CN109423466B - Compound fermentation inoculant and application thereof - Google Patents

Abstract

The invention discloses a composite fermentation microbial inoculum and application thereof, wherein bacillus subtilis, bacillus licheniformis and enterococcus faecium are used as active ingredients, and the ratio of the bacillus subtilis to the bacillus licheniformis to the enterococcus faecium is 1-5: 1-5: 0.5-5, and the total bacteria count is 20-200 hundred million CFU/g. The invention produces the fermented feed, realizes multiple functions of antibiosis, acidification, predigestion, mycotoxin reduction and the like, and has wide application prospect.

Description

Compound fermentation inoculant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a high-efficiency solid composite fermentation inoculant and application thereof.

Background

The fermented feed is an organic biological feed with biological activity function, which is finally generated by scientifically compounding a plurality of beneficial microorganisms, inoculating the beneficial microorganisms into feed raw materials in a certain proportion, and fermenting under proper conditions. The fermented feed can reduce anti-nutritional factors in feed raw materials (such as cottonseed meal, soybean meal and the like) and improve the utilization value of the anti-nutritional factors; also can utilize unconventional feed resources (such as potato residue, distiller's grains, apple residue, etc.) to improve the nutrient content and palatability. Therefore, the fermented feed can not only relieve the problem of feed resource shortage, but also save resources and even discharge of wastes.

China takes biotechnology as a national emerging development strategy industry in 12 months in 2012 and certainly promotes the overall development of the biotechnology field. At present, the animal husbandry in China is in the key period of accelerating the change of the development mode, and the demonstration and popularization of the green, environment-friendly, safe and efficient probiotic preparation have important significance for ensuring the effective supply of animal products, promoting the income increase of farmers, protecting the ecological environment and promoting the good and fast development of the animal husbandry. The Ministry of agriculture standardizes the types and applications of microecologics allowed to be used in China in a newly revised feed additive variety catalog (2013), expands the types and use objects of probiotics allowed to be used in feeds, encourages the research and development and application of green and safe microecological feeds, and politically promotes the popularization and application of microecological preparations in the animal husbandry. Therefore, according to the national policy support and development direction, the research on the low-cost high-efficiency microecological feed has the epoch characteristics, conforms to the national industry development direction, conforms to the current development requirement of animal husbandry, and has future development and application prospects and potentials.

Disclosure of Invention

The invention aims to provide a solid composite fermentation microbial inoculum, and the fermented feed produced by the microbial inoculum can reduce the using amount of antibiotics, reduce the content of aflatoxin and has a pre-digestion effect on the feed in the fermentation process.

In order to achieve the purpose, the invention adopts the following technical scheme:

a composite fermentation microbial inoculum takes bacillus subtilis, bacillus licheniformis and enterococcus faecium as active ingredients, and the bacillus subtilis, the bacillus licheniformis and the enterococcus faecium are mixed according to the ratio of bacteria of 1-5: 1-5: 0.5-5, and the total bacteria count is 20-200 hundred million CFU/g.

The Enterococcus Faecium high-yield lactic acid is preserved with the number of CGMCC No. 9666; the high-yield neutral protease and amylase of the bacillus licheniformis are CGMCC No. 14441; the Bacillus subtilis can reduce the content of aflatoxin in feed during solid fermentation, and the preservation number is CGMCC No. 14442.

The Bacillus subtilis and the Bacillus licheniformis are preserved in 19.7.2017 in the common microorganism center of China Committee for culture Collection of microorganisms (No. 3 Hosieboldo No.1 of Beijing Ind. district of China, Microbiol research institute of China academy of sciences, 100101), and the preservation numbers are CGMCC NO.14442 and CGMCC NO.14441 respectively. Enterococcus Faecium (Enterococcus Faecium) has been deposited in the China general microbiological culture Collection center (CGMCC) at 9/16/2014 with the collection number of CGMCC NO. 9666.

The invention mainly relates to enterococcus faecium with high lactic acid yield, a bacillus licheniformis with high neutral protease and amylase yield; a bacillus subtilis capable of reducing the content of aflatoxin in feed during solid fermentation.

