CN105985916A - Composite microorganism preparation and preparation method thereof - Google Patents

Abstract

The invention discloses a composite microorganism preparation including the active components as follows: at least two of enterococcus faecalis, saccharomyces cerevisiae and bacillus subtilis, and as well as zymosan and corn starch. The preparation is composed of, by weight, 0.1-15 parts of powder of the enterococcus faecalis, 0.1-15 parts of powder of the saccharomyces cerevisiae, 0.1-10 parts of powder of the bacillus subtilis, 0.1 parts of the zymosan and 59.9-74.9 parts of the corn starch. Compared with the prior art, the enterococcus faecalis, saccharomyces cerevisiae and bacillus subtilis are organically combined so that advantages of the bacteria can be achieved completely. The preparation can adapt different hosts and conditions, can partially, even completely replace antibiotics, and can regulate ecologic balance in gastrointestinal tracts, promote growth, increase conversion rate of feeds and enhance immunity. The preparation can improve enteric microorganism environment in animals, inhibit growth of harmful bacteria and promote reproduction of beneficial bacteria, improves animal immunity and enhances disease resistance, supplies nutrients to animals, improves utilization rate of feed and promotes growth of the animals.

Description

A kind of complex microorganism preparations and preparation method thereof

Technical field

The invention belongs to microbiological feed technical field, be specifically related to a kind of complex microorganism preparations and preparation method thereof.

Background technology

Antibiotic is as medicine or feed additive, significantly promote the development of animal husbandry, but antibiotic large-scale use is in animal husbandry, especially as feed additive, its all drawback is more and more obvious, has drug residue, the shortcoming such as Resistant strain, destruction animal intestinal normal flora occurs.For overcoming the drawback of antibiotic, countries in the world strengthen research and the application of novel green feed additive, and particularly additive for microbe feedstuff is subject to the people's attention, and fast development is got up.

Enterococcus faecalis (Enterococcus faecalis) it is gram positive bacteria, this bacterium is classified as Enterococcus by " the primary Jie Shi Bacteria Identification handbook " within 1994, published.Enterococcus faecalis is present in the intestinal of people and most animals, is the critical bacterial populations in human body intestinal canal, is resistant to gastric acid and bile acid.Enterococcus faecalis, as microorganism formulation, can carry out Tiny ecosystem regulation to host gastrointestinal tract, strengthens animal immunizing power.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) belong to fungus, an amphimicrobian quasi-microorganism.Yeast proportion in human or animal's gastrointestinal tract is less, but it has abundant nutrient substance.Yeast tropina accounts for the 32% ~ 75% of dry, and containing 20 several amino acids in yeast cells, the ribonucleic acid of 4.5% ~ 8.5%, and multivitamin, wherein vitamin B group content is the abundantest.Yeast cell wall main component has mannooligo saccharide (6.6%), beta glucan (57%), glycoprotein 22%, and the most a small amount of other protein, chitin, lipid and ash grade.Abundant enzyme is also had, such as amylase, protease, cellulase, phytase, glucanase, chitinase etc. in yeast cell.Saccharomyces cerevisiae, as microorganism formulation, can be that host provides nutrient substance, promote the propagation of probiotics, regulating intestinal canal microecological balance, it is provided that animal digestion ability, promote the growth of animal.

Bacillus subtilis (Bacillus subtilis) belong to bacillus category probiotics, have the advantages that other probiotic bacteria does not has, (1), when bacillus cereus is presented in spore, can tolerate gastric acid and cholate, and keep high activity when entering human or animal's gastrointestinal tract；(2) existence due to spore can tolerate the high temperature of 100 DEG C, and producing, loss rate during preparation is pelletized is smaller, and activity is high, and the holding time is long；(3) bacillus category can produce the multiple enzyme materials such as the strongest amylase of activity, protease, lipase, cellulase, it is also possible to producing some small cyclopeptide bacterioid element or some polypeptides matters, it all has antagonism to the pathogenic bacterium in intestinal.

Summary of the invention

The technical problem to be solved in the present invention is that antibiotic is as medicine or feed additive, there is drug residue, the shortcoming such as Resistant strain, destruction animal intestinal normal flora occurs, for solving the problems referred to above, the present invention provides a kind of complex microorganism preparations and preparation method thereof.

