake to be gradually reduced.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the bacterium enzyme combined compound compatibility which takes the viable bacteria as the main part and the glucose oxidase as the auxiliary part, has scientific and reasonable proportion and can powerfully play the synergistic effect of the bacterium enzyme. The coating lactic acid bacteria can endure adverse environments such as high-temperature granulation, gastric acid, cholate and the like after being subjected to multi-layer microencapsulation coating treatment, and the selected coating material can be rapidly disintegrated and released in the intestinal tract to occupy the structural site of the intestinal mucosa, so that the dominant intestinal flora can be rapidly established and the balance of the intestinal flora can be adjusted in the whole intestinal tract after field planting and distribution; in addition, the bacteriostatic bacillus subtilis selected by the invention has definite bacteriostatic action, so that harmful bacteria in the front section of the intestinal tract (mainly small intestine) are less adhered, the intestinal mucosa is less damaged, the intestinal barrier is healthy, the obvious bacteriostatic and antibacterial action is realized, the digestion and absorption of nutrient substances are promoted, and the feed utilization rate is improved; after entering the body, clostridium butyricum proliferates in a large amount in the rear section of the intestinal tract (mainly large intestine and caecum), short-chain fatty acids such as butyric acid and the like are generated, energy is rapidly supplied, damaged intestinal mucosa is repaired, the generation of endogenous defense peptides is promoted, the capability of animals for defending invasion and damage of pathogenic bacteria is enhanced, and the effects of promoting the growth of intestinal villi and reducing the diarrhea of the animals are achieved; on one hand, glucose oxidase can reduce the acid environment in the gastrointestinal tract by oxidizing glucose, so that the health of the intestinal tract is promoted, meanwhile, an anaerobic environment created by consuming a large amount of oxygen has the effects of inhibiting and even killing aerobic bacteria (most pathogenic bacteria grow under the aerobic condition) and facultative bacteria, and on the other hand, metabolites (the gluconic acid and hydrogen peroxide) generated by the oxidative decomposition of the glucose have the broad-spectrum bactericidal effect, so that the quantity of pathogenic microorganisms in the intestinal tract is reduced, and thus beneficial bacteria form the microecological competitive advantage, and the glucose oxidase has better effects on preventing and treating the livestock and poultry physiological diarrhea and intractable diarrhea. Therefore, the bacterial enzyme compound compatibility scheme taking the live bacteria as the main part and the glucose oxidase as the auxiliary part has strong combination and synergistic interaction of the live bacteria and the enzyme, and can greatly improve the effects of preventing diarrhea, promoting growth and improving the production performance of the compound feed additive in nonreactive culture.
The 3 kinds of viable bacteria and glucose oxidase selected by the invention have exact bacteriostatic action on common pathogenic bacteria in intestinal tracts, for example, the bacteriostatic action of the coated lactic acid bacteria on coliform and clostridium perfringens is respectively medium-sensitive and high-sensitive (the diameters of bacteriostatic rings are respectively 18mm and 23mm), the bacteriostatic action of the bacteriostatic bacillus subtilis and the glucose oxidase on clostridium perfringens can achieve high-sensitive effect (the diameters of bacteriostatic rings are respectively 24mm and 25mm), the bacteriostatic action of clostridium butyricum on salmonella, staphylococcus aureus and escherichia coli can achieve medium-sensitive (the diameters of bacteriostatic rings are respectively 16mm, 15mm and 12mm), and the bacteriostatic action on clostridium perfringens can achieve high-sensitive effect (the diameter of the bacteriostatic ring is 23 mm).
The bacterial enzyme compound feed additive has less dosage, for example, 200g/t of the bacterial enzyme compound feed additive can play a significant role when being added into a weaned pig compound feed, and the bacterial enzyme compound feed additive is specifically represented as follows: the daily gain of the weaned piglets is increased by 10 percent, the feed meat is 0.14 percent lower than that of a control group, and the diarrhea rate is reduced by 1/2; for example, 100g/t of compound feed for 50-week-old laying hens can slowly reduce laying rate, egg breakage rate and death and culling rate by half.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The percent in the present invention means mass percent unless otherwise specified; but the percent of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of the solution.