The production method of the bacillus subtilis or the bacillus licheniformis comprises the following steps:

primary slant culture: inoculating the bacillus on a first-level slant culture medium, and culturing for 24-36 hours at 24-37 ℃ to obtain a first-level bacillus colony; the culture medium comprises the following components: 2.5-5.0 g of beef extract, 5.0-15.0 g of soybean peptone, 1.0-5.0 g of yeast extract, 1.0-5.0 g of sodium chloride, 5.0-10.0 g of glucose, 5.0-20 g of agar, 1000ml of distilled water and pH of 6.5-7.5;

secondary liquid culture: inoculating the cultured first-stage bacillus colony into a second-stage culture solution, wherein the liquid loading amount is 150ml/500ml, and culturing for 15-20 hours at the temperature of 30-37 ℃ and at the speed of 110-220 r/min to obtain a second-stage bacillus seed solution; preparing a second-stage culture solution in proportion: 5.0-15 g of peptone, 1.0-5.0 g of beef extract, 1.0-5.0 g of sodium chloride, 1000mL of distilled water and pH of 6.5-7.5;

third-stage amplification culture: inoculating the second-level bacillus seed solution into a fermentation tank according to the weight ratio of 0.1-0.5%, and culturing for 24-36 hours on a culture medium by adopting a conventional method at the temperature of 30-37 ℃ and the speed of 110-220 r/min to obtain a third-level bacillus seed solution; the culture medium comprises the following components: 0.1-0.5% of corn flour, 0.1-1.0% of glucose, 0.1-5.0% of bean cake powder, 0.1-0.5% of fish meal, 1.0-5.0% of calcium carbonate, 0.01-0.05% of ammonium sulfate, 0.01-0.05% of dipotassium hydrogen phosphate, 0.01-0.05% of magnesium sulfate and 0.01-0.05% of manganese sulfate;

liquid fermentation: introducing steam into a fermentation tank, preserving heat and sterilizing at 100-150 ℃ for 40-60 minutes, crushing a fermentation substrate to 80 meshes, putting the crushed fermentation substrate into the fermentation tank, and adding water, wherein the weight ratio of the added water to the fermentation substrate is 80-120: 1-10, introducing high-pressure steam to raise the temperature to 100-150 ℃, preserving the heat for 40-60 minutes, naturally cooling for 40-60 minutes,cooling water is introduced to the mixture until the temperature is 20-40 ℃, third-stage bacillus subtilis seed liquid is inoculated under the aseptic condition, the inoculation amount is 0.1-1.0% by weight, the tank pressure is 0.05MPa, the stirring rotating speed is 120-220 rpm, and the ventilation ratio is 0.5-1 by volume: 0.5-1, and the fermentation time is 24-48 hours; detecting the viable count of fermentation liquor to be l.35 multiplied by 10¹⁰CFU/ml; the fermentation substrate comprises the following components in percentage by weight: 1 to 5 percent of yeast powder, 1 to 5 percent of 3 percent of soybean meal hydrolysate, 0.1 to 1.5 percent of glucose, 0.1 to 1.5 percent of corn starch, 0.1 to 1.0 percent of calcium carbonate and 7.0 to 7.5 percent of pH; the fermentation liquor is filtered to obtain the product with the content of 10¹⁰CFU/ml liquid bacillus.

The invention provides an enterococcus faecium fermentation method, which comprises the following steps:

using MRS culture medium as seed culture medium, culturing enterococcus faecium for 18-24 hours under aerobic or facultative condition at 35-45 ℃ to form first-grade seed;

inoculating the primary seed liquid into a seed tank for amplification culture, adopting an MRS culture medium as a seed culture medium, and culturing for 18-24 hours under the aerobic or facultative condition at 35-45 ℃ to obtain secondary seeds;

inoculating the obtained secondary seed liquid into a fermentation tank for fermentation culture, adopting an improved MRS culture medium as a fermentation culture medium, and culturing for 18-36 hours in a timed or continuous fed-batch fermentation mode under the aerobic or facultative condition at 30-45 ℃;