It is an object of the invention to realize in the following manner:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

It is made up of the raw material of following weight portion: enterococcus faecalis mycopowder 0.1 ~ 15 part, saccharomyces cerevisiae mycopowder 0.1 ~ 15 part, bacillus subtilis mycopowder 0.1 ~ 10 part, zymosan 0.1 part, corn starch 59.9 ~ 74.9 parts.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.1 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 18-20 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 30 ~ 34 g/L, yeast powder 9.0 ~ 11 g/L, glucose 26 ~ 30 G/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 g/L, calcium carbonate 10 g/L；Regulate pH to 7.1 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 2.5 ~ 5.0%, primary seed solution be seeded in liquid fermentation medium, is 34 ~ 37 in temperature DEG C, hunting speed is fermentation 12 ~ 14 h under conditions of 120 ~ 150 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:20 ~ 40 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.0 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is cultivation 22 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 110 ~ 120 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 28 ~ 32 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.0 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0% ~ 10%, primary seed solution be seeded in liquid fermentation medium, is 27 ~ 30 in temperature DEG C, hunting speed is 180 ~ 200 r/min bottom fermentation 16 ~ 18 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:5 ~ 10 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(3) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.0 ± 0.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is cultivation 18 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: 80.5 ~ 85.5 parts of wheat bran, Semen Maydis powder 10 ~ 15 parts, glucose 0.5 part, peptone 2 parts, NaCl 0.5 part, manganese sulfate 0.5 part, 0.5 part of magnesium sulfate, calcium carbonate 0.5 part, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 2.5% ~ 5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 35 ~ 38 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(4) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

Relative to prior art, enterococcus faecalis, saccharomyces cerevisiae and bacillus subtilis are carried out organic assembling by the present invention, give full play to the advantage of each strain, can adapt to different hosts and multiple condition, there is part substitute antibiotics the most completely, adjust gastrointestinal tract ecological balance, promote growth, improve food conversion ratio and the several functions such as enhancing immunity, animal intestinal microbial environment, suppression harmful bacteria growth can be improved, promote probiotics propagation；Improve animal immunizing power, strengthen resistance against diseases；There is provided nutrient substance for animal, improve efficiency of feed utilization, promote growth of animal.

Strain enterococcus faecalis used in the present invention, saccharomyces cerevisiae and bacillus subtilis are and independently separate from health pig intestinal, screen, and have excellent probiotic properties and fermentation character, definite functions, and effect is obvious, good stability.Strain used is strain listed by Ministry of Agriculture's " feed additive kind catalogue ", can be used safely in poultry, aquatic animal.

Detailed description of the invention

Embodiment 1:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

It is made up of the raw material of following weight portion: enterococcus faecalis mycopowder 0.1 ~ 15 part, saccharomyces cerevisiae mycopowder 0.1 ~ 15 part, bacillus subtilis mycopowder 0.1 ~ 10 part, zymosan 0.1 part, corn starch 59.9 ~ 74.9 parts.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.1 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 18-20 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 30 ~ 34 g/L, yeast powder 9.0 ~ 11 g/L, glucose 26 ~ 30 G/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 g/L, calcium carbonate 10 g/L；Regulate pH to 7.1 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 2.5 ~ 5.0%, primary seed solution be seeded in liquid fermentation medium, is 34 ~ 37 in temperature DEG C, hunting speed is fermentation 12 ~ 14 h under conditions of 120 ~ 150 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:20 ~ 40 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.0 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is cultivation 22 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 110 ~ 120 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 28 ~ 32 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.0 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0% ~ 10%, primary seed solution be seeded in liquid fermentation medium, is 27 ~ 30 in temperature DEG C, hunting speed is 180 ~ 200 r/min bottom fermentation 16 ~ 18 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:5 ~ 10 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(3) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.0 ± 0.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is cultivation 18 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: 80.5 ~ 85.5 parts of wheat bran, Semen Maydis powder 10 ~ 15 parts, glucose 0.5 part, peptone 2 parts, NaCl 0.5 part, manganese sulfate 0.5 part, 0.5 part of magnesium sulfate, calcium carbonate 0.5 part, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 2.5% ~ 5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 35 ~ 38 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(4) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

Embodiment 2:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 18 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 30 g/L, yeast powder 9.0 g/L, glucose 26 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.0, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 2.5%, primary seed solution be seeded in liquid fermentation medium, is 34 DEG C in temperature, and hunting speed is 12 h that ferment under conditions of 120 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:20 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 5.9,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 22 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 110 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 28 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 G/L, regulates pH to 5.9, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, is fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0% ~ 10%, primary seed solution be seeded in liquid fermentation medium, is 27 DEG C in temperature, and hunting speed is 180 r/min bottom fermentation 16 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:5 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(3) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 0.1kg, saccharomyces cerevisiae mycopowder 0.1 kg, zymosan 0.1 kg, corn starch 59.9 kg.