Example 1: preparation method of coated lactic acid bacteria
1.1 preparation of enterococcus faecium HEW-A588 fermentation liquor comprises the following steps:
taking an enterococcus faecium HEW-A588 glycerol tube preserved in liquid nitrogen, quickly dissolving in a water bath kettle at 37 ℃ for 25 minutes, separating and purifying by streaking on an MRS plate, and culturing at 37 ℃ for 48 hours; picking out a single colony growing vigorously on a flat plate in a sterile room, inoculating the single colony on a fresh MRS inclined plane by using an inoculating loop, and culturing for 18h at 37 ℃; adding 2.0mL of sterilized normal saline into a test tube in a sterile room, scraping lawn by using an inoculating loop, and preparing bacterial suspension; inoculating 0.8mL of the bacterial suspension into a 1L triangular flask containing 300mL of shake flask culture medium, performing shake culture at 37 deg.C and 150r/min for 6.5 hr to obtain seed solution with viable count greater than 9.0 × 108CFU/mL。
After the shake flask fermentation is finished, performing a pilot test on a fermentation tank, inoculating 100mL of shake flask fermentation seed liquid into a 50L fermentation tank, wherein the liquid loading amount is 20L of culture medium, the fermentation temperature is 37 ℃, the pH value is 6.8, the stirring speed is 120r/min, and the fermentation time is 10h to prepare a secondary seed liquid; transfer of secondary seed liquid to 2X 104In the L fermentation tank, the liquid loading amount is 70% (v/v), then the fermentation is carried out for 10h at the pH of 6.8 and the temperature of 37 ℃ and the rotating speed of 120r/min, and the enterococcus faecium HEW-A588 fermentation liquid is obtained, wherein the viable count is more than 4.0 multiplied by 109CFU/mL。
Wherein the shake flask culture medium comprises the following components in percentage by weight: sucrose 1.5%, glucose 0.5%, peptone 1.6%, yeast extract 0.8%, MgSO4·7H2O 0.3％，MnSO4·4H20.02% of O, 0.5% of NaCl, 0.1% of diammonium citrate and CaCO30.05 percent, the balance being water, and the pH value being 6.8 plus or minus 0.2.
The test medium in the 50L fermentation tank comprises the following components in percentage by weight: 0.8% of soft sugar, 1.0% of sodium citrate, 1.5% of yeast extract, 0.06% of magnesium chloride, 0.1% of sodium chloride, 0.03% of manganese sulfate, 0.1% of dipotassium phosphate, 0.004% of ferric ammonium citrate, 800.1% of tween-800 and the balance of water, wherein the pH value is 7.0 +/-0.2.
1.2 preparation method of enterococcus faecium HEW-A588 active bacterial mud:
and after the fermentation is finished, centrifuging the fermentation liquid of the enterococcus faecium HEW-A588 at 12000r/min for 40min to obtain the active bacterial sludge of the enterococcus faecium HEW-A588.
1.3 coating procedure of enterococcus faecium HEW-A588 is as follows:
1) micro-capsule coating: sequentially adding the active bacterium mud of the enterococcus faecium HEW-A588 and the protection solution into a stirring tank according to the weight ratio of 0.3 to 1, stirring at the rotating speed of 50r/min for 40min, adding water, and adjusting the humidity of the material to 30% to obtain micro-capsule wet powder;
2) granulating and coating: putting the wet micro-capsule bacteria powder and the isolated soybean protein into a granulator according to the weight ratio of 1: 0.2, and adjusting the particle size to be 30 meshes to obtain a semi-finished product in a particle shape;
3) enteric coating: putting the granular semi-finished product into a granulating and coating machine of a cyclone fluidized bed for drying, controlling the temperature of inlet dry air at 60 +/-5 ℃, controlling the temperature of outlet air at 32 +/-5 ℃ and drying for 45 min; then coating treatment is carried out: starting a side-spraying coating device, controlling the air inlet temperature at 70 ℃ and the atomization pressure at 0.15MPa, spraying the coating agent solution to the material in the rotational flow state at the speed of 28mL/min to form a coating film layer on the surface of the particulate material, and obtaining a 30-mesh particle product after 60min to obtain the coated lactic acid bacteria.