preferably, 5-30 g of soybean peptone, 1-10 g of glucose, 1-10 g of yeast powder, 1-10 g of sodium acetate, 0.1-8 g of diamine citrate, 800.1-5 g of tween, 0.1-5 g of dipotassium hydrogen phosphate, 0.05-lg of magnesium sulfate, 0.001-0.2 g of manganese sulfate, 1-30 g of calcium carbonate and 1 liter of purified water are added into each liter of MRS culture medium for fermenting enterococcus faecium, and the pH value is adjusted to be 5.5-7.5.

The invention also provides application of the composite fermentation inoculant in a fermentation solid culture medium.

Preferably, the composite fermentation inoculant is added into a solid fermentation material according to the inoculation amount of 5-15% by mass ratio for solid fermentation.

Further, a specific production method for solid fermentation comprises the steps of:

(1) adding fermentation strain and carbon source into 30-40 deg.C water, stirring, and standing for 5-12 hr;

(2) adding the solution obtained in the step (1) into the solid fermentation material, and supplementing water to ensure that the water content of the solid fermentation material is between 35 and 40 percent, and fermenting for 12 to 36 hours.

According to a preferred technical scheme of the invention, the solid fermentation is carried out by adopting the fermentation strain provided by the invention, and the steps are as follows:

preparing a plastic barrel capable of containing 100 kilograms of water and 300 kilograms of fermentation strains, putting the fermentation strains and 1000 grams of brown sugar into the plastic barrel, then filling the plastic barrel with water, fully stirring the mixture at the optimal water temperature range of 30-40 ℃, and standing the mixture for 5-12 hours. Clean water source without impurities and pollution is adopted as much as possible, and the external contact is reduced in the placing process.

Firstly, calculating the weight of water to be added, wherein the water content of the material after the water is added is 35-40%, taking one ton of feed as an example, the water content in the normal feed is 14%, so that the weight of the added water required by each ton of feed is 400-430 kg, the water adding amount is reduced when the temperature is high, and the water adding amount is increased when the temperature is low. And (3) mixing the water to be added with the activated water and then uniformly mixing the water to be added with the feed. The water temperature is as close to 30 ℃ as possible, which is beneficial to the rapid fermentation of microorganisms.

The fodder is piled on clean cement floor without water seepage to prevent water from seeping into the floor, and the fodder is piled to about 1m thickness and covered with one layer of plastic paper. The feed stacking position is as clean as possible, warm, windless and rainproof, the temperature is kept as much as possible in winter, and the direct sunlight is avoided in summer. The fermentation time is generally 12 hours to 36 hours, and is closely related to the specific ambient temperature, the summer time is shortened, and the winter time is prolonged. The temperature inside the feed will rise as the degree of fermentation increases. After fermenting for 12 hours (24 hours in winter), the feed can be turned over once every 10 hours to promote uniform fermentation.

The bacillus subtilis used in the invention is a bacillus capable of reducing the content of aflatoxin in feed during solid fermentation, and the preservation number is CGMCC No. 14442. The invention provides a method for producing efficient solid fermentation by combining enterococcus faecium with bacillus subtilis and bacillus licheniformis. The fermented feed produced by the strain can reduce the using amount of antibiotics, reduce the content of aflatoxin and has a predigestion effect on the feed in the fermentation process. The bacillus used in the invention can simultaneously produce neutral protease and amylase with high yield, and can promote the proliferation of lactic acid bacteria. The enterococcus faecium screened by the method can quickly proliferate to generate organic acid, quickly reduce the pH value in the fermentation process and inhibit the growth of mixed bacteria. The bacillus licheniformis used in the invention can reduce the content of aflatoxin in the feed during solid fermentation. Preferably, the special high-efficiency solid fermentation strain provided by the invention is prepared by matching two strains of bacillus and enterococcus faecium according to the ratio of bacteria to bacteria of 2:2:1, is used for producing fermented feed, realizes multiple functions of resisting bacteria, acidifying, predigesting, reducing mycotoxin and the like, and has a wide application prospect.