Embodiment 3:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 19 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 32 g/L, yeast powder 10 g/L, glucose 28 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 4.0%, primary seed solution be seeded in liquid fermentation medium, is 36 DEG C in temperature, and hunting speed is 13 h that ferment under conditions of 135 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:30 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 23 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 115 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 30 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.0, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 7.5%, primary seed solution be seeded in liquid fermentation medium, is 28 DEG C in temperature, and hunting speed is 190 r/min bottom fermentation 16 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:7.5 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(3) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 3kg, saccharomyces cerevisiae mycopowder 3 kg, zymosan 0.1 kg, corn starch 63 kg.

Embodiment 4:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 20 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 34 g/L, yeast powder 11 g/L, glucose 30 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.2, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0%, primary seed solution be seeded in liquid fermentation medium, is 37 DEG C in temperature, and hunting speed is 14 h that ferment under conditions of 150 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:40 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 24 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 120 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 32 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 10%, primary seed solution be seeded in liquid fermentation medium, is 30 DEG C in temperature, and hunting speed is 200 r/min bottom fermentation 18 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:10 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(3) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 6kg, saccharomyces cerevisiae mycopowder 6 kg, zymosan 0.1 kg, corn starch 66 kg.

Embodiment 5:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 9kg, saccharomyces cerevisiae mycopowder 9 kg, zymosan 0.1 kg, corn starch 69 kg.

Other are with embodiment 2.

Embodiment 6:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 12kg, saccharomyces cerevisiae mycopowder 12 kg, zymosan 0.1 kg, corn starch 72 kg.

Other are with embodiment 2.

Embodiment 7:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, saccharomyces cerevisiae, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 15kg, saccharomyces cerevisiae mycopowder 15kg, zymosan 0.1 kg, corn starch 74.9 kg.

Other are with embodiment 2.

Embodiment 8:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 18 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 30 g/L, yeast powder 9.0g/L, glucose 26 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.0, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 2.5%, primary seed solution be seeded in liquid fermentation medium, is 34 DEG C in temperature, and hunting speed is 12 h that ferment under conditions of 120 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:20 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 6.8,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 18 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 80.5kg, Semen Maydis powder 10 kg, glucose 0.5 kg, peptone 2 kg, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 2.5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 35 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by enterococcus faecalis mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 0.1 Kg, bacillus subtilis mycopowder 0.1 kg, zymosan 0.1 Kg, corn starch 59.9 kg.

Embodiment 9:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 19 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 32 g/L, yeast powder 10 g/L, glucose 28 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 4%, primary seed solution be seeded in liquid fermentation medium, is 35 DEG C in temperature, and hunting speed is 13 h that ferment under conditions of 135 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:30 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 21 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 83kg, Semen Maydis powder 12 kg, glucose 0.5 kg, peptone 2 kg, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 4%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 36 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by enterococcus faecalis mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 3 kg, bacillus subtilis mycopowder 2 kg, zymosan 0.1 kg, corn starch 63 kg.

Embodiment 10:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 G/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 G/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 20 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 34 g/L, yeast powder 11 g/L, glucose 30 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 G/L, calcium carbonate 10 g/L；Regulate pH to 7.2, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0%, primary seed solution be seeded in liquid fermentation medium, is 37 DEG C in temperature, and hunting speed is 14 h that ferment under conditions of 150 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:40 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 24 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 85.5 kg, Semen Maydis powder 15 kg, glucose 0.5 kg, peptone 2 kg, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 38 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by enterococcus faecalis mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 6 kg, bacillus subtilis mycopowder 4kg, zymosan 0.1 kg, corn starch 66 kg.

Embodiment 11:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 9 kg, bacillus subtilis mycopowder 6kg, zymosan 0.1 kg, corn starch 69 kg.

Other are with embodiment 8.

Embodiment 12:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 12 kg, bacillus subtilis mycopowder 8kg, zymosan 0.1 kg, corn starch 72 kg.

Other are with embodiment 8.

Embodiment 13:

A kind of complex microorganism preparations, including following active component: enterococcus faecalis, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: enterococcus faecalis mycopowder 15 kg, bacillus subtilis mycopowder 10kg, zymosan 0.1 kg, corn starch 74.9 kg.