Wherein the protective solution in the step 1) comprises the following components in percentage by weight: 2.5% of glycerol, 1.5% of maltodextrin, 4% of trehalose, 35% of corn starch, 1.5% of microcrystalline cellulose, 0.5% of polyvinylpyrrolidone, 3% of vegetable oil and the balance of water;
the coating agent solution in the step 3) comprises the following components in percentage by weight: 10% of sodium alginate, 4% of sodium carboxymethylcellulose, 1.5% of hydroxypropyl methylcellulose, 2.8% of pectin, 2.2% of carrageenan, 3.5% of glucan, 5% of malto-oligosaccharide, 12% of skimmed milk powder and the balance of water.
Example 2: bacteriostasis experiment of 3 kinds of live bacteria and glucose oxidase in fungus enzyme composite additive
2.1 preparation of a suspension of pathogenic bacteria
Respectively taking 20 mu L of externally purchased pathogenic escherichia coli, salmonella and staphylococcus aureus from a glycerol tube in a sterile room, inoculating the 20 mu L of externally purchased pathogenic escherichia coli, salmonella and staphylococcus aureus to 50mL of liquid nutrient medium (the components of the culture medium according to mass percentage comprise 0.3% of peptone, 1% of beef extract, 0.5% of sodium chloride and the balance of water, adjusting the pH value to 7.0, sterilizing at 121 ℃ for 30min), dynamically culturing the mixture in a shaking table at 200r/min for 18h, respectively preparing escherichia coli, salmonella and staphylococcus aureus bacterial suspensions, and storing the suspensions in a refrigerator at 4 ℃ for later use.
The method comprises the steps of firstly, drawing a commercially available clostridium perfringens to a TSC (tryptone-sulfite-cycloserine agar) plate from a glycerol tube by using an inoculating loop in a sterile room, carrying out standing anaerobic culture for 20 hours to obtain a single colony, then, inoculating the single colony to 50mLRCM (reinforced clostridium culture medium) liquid, carrying out standing anaerobic culture at 37 ℃ for 18 hours to prepare clostridium perfringens suspension, and storing the clostridium perfringens in a refrigerator at 4 ℃ for later use.
2.2 preparation of antibacterial substances
Preparation of a lactic acid bacteria suspension: collecting glycerol tube of lactobacillus (enterococcus faecium HEW-A588) preserved in liquid nitrogen, rapidly dissolving in water bath at 37 deg.C for 30min, separating and purifying by streaking on MRS plate, and culturing at 37 deg.C for 48 hr; picking a single colony growing vigorously on a flat plate in a sterile room by using an inoculating needle, inoculating the single colony on a fresh MRS inclined plane, and culturing for 18h at 37 ℃; adding 2.0mL of sterilized normal saline into a test tube in a sterile room, scraping lawn by using an inoculating loop, and preparing bacterial suspension; inoculating 0.5mL of the suspension into MRS liquid medium in shake flask culture (50mL/250mL triangular flask), and shake culturing at 37 deg.C and 180r/min for 24h to obtain a suspension of lactic acid bacteria with viable count greater than 8 × 108CFU/mL。
Preparing bacteriostatic bacillus subtilis suspension: 0.05g of antibacterial bacillus subtilis powder is put into 5mL of sterilized normal saline with the weight percent of 0.85 to be mixed evenly, then the mixture is marked on an NA (nutrient agar medium) solid plate in a sterile room, after a single bacterium grows out after being cultured for 24h at 37 ℃, the single bacterium is cultured by using an inoculating loopInoculating into 50 mL/bottle liquid nutrient medium (composed of peptone 0.3%, beef extract 1%, sodium chloride 0.5%, and water in balance, adjusting pH to 7.0, sterilizing at 121 deg.C for 30min), dynamically culturing at 37 deg.C for 24 hr at 200r/min in shaking table to obtain antibacterial Bacillus subtilis suspension with viable count greater than 1.0 × 109CFU/mL。
Preparation of clostridium butyricum bacterial suspension: 0.05g of clostridium butyricum powder is put into 5mL of sterilized normal saline with the weight percent of 0.85 percent and evenly mixed, then the mixture is marked on a TSC solid plate in a sterile room, after a single bacterium grows out after anaerobic culture for 24h at 37 ℃, the single bacterium is put into a 50 mL/bottle RCM liquid culture medium and is put into an incubator at 37 ℃ for anaerobic static culture for 24h to prepare clostridium butyricum suspension, the viable count of which is more than 1.0 multiplied by 10, the clostridium butyricum suspension is prepared8CFU/mL。
Preparation of glucose oxidase metabolite:
(1) phosphate buffer solution and 20 wt% glucose solution required in a glucose reaction system are prepared first, and the mixture is sterilized for 30min at 121 ℃ for later use.