Drawings

FIG. 1 shows the results of screening protease-producing strains;

FIG. 2 shows the results of screening for alpha-amylase producing strains;

FIG. 3 shows the colony morphology of enterococcus faecium according to the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example-screening of protease and Amylase producing strains

Test method 1

1.1 screening of protease-producing strains

Primary screening: the isolated and purified strain was spotted on a casein medium plate by the casein plate hydrolysis cuvette method, cultured in a thermostat for 48 hours, colonies that produced hydrolysis cuvette were picked up (as shown in fig. 1), and the L value (L value ═ cuvette diameter/colony diameter) was measured and stored at 4 ℃ for further use.

Re-screening: the bacterial strain producing the transparent circle is obtained by primary screening, and 1mL of bacterial suspension (10) is absorbed⁸CFU/mL) is inoculated in a fermentation medium, stirred uniformly, the pH value is natural, and the protease activity is measured after the culture is carried out for 24 hours in an incubator at 37 ℃. And (3) enzyme activity determination: taking the enzyme solution to be detected. Adding 2mL (shaking) of casein into a thermostatic water bath at 40 ℃ for 2min, adding 4mL (shaking) of trichloroacetic acid into the thermostatic water bath at 40 ℃ for 10min, centrifuging to obtain a supernatant, and measuring the OD275nm value. At the same time, blank control was performed.

1.2 screening of alpha-Amylase-producing strains

Primary screening: the strain is inoculated on a sterilized starch agar culture medium, is statically cultured for 24 hours at 37 ℃, is transferred into a liquid triangular flask filled with 25mL of the culture medium, and is cultured for 24 hours at 37 ℃ and 220r/min in a shaking way. Diluting 1mL culture solution with sterile water, respectively spreading 0.15mL bacterial suspension on plate separation culture medium, culturing at 37 deg.C for 24h, spraying iodine solution on the plate, picking out single colony with transparent ring (as shown in FIG. 2), streaking, separating, picking to slant preservation culture medium, culturing at 37 deg.C for 24h, and storing at 4 deg.C.

Re-screening: inoculating the 1-ring primary-screened strain into a seed culture medium, and performing shake cultivation at 37 ℃ and 220r/min for 24 h. Inoculating 1mL of the culture medium into a solid enzyme production medium, culturing at a constant temperature of 30 ℃ for 72h, determining the activity of the alpha-amylase, and storing the strain with the best activity at 4 ℃.

The method for measuring the enzyme activity comprises the following steps: preheating substrate 5mL in 40 deg.C water bath for 10min, adding enzyme solution 0.5mL, maintaining temperature for 5min, adding mixed solution 0.5mL into H5 mL with 0.1mol/L₂SO₄The reaction was terminated, 0.5mL of the resulting solution was added to 5mL of a dilute iodine solution to develop color, and the absorbance at 660nm was measured. 0.5mL of buffer was used as a control in place of 0.5mL of enzyme solution, and distilled water was used as a blank for color comparison.

2 results of the experiment

2.1 Primary screening 15 strains of the Casein-producing circle of hydrolysis strain were obtained from 30 strains of bacteria, accounting for 50% of all tested strains. The L value was initially determined (see Table 1) and the data shows that the L value was maximal for strain Y27, at 3.01.

2.2 rescreening and enzyme activity determination 5 strains with larger L value are selected, and protease activity (shown in table 2) is determined after fermentation culture, wherein in the 5 strains, the strain with larger L value and enzyme activity is Y27, the L value is 3.01, and the protease activity is 324.72U/mL.

2.3 screening of strains producing alpha-amylase it was found by amylase production experiments that a total of 9 strains produced alpha-amylase, accounting for 30% of the tested strains. Further re-screening finds that Y8, Y20 and Y27 have good alpha-amylase producing activity, wherein Y27 strain has high enzyme producing activity and the enzyme activity reaches 225.76U/g, Y30 strain with low enzyme activity has the enzyme activity of 67.9U/g, and the results are shown in Table 3. When the enzyme-producing abilities of the test strains are compared in combination, the strain Y27 having the highest protease and alpha-amylase activities is selected.