Wherein the preparation process of the phosphate buffer solution comprises the following steps: weighing 13.5g of sodium dihydrogen phosphate dihydrate and 3.96g of disodium hydrogen phosphate dodecahydrate, adding 800ml of deionized water for dissolving, then fixing the volume to 1L, and adjusting the pH value to 6.0.
Wherein the preparation process of the 20 wt% glucose solution comprises the following steps: weighing 20g of glucose, adding phosphate buffer solution, and fixing the volume to 100 mL.
(2) Dilution of 15000U/g glucose oxidase powder: weighing 0.5g of glucose oxidase powder, adding the glucose oxidase powder into a sterilized 250mL triangular glass bottle, adding 50mL of phosphate buffer in a sterile room, and preparing glucose oxidase diluent (with the enzyme activity of 150U/mL) with the volume ratio of 1: 100; then 0.5mL of glucose oxidase diluent with the volume ratio of 1:100 is absorbed and injected into a 4.5mL phosphate buffer centrifugal tube, and the glucose oxidase diluent with the volume ratio of 1:1000 (the enzyme activity is 15U/mL) is obtained by vortex mixing; and then 0.5mL of glucose oxidase diluent with the volume ratio of 1:1000 is absorbed and injected into a 4.5mL phosphate buffer centrifuge tube, and the glucose oxidase diluent with the volume ratio of 1:10000 (the enzyme activity is 1.5U/mL) is obtained by vortex and uniform mixing for later use.
(3) Preparation of 0.3U/mL glucose oxidase metabolite: and (3.5 mL) of sterile phosphate buffer solution prepared in the step (1) is added into a 10mL sterile centrifuge tube, 0.5mL of sterile 20 wt% glucose solution prepared in the step (1) is added, and finally 1.0mL of glucose oxidase diluent with the enzyme activity of 1.5U/mL prepared in the step (2) is added, so that a 5mL reaction system is obtained, and the reaction system is used immediately after the reaction is carried out for 10min at 37 ℃.
(4)2.0U/mL preparation of glucose oxidase metabolite: and (3.8 mL) of sterile phosphate buffer solution prepared in the step (1) is added into a 10mL sterile centrifuge tube, 0.5mL of sterile 20 wt% glucose solution prepared in the step (1) is added, and finally 0.7mL of glucose oxidase diluent with the enzyme activity of 15U/mL prepared in the step (2) is added, so that a 5mL reaction system is obtained, and the reaction system is used immediately after the reaction is carried out for 10min at 37 ℃.
2.3 antibacterial Experimental procedures
2.3.1 lactic acid bacteria bacteriostasis experiment
Respectively taking 0.1mL of pathogenic bacteria suspension (concentration of 1.0 × 10) on a sterile operating platform9Coating CFU/mL salmonella, escherichia coli, staphylococcus aureus and clostridium perfringens on an air-cultured NA plate (coating clostridium perfringens on an RCM plate), punching holes on the plate coated with pathogenic bacteria by using a sterilized 1mL pipette tip, punching four holes (2 holes for comparison and test) on each plate, adding 200 muL of the lactic acid bacteria suspension and MRS liquid culture medium in the step 2.2 into each hole respectively for comparison, preventing the lactic acid bacteria suspension from overflowing, culturing in a 37 ℃ culture box (the clostridium perfringens needs anaerobic culture) for 48 hours, measuring the diameter of a bacteriostatic circle by using a vernier caliper, and taking an average value.
2.3.2 bacteriostatic experiment of bacteriostatic Bacillus subtilis
Respectively diluting 0.1mL of pathogenic bacteria suspension (Salmonella, Escherichia coli, and Staphylococcus aureus to 1.0 × 10) on aseptic operation table7CFU/mL, Clostridium perfringens concentration of 109CFU/mL) were coated onto air-cultured NA plates (clostridium perfringens coated on RCM plates), plates coated with pathogenic bacteria were perforated with sterilized 1mL pipette tips, four wells (2 wells for control and test) were drilled on each plate, and wells were added to each well separatelyAdding 200 μ L of the bacteriostatic Bacillus subtilis suspension obtained in 2.2 step and normal saline as control, culturing in 37 deg.C incubator (Clostridium perfringens needs anaerobic culture) for 24 hr, measuring the diameter of the bacteriostatic circle with vernier caliper, and averaging.
2.3.3 Clostridium butyricum bacteriostatic test
Respectively diluting 0.1mL of pathogenic bacteria suspension (containing Salmonella, Escherichia coli, Staphylococcus aureus, and Clostridium perfringens at concentrations of 1.0 × 10)6CFU/mL) is coated on an NA flat plate which is subjected to air culture (the clostridium perfringens is coated on an RCM flat plate), a sterilized 1mL pipette tip is used for punching on the flat plate coated with pathogenic bacteria, four holes (2 holes for comparison and test) are punched on each flat plate, 200 muL of the clostridium butyricum bacterial suspension and physiological saline which are obtained in the step 2.2 are respectively added into each hole for comparison, the bacteria do not overflow, the bacteria are placed into an incubator at 37 ℃ for culture (the clostridium perfringens needs anaerobic culture) for 24 hours, the diameter of a bacteriostatic circle of the bacteria is measured by a vernier caliper, and an average value is obtained.
2.3.4 bacteriostatic test with glucose oxidase
Respectively taking 0.1mL of pathogenic bacteria suspension (the concentration is 1.0X 10) on a sterile operating platform9Coating CFU/mL salmonella, escherichia coli, staphylococcus aureus and clostridium perfringens on an air-cultured NA plate (coating clostridium perfringens on an RCM plate), punching holes on the plate coated with pathogenic bacteria by using a sterilized 1mL pipette tip, punching four holes on each plate (2 holes for comparison and test), respectively adding 200 muL of 0.3U/mL glucose oxidase metabolite and phosphate buffer solution which are obtained in the step 2.2 into each hole of the plate coated with salmonella, staphylococcus aureus and clostridium perfringens as comparison, simultaneously adding 200 muL of 2.2U/mL glucose oxidase metabolite and phosphate buffer solution which are obtained in the step 2.2 into each hole of the plate coated with escherichia coli as comparison, and placing the plate into a 37 ℃ incubator (the clostridium perfringens needs anaerobic culture) for 24h and then measuring the diameter of a bacteriostatic circle by using a vernier caliper, the average values were taken and the test results are shown in tables 1-4.