TABLE 1 Primary screening of protease producing strains

TABLE 2 rescreening of the strains

TABLE 3 measurement results of alpha-amylase activity

2.4 identification of the strains

The similarity of the gene sequence (SEQ ID NO.1) of the Y27 strain to Bacillus licheniformis (Bacillus licheniformis) was 99% as identified by the gyrB gene sequence, and it was identified as Bacillus licheniformis (Bacillus licheniformis). The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of the institute of microbiology of the national academy of sciences of China, No. 100101 of the national institute of microbiology, North Chen West Lu No.1 of the south-rising area of Beijing) in 2017, 19 months, and the preservation number is CGMCC NO. 14441.

EXAMPLE 2 screening of high-yielding acid acidophilus bacteria

Test method 1

1.1 acid-producing Strain screening

Separating 5 acid-producing strains from healthy chicken intestinal tracts by combining an enrichment culture method and a calcium-solubilizing ring test, inoculating the purified strains into 5mL of sterilized MRS broth culture medium, performing anaerobic fermentation at 37 ℃ for 24h, and then detecting the pH and the lactic acid content of the fermentation broth centrifugation supernatant (4 ℃, 5000g and 10 min).

When the content of the lactic acid is measured, the absorbance values of the lactic acid standard solutions (10, 20, 30, 40 and 50mg/L) are measured under 560nm ultraviolet light, and then a curve is drawn by taking the concentration of the lactic acid standard solution as an abscissa and the absorbance value as an ordinate. When the sample liquid is treated, 4g of activated carbon is added into 40mL of centrifugal supernatant liquid, the mixture is stirred in a water bath at 65 ℃ for 40min and decolored, then the mixture is filtered by filter paper, then the filtrate is repeatedly treated for a plurality of times until the filtrate becomes clear, the clear solution is MRS broth fermentation supernatant liquid which is pretreated by lactic acid determination methods developed by referring to beams and the like for the crude extract liquid of organic acid, then distilled water is used as a blank control, and the absorbance value of the sample fermentation liquid is determined at 560 nm.

TABLE 4 pH of the lactic acid bacteria broth and lactic acid content (mg/L) in the broth

1.2 molecular biological identification of high-yielding lactic acid bacteria strains

16SDNA sequence analysis: the YB-5 bacteria total DNA is extracted by adopting a bacteria genome DNA extraction kit (Tiangen DP302-02, Beijing technology, Ltd.). The 16S rDNA amplification primer adopts a bacterial universal primer, and the primer sequence is as follows: the forward primer was 27F (corresponding to the bases 8-27 of Escherichia coil): 5'-AGAGTTTGATCCTGGCTC AG-3', respectively; the reverse primer was 1495R (corresponding to the bases 1495-1515 of Escherichia coil): 5'-CTACGGCTACCTTGTTACGA-3' are provided. The reaction system (50. mu.L) is shown in Table 2:

TABLE 5 PCR amplification System

The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 58 deg.C for 1 min; extension at 72 ℃ for 2min, 30 cycles, and finally extension at 72 ℃ for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, the positive product with a fragment length of about 1500bp was purified and subjected to sequence determination by Bordeteceae Biotechnology (Beijing) Ltd, and the nucleotide sequence of the 16S rRNA coding gene of YB-5 bacteria was the sequence in the sequence table.

The resulting gene sequence (SEQ ID NO.2) was subjected to BLAST (http:// blast.ncbi. nlm. nih. gov/BLAST. cgi) homology alignment in GenBank database. As a result of the identification, it was found that YB-5 has a homology of 99% with Enterococcus faecium (Enterococcus faecalis), and was identified as Enterococcus faecalis. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 of West Lu No.1 of Beijing republic of south China, institute of microbiology, China academy of sciences, zip code 100101) at 9 months and 16 days in 2014, the preservation number is CGMCC No.9666, the strain is classified and named as Enterococcus Faecium (Enterococcus faecalis), and the colony morphology is shown in figure 3.

Example 3 study of the combination of highly effective solid fermentation strains

(I) experiment of combination ratio of bacillus subtilis and bacillus licheniformis solid fermentation material

The corn bean pulp is used as a culture medium, different proportion combinations of bacillus subtilis and bacillus licheniformis are inoculated, and the optimal addition proportion between the two strains is determined by measuring the change of the number of bacteria after solid fermentation. The mixture ratio of 1 ton of fermentation substrate and 100g of 200 billion total bacteria powder is shown in the following table 6. Accurately weighing 7 parts of 100g corn bean pulp (7:3), wrapping with newspaper, and sterilizing at 120 deg.C for 30 min. Adding bacillus subtilis and bacillus licheniformis into sterilized corn bean pulp according to a corresponding proportion, fully and uniformly stirring, and adding water to ensure that the water content of the final corn bean pulp culture medium is 40%. Placing into an incubator at 37 ℃ and culturing for 36 h. The total number of bacteria in each test group was calculated by the mixed plate counting method.

TABLE 6 grouping design

As can be seen from tables 6 and 7, the total number of bacteria after fermentation in the fourth group is the highest in the seven combinations, and the results show that the ratio of the bacillus subtilis to the bacillus licheniformis in the fourth group is the most suitable, and the bacillus subtilis and the bacillus licheniformis can be mutually compatible to improve the multiplication capacity of the strains in solid fermentation.

TABLE 7 viable cell count after fermentation

(II) experiments on combination ratio of enterococcus faecium and bacillus in solid fermentation material

The method comprises the steps of combining three kinds of bacteria in different proportions in a laboratory by screening enterococcus faecium with high lactic acid yield, combining bacillus subtilis with high neutral protease and amylase with bacillus licheniformis, fermenting the feed, evaluating the fermentation decomposition effect of different strain proportions on the feed through indexes such as pH, total bacteria number, feed decomposition rate and the like after fermentation, and determining the optimal combination compatibility of the fermented feed type composite microecological preparation. The mixture ratio of 1 ton of fermentation substrate and 100g of bacterial powder with 200 hundred million CFU/g of bacteria is shown in the following table 8.

TABLE 8 grouping design

Blank control group: sterile water in the same amount as the test group was added to the feed sample, and the water and the feed were sufficiently stirred.

Test groups: and pouring the sterilized feed sample and the bacterial liquid fully dissolved in proportion into a beaker, washing the triangular flask with 50ml of water, completely washing the bacterial liquid, transferring the bacterial liquid into the beaker, and fully and uniformly stirring the water and the feed. Placing into an incubator at 37 ℃ and culturing for 36 h.

TABLE 9 number of bacteria and pH value variation after fermentation of each group

TABLE 10 feed decomposition rates

Feed soluble substance/%, which is the weight of feed substance per se to the weight of blank dry substance, and feed dry substance weight x 100%; the decomposition rate of the microorganism to the feed/% (blank dry matter weight-sample dry matter weight)/dry matter weight of the feed per se x 100%;

the results of the combined compatibility studies of the complex probiotics in tables 3-10 show that the ratio of lactobacillus to bacillus is 2:8, which can exert the synergistic effect to the maximum extent.

Example 4 application of feed fermented by high-efficiency solid fermentation strain to piglets

The experiment selects 100 weaned Du multiplied by long multiplied by big ternary hybrid pigs of 28 days old with similar gestational times, body weight and good health condition, randomly divides the pigs into 2 groups, each group is divided into 5 repeats, and each repeat is 10. Control group: basal diet feeding, test group: feeding solid fermented material obtained by fermenting with high-efficiency fermentation strain.

The test period is 21 days from 28 days of the weaned piglets. The pigpen was thoroughly rinsed and rigorously disinfected prior to testing. During the test period, the feed is fed for 5 times every day, and dry mixed materials are adopted, and the principle that the rest materials in the crib are used as the principle. The test pigs had free food intake and free water intake. Naturally ventilating the colony house, periodically cleaning, and conventionally inoculating and immunizing.