2.4 results of bacteriostasis
TABLE 1 bacteriostatic results of lactic acid bacteria
TABLE 2 bacteriostatic results of bacteriostatic Bacillus subtilis
TABLE 3 bacteriostatic results of Clostridium butyricum
TABLE 4 bacteriostatic results of glucose oxidase
As can be seen from tables 1 to 4, the lactic acid bacteria, the bacteriostatic bacillus subtilis, the clostridium butyricum and the glucose oxidase have bacteriostatic effects on four common pathogenic bacteria in different degrees, and particularly, the inhibition of clostridium perfringens achieves high-sensitivity bacteriostatic effects. The bacteriocin generated by the metabolism of the lactic acid bacteria is a polypeptide or protein substance with activity, and has the functions of inhibiting and killing most gram-positive pathogenic bacteria and putrefying bacteria; the bacteriostatic bacillus subtilis is selected from a strain with bacteriostatic action, and active substances such as subtilin, polymyxin, gramicidin and the like generated in the growth process of the strain have obvious inhibitory action on pathogenic bacteria or conditional pathogenic bacteria of endogenous infection; the clostridium butyricum can maintain or recover the dominant flora of the host intestinal tract in vivo as probiotics, and inhibit the growth of common intestinal pathogenic bacteria such as escherichia coli, salmonella, clostridium perfringens and the like by secreting short-chain fatty acid, antibacterial peptide, clostridium butyricum and the like; glucose oxidase is used for killing and inhibiting bacteria through a non-drug mechanism and a non-drug way, and is mainly embodied in that: the glucose oxidase is helpful for inhibiting harmful bacteria after consuming oxygen in the enzymatic reaction process, the gluconic acid generated by the enzymatic reaction can improve the pH value of an intestinal tract, the generated hydrogen peroxide has a broad-spectrum bactericidal effect, and the growth and the propagation of bacteria such as escherichia coli, salmonella and the like can be inhibited.
Example 3: preparation method of bacterial enzyme compound feed additive
The bacterial enzyme compound feed additive comprises the following components in percentage by weight: 20% of coating lactic acid bacteria, 15% of bacteriostatic bacillus subtilis, 5% of clostridium butyricum, 10% of glucose oxidase, 30% of bentonite and 20% of talcum powder.
The viable count of the coated lactic acid bacteria is 2.0 × 1010The viable count of CFU/g and bacteriostatic bacillus subtilis is 1.0 multiplied by 1011The viable count of the Clostridium butyricum is 1.0 multiplied by 1010CFU/g; the enzyme activity of the glucose oxidase is 15000U/g.
The bacteriostatic bacillus subtilis, the clostridium butyricum and the glucose oxidase are all commercially available products.
The specific preparation of the coated lactic acid bacteria is shown in example 1.
The preparation steps of the bacterial enzyme compound feed additive are as follows: respectively carrying out primary premixing on 10 wt% of glucose oxidase powder, 5 wt% of clostridium butyricum powder and 15 wt% of bentonite by using a V-shaped mixer (the rotating speed of the mixer is 30 rpm, and the mixing time is 240 seconds) to prepare small materials, then putting the small materials into a high-efficiency paddle mixer, carrying out secondary mixing on the small materials, 20 wt% of coating lactic acid bacteria, 15 wt% of antibacterial bacillus subtilis, 20 wt% of talcum powder and 15 wt% of bentonite (the rotating speed of the mixer is 50 rpm, and the mixing time is 145 seconds), fully mixing uniformly, and quantitatively packaging to obtain the product.
Example 4: application of bacterium-enzyme compound additive to piglets
144 healthy (Duroc, Changbai and Dabai) three-element commercial piglets with consistent male and female proportions and one week (about 35 days old) after weaning are selected and randomly divided into 2 treatment groups (a control group and a test group), wherein each treatment group has 8 repetitions (8 pigs in the column) and each treatment group has 9 repetitions. The control group daily ration is basic daily ration (prepared according to Chinese pig feeding standard). The daily ration of the test group is obtained by adding 200g of the bacterial enzyme compound feed additive prepared in the embodiment 3 into each ton of basic daily ration. The test pigs all adopt a closed colony house, and are fed and drunk freely, and the colony house is cleaned at least 2 times every day. The feeding management and the immunization program are the same as the daily management of a pig farm, the pigs are disinfected at regular intervals, the pigs are eliminated and selected in time when being found to be sick, and test pigs are not supplemented any more. The test period is 36 days and ends when the piglets are 70 days old. During the test period, the mental state, the ingestion condition, the diarrhea, the fecal state and the fur color of the piglets are observed every day, the weight of piglets is weighed on an empty stomach (initial weight) in the morning of the first day of the test, the weight of piglets is weighed on an empty stomach (final weight) in the morning of the next day after the test is finished, the feeding amount and the residual amount of piglets are recorded every day, and the diarrhea occurrence condition of each group is recorded; the daily feed intake, daily gain, feed-to-weight ratio and diarrhea rate were calculated and are shown in Table 5.