The results of the effect of the solid fermented feed on the growth performance and diarrhea rate of piglets are shown in Table 11.

TABLE 11 influence of solid fermented feed on growth performance and diarrhea rate of piglets

Note: the data in the same row followed by different letters indicates significant difference (P <0.05), and the same letters indicate insignificant difference (P > 0.05). Results are expressed as mean ± standard deviation, as follows.

As shown in table 11, the solid fermented feed fermented by the fermentation strain can significantly increase the average daily gain and average daily feed intake of piglets, and simultaneously reduce the diarrhea rate.

The results of the effect of the solid fermented feed on the dry matter digestibility of the piglets are shown in Table 12

TABLE 12 comparison of pig Dry matter digestibility test results

As can be seen from Table 12, the dry matter digestibility of the test group fed with the solid fermentation feed was improved as compared to the control group.

The feed fermented by the high-efficiency solid fermentation strain can obviously improve the feed intake and average daily gain of piglets, has the tendency of reducing feed-meat ratio, reduces the diarrhea rate of piglets, improves the digestion of dry matters, promotes the absorption and utilization of nutrient substances, improves the immunity of organisms, further improves the breeding benefit of pig farms, and reduces the environmental pollution.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

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gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tgggaagtac 1200

aacgagttgc gaagtcgcga ggctaagcta atctcttaaa gcttctctca gttcggattg 1260

caggctgcaa ctcgcctgca tgaagccgga atcgctagta atcgcggatc agcacgccgc 1320

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380

ccgaagtcgg tgaggtaacc tttttggagc cagccgccta aggtgggata gatgattggg 1440

gtgaagtcgt aacaaggtaa cca 1463

Claims (7)

1. The composite fermentation microbial inoculum is characterized by taking bacillus subtilis, bacillus licheniformis and enterococcus faecium as active ingredients, wherein the bacillus subtilis, the bacillus licheniformis and the enterococcus faecium are mixed according to the microbial ratio of 1-5: 1-5: 0.5-5, and the total number of bacteria is 20-200 hundred million CFU/g;

the Enterococcus Faecium high-yield lactic acid is preserved with the number of CGMCC No. 9666; the high-yield neutral protease and amylase of the bacillus licheniformis have the preservation number of CGMCC No. 14441; the bacillus subtilis can reduce the content of aflatoxin in feed during solid fermentation, and the preservation number is CGMCC No. 14442.

2. The complex fermentation inoculant according to claim 1, wherein the production method of bacillus subtilis or bacillus licheniformis comprises the following steps:

1) inoculating bacillus subtilis or bacillus licheniformis on a primary slant culture medium, and culturing at 24-37 ℃ for 24-36 hours to obtain a primary bacillus colony; the culture medium comprises the following components: 2.5-5.0 g of beef extract, 5.0-15.0 g of soybean peptone, 1.0-5.0 g of yeast extract, 1.0-5.0 g of sodium chloride, 5.0-10.0 g of glucose, 5.0-20 g of agar, 1000ml of distilled water and pH of 6.5-7.5;

2) inoculating the cultured first-stage bacillus colony into a second-stage culture solution, wherein the liquid loading amount is 150ml/500ml, and culturing for 15-20 hours at the temperature of 30-37 ℃ and at the speed of 110-220 r/min to obtain a second-stage bacillus seed solution; preparing a second-stage culture solution in proportion: 5.0-15 g of peptone, 1.0-5.0 g of beef extract, 1.0-5.0 g of sodium chloride, 1000mL of distilled water and pH of 6.5-7.5;

3) inoculating the second-level bacillus seed solution into a fermentation tank according to the weight ratio of 0.1-0.5%, and culturing for 24-36 hours on a culture medium by adopting a conventional method at the temperature of 30-37 ℃ and the speed of 110-220 r/min to obtain a third-level bacillus seed solution; the culture medium comprises the following components: 0.1-0.5% of corn flour, 0.1-1.0% of glucose, 0.1-5.0% of bean cake powder, 0.1-0.5% of fish meal, 1.0-5.0% of calcium carbonate, 0.01-0.05% of ammonium sulfate, 0.01-0.05% of dipotassium hydrogen phosphate, 0.01-0.05% of magnesium sulfate and 0.01-0.05% of manganese sulfate;

4) introducing steam into a fermentation tank, preserving heat and sterilizing at 100-150 ℃ for 40-60 minutes, crushing a fermentation substrate to 80 meshes, putting the crushed fermentation substrate into the fermentation tank, and adding water, wherein the weight ratio of the added water to the fermentation substrate is 80-120: 1-10, introducing high-pressure steam to raise the temperature to 100-150 ℃, preserving the heat for 40-60 minutes, naturally cooling for 40-60 minutes, and introducing cooling water to cool toInoculating a third-stage bacillus subtilis seed solution at 20-40 ℃ under an aseptic condition, wherein the inoculation amount is 0.1-1.0% by weight, the tank pressure is 0.05MPa, the stirring speed is 120-220 rpm, and the ventilation ratio is 0.5-1 by volume: 0.5-1, and the fermentation time is 24-48 hours; detecting the viable count of fermentation liquor to be l.35 multiplied by 10¹⁰CFU/ml; the fermentation substrate comprises the following components in percentage by weight: 1 to 5 percent of yeast powder, 1 to 5 percent of 3 percent of soybean meal hydrolysate, 0.1 to 1.5 percent of glucose, 0.1 to 1.5 percent of corn starch, 0.1 to 1.0 percent of calcium carbonate and 7.0 to 7.5 percent of pH; the fermentation liquor is filtered to obtain the product with the content of 10¹⁰CFU/ml liquid bacillus.

3. The complex fermentation inoculant according to claim 1, wherein the enterococcus faecium is produced by a method comprising the following steps:

1) using MRS culture medium as seed culture medium, culturing enterococcus faecium for 18-24 hours under aerobic or facultative condition at 35-45 ℃ to form first-grade seed;

2) inoculating the primary seed liquid obtained in the step 1) into a seed tank for amplification culture, adopting an MRS culture medium as a seed culture medium, and culturing for 18-24 hours under the aerobic or facultative condition at 35-45 ℃ to form secondary seeds;

3) inoculating the secondary seed liquid obtained in the step 2) into a fermentation tank for fermentation culture, adopting an improved MRS culture medium as a fermentation culture medium, and culturing for 18-36 hours in a timed or continuous fed-batch fermentation mode under the aerobic or facultative condition at 30-45 ℃.

4. The composite fermentation bacteria agent of claim 3, wherein the modified MRS medium in step 3) is prepared by adding 5-30 g of soybean peptone, 1-10 g of glucose, 1-10 g of yeast powder, 1-10 g of sodium acetate, 0.1-8 g of diamine citrate, 800.1-5 g of Tween, 0.1-5 g of dipotassium phosphate, 0.05-lg of magnesium sulfate, 0.001-0.2 g of manganese sulfate, 1-30 g of calcium carbonate, 1L of purified water and adjusting the pH value to 5.5-7.5.

5. Use of the complex fermentation inoculant according to any one of claims 1 to 4 in fermenting solid media.

6. The application of the composite fermentation inoculant according to claim 5 in fermenting a solid medium, wherein the composite fermentation inoculant is added into the solid fermentation material according to an inoculation amount of 5-15% by mass for solid fermentation.

7. The application of the composite fermentation inoculant in the fermentation of solid media according to claim 6, wherein the specific production method for solid fermentation comprises the following steps:

1) adding a fermentation strain and a carbon source into water with the temperature of 30-40 ℃, fully stirring, and standing for 5-12 hours;

2) adding the solution obtained in the step 1) into the solid fermentation material, and supplementing water to ensure that the water content of the solid fermentation material is between 35 and 40 percent, and fermenting for 12 to 36 hours.

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