TABLE 5 influence of the fungal enzyme Compound feed additive on the growth Performance of weaned piglets
| Item | Control group | Test group |
| Average initial weight kg/head | 8.20±0.15 | 8.27±0.20 |
| Average end weight kg/head | 22.55±0.45 | 24.12±0.41 |
| Average daily food intake g/d | 698.45±11.98 | 706.63±11.81 |
| Average daily gain g/d | 398.75±9.85 | 440.28±10.39 |
| Feed conversion ratio | 1.75±0.05 | 1.61±0.03 |
| The diarrhea rate% | 8.96 | 4.81 |
As can be seen from table 5, the difference between the feed intake of the piglets in the experimental group and the feed intake of the piglets in the control group is small, but the daily gain is 10% higher than that of the piglets in the control group, the feed and meat are 0.14% lower than that of the piglets in the control group, and the diarrhea rate is 1/2% lower than that of the piglets in the control group, so that the microbial enzyme compound feed additive prepared in the embodiment 3 can be added into the basic ration to effectively inhibit pathogenic microorganisms, improve the balance of intestinal flora, protect the intestinal health of the piglets, improve the digestion, absorption and utilization rate of nutrient substances, and reduce the diarrhea of the piglets. The enterococcus faecium in the coated lactobacillus has the characteristics of whole-intestinal-tract field planting and distribution, so that the lactobacillus in the front section, the middle section and the rear section of the intestinal tract can be rapidly proliferated to become dominant flora, the growth of harmful bacteria is inhibited, and the good effect of regulating the balance of intestinal flora is achieved; the antibacterial bacillus subtilis and the glucose oxidase can strengthen the antibacterial function and reduce diarrhea of piglets; the clostridium butyricum can promote intestinal development of healthy piglets, improve digestive absorption of feed, repair damaged intestinal mucosa of diarrhea piglets, rebuild the quantity of intestinal villi on the intestinal mucosa and increase the height of the villi, improve the utilization rate of the piglets on the feed and improve the daily gain of the piglets.
Example 5: application of bacterium-enzyme compound additive in laying hens
1152-feather 50-week-old Hailan-brown laying hens fed in the same house are randomly selected and divided into 2 treatment groups (a control group and a test group), each treatment group has 6 repetitions, each repetition has 96-feather laying hens, and the experimental period is 60 days. The control group daily ration is basic daily ration; the daily ration of the test group is that 100g of the bacterial enzyme compound feed additive prepared in the embodiment 3 is added into each ton of basic daily ration. The test chicken group adopts a closed chicken house, freely takes food and water, and the feeding management and the immunization program are the same as the daily management of a chicken farm, and eggs are picked regularly 2 times a day. During the test period, the mental state, the ingestion condition, the excrement state, the death and culling condition and the appearance quality of the eggshells of the chicken flocks are observed every day, and the egg number, the broken egg number, the egg weight of each box and the death and culling number of each group are recorded; daily average feed intake, laying rate, egg weight, feed-egg ratio, egg breaking rate and death and culling rate were calculated and shown in table 6.
TABLE 6 influence of fungus-enzyme compound feed additive on egg laying performance of laying hens
| Item | Control group | Test group |
| Daily average feed intake g/d | 130.83±0.88 | 129.74±0.93 |
| Laying rate% | 90.32±0.45 | 92.55±0.37 |
| Egg weight g/piece | 62.08±0.87 | 62.81±0.70 |
| Material to egg ratio | 2.11±0.02 | 2.07±0.02 |
| Breaking rate% | 0.82±0.04 | 0.46±0.05 |
| The death and culling rate% | 4.21±0.05 | 1.89±0.18 |
As can be seen from Table 6, the daily average feed intake, the egg weight and the feed-egg ratio of the laying hens in the test group are not different from those in the control group, but the laying rate is 2.2 percentage points higher than that of the control group, and the egg breaking rate and the death rate are about half lower than those in the control group, so that the microbial enzyme compound feed additive added into the basic ration can effectively inhibit pathogenic microorganisms in intestinal tracts, oviducts and the like of the laying hens, improve the health degree and the eggshell hardness of the laying hens, improve the utilization rate of nutrient substances, increase the egg weight, reduce the egg breaking rate and reduce the death and culling of the laying hens caused by enteritis, salpingitis, rectocele and the like. The enterococcus faecium in the coated lactic acid bacteria enters intestinal tracts of the laying hens, is planted and distributed in the whole intestine, is rapidly proliferated to form dominant flora, inhibits the growth of enteric pathogenic bacteria such as salmonella and escherichia coli, reduces ulcer and erosion caused by myogastric gastritis of the laying hens, improves the absorption and utilization of the laying hens on feed nutrients, improves the laying rate, and simultaneously improves the thickness and hardness of eggshells and reduces the egg breakage rate by increasing the absorption of calcium and phosphorus of the laying hens; the antibacterial bacillus subtilis and the glucose oxidase can strengthen the antibacterial function of the laying hens on harmful bacteria in intestinal tracts and oviducts of the laying hens, and reduce death and culling of the laying hens caused by diarrhea, anal fissure, anal prolapse and the like; the clostridium butyricum can promote the growth and development of intestinal villi, increase the absorption area of intestinal tracts, improve the absorption and utilization rate of feed nutrition of laying hens, and improve the laying rate and the hardness and thickness of eggshells.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The microbial enzyme compound feed additive is characterized by comprising the following components in percentage by weight: 3% -20% of glucose oxidase; coating 11% -25% of lactobacillus; 15% -35% of bacteriostatic bacillus subtilis; 1.0 to 6.0 percent of clostridium butyricum; 15 to 30 percent of bentonite and 10 to 20 percent of talcum powder.
2. The bacterial-enzyme composite feed additive as claimed in claim 1, wherein the coating lactic acid bacteria is enterococcus faecium HEW-A588, and the granularity of the coating lactic acid bacteria is 30 meshes.
3. The microbial enzyme compound feed additive of claim 1, wherein the number of viable bacteria of the coated lactic acid bacteria is not less than 2.0 x 109CFU/g, the viable count of the bacteriostatic bacillus subtilis is not less than 1.0 multiplied by 1010CFU/g, the viable count of the clostridium butyricum is not less than 1.0 multiplied by 108CFU/g。
4. The microbial enzyme complex feed additive according to claim 1, wherein the activity of the glucose oxidase in the microbial enzyme complex additive is not less than 750U/g.
5. A method for preparing a bacterial-enzyme compound feed additive according to any one of claims 1 to 4, which is characterized by comprising the following steps:
primarily premixing glucose oxidase and clostridium butyricum in formula amount with one half of bentonite in formula amount to obtain a primary premix;
and then the primary premix is mixed with the coating lactic acid bacteria, the bacteriostatic bacillus subtilis and the talcum powder in formula amount and the bentonite in the rest half formula amount for the second level to obtain the bacterial enzyme compound feed additive.
6. The method for preparing the bacterial enzyme compound feed additive as claimed in claim 5, wherein the rotation speed of the primary premixing is 20-40 rpm, and the mixing time is 200-300 seconds.
7. The method for preparing bacterial enzyme compound feed additive as claimed in claim 5, wherein the rotation speed of the secondary mixing is 40-60 rpm, and the mixing time is 120-180 seconds.
8. The use of the bacterial enzyme compound feed additive as defined in any one of claims 1-4 in the antibiotic-free feed for livestock and poultry.
9. The application of the bacterial-enzyme compound feed additive in the livestock antibiotic-free feed according to claim 8, which comprises the following steps: the bacterial enzyme compound feed additive is directly added into the livestock feed.
10. The application of the bacterial enzyme compound feed additive in livestock and poultry antibiotic-free feed according to claim 9, wherein the addition proportion in the livestock feed is 0.15-0.3 wt%, and the addition proportion in the poultry feed is 0.1-0.2 wt%.
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| CN116875483A (en) * | 2023-05-30 | 2023-10-13 | 大理州大台农台标饲料有限公司 | Composite microbial agent, fermented feed and preparation method thereof |
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