Other are with embodiment 8.

Embodiment 14:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 5.9,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 22 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 110 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 28 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 5.9, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0%, primary seed solution be seeded in liquid fermentation medium, is 27 DEG C in temperature, and hunting speed is 180 r/min bottom fermentation 16 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:5 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 6.8,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 18 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 80.5kg, Semen Maydis powder 10 kg, glucose 0.5 kg, peptone 2 parts, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 2.5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 35 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 0.1 Kg, bacillus subtilis mycopowder 0.1 kg, zymosan 0.1 Kg, corn starch 59.9 kg.

Embodiment 15:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 23 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 115 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 30 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.0, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 7.5%, primary seed solution be seeded in liquid fermentation medium, is 28 DEG C in temperature, and hunting speed is 190 r/min bottom fermentation 17 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:7.5 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.0,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 21 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 83kg, Semen Maydis powder 12 kg, glucose 0.5 kg, peptone 2 kg, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 4%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 37 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 3kg, bacillus subtilis mycopowder 2 kg, zymosan 0.1 kg, corn starch 63 kg.

Embodiment 16:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

Specifically comprise the following steps that

(1) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is to cultivate 24 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 120 g/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 32 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L、NaCl 1.0 g/L, regulate pH to 6.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 10%, primary seed solution be seeded in liquid fermentation medium, is 30 DEG C in temperature, and hunting speed is 200 r/min bottom fermentation 18 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:10 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰ More than cfu/g；

(2) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is to cultivate 24 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: wheat bran 85.5 kg, Semen Maydis powder 15 kg, glucose 0.5 kg, peptone 2 kg, NaCl 0.5 kg, manganese sulfate 0.5 kg, magnesium sulfate 0.5 kg, calcium carbonate 0.5 kg, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 5%, primary seed solution be seeded in solid medium, stir, seals with 8 layers of gauze, standing for fermentation 48 h at 38 DEG C, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(3) by saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 6 kg, bacillus subtilis mycopowder 4 kg, zymosan 0.1 kg, corn starch 66 kg.

Embodiment 17:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 9 kg, bacillus subtilis mycopowder 6 kg, zymosan 0.1 kg, corn starch 69 kg.

Other are with embodiment 14.

Embodiment 18:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 12 kg, bacillus subtilis mycopowder 8 kg, zymosan 0.1 kg, corn starch 72 kg.

Other are with embodiment 14.

Embodiment 19:

A kind of complex microorganism preparations, including following active component: saccharomyces cerevisiae, bacillus subtilis, zymosan and corn starch.

In step (3), the parts by weight of each component are: saccharomyces cerevisiae mycopowder 15 kg, bacillus subtilis mycopowder 10 kg, zymosan 0.1 kg, corn starch 74.9 kg.

Other are with embodiment 14.

Embodiment 20:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 0.1 Kg, saccharomyces cerevisiae mycopowder 0.1 kg, bacillus subtilis mycopowder 0.1 Kg, zymosan 0.1 kg, corn starch 59.9 kg。

Other are with embodiment 1.

Embodiment 21:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 3 kg, saccharomyces cerevisiae mycopowder 3 kg, bacillus subtilis mycopowder 2 kg, zymosan 0.1 kg, corn starch 63 kg.

Other are with embodiment 1.

Embodiment 22:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 6 kg, saccharomyces cerevisiae mycopowder 6 kg, bacillus subtilis mycopowder 4 kg, zymosan 0.1 kg, corn starch 66 kg.

Other are with embodiment 1.

Embodiment 23:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 9 kg, saccharomyces cerevisiae mycopowder 9 kg, bacillus subtilis mycopowder 6 kg, zymosan 0.1 kg, corn starch 69 kg.

Other are with embodiment 1.

Embodiment 24:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 12 kg, saccharomyces cerevisiae mycopowder 12 kg, bacillus subtilis mycopowder 8 kg, zymosan 0.1 kg, corn starch 72 kg.

Other are with embodiment 1.

Embodiment 25:

A kind of complex microorganism preparations, including following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

In step (4), the parts by weight of each component are: enterococcus faecalis mycopowder 15 kg, saccharomyces cerevisiae mycopowder 15 kg, bacillus subtilis mycopowder 10 kg, zymosan 0.1 kg, corn starch 74.9 kg.

Other are with embodiment 1.

Clinical trial

Test example 1: complex microorganism preparations application in broiler

The complex microorganism preparations of this test is the product of embodiment 7 preparation, selects young 3000 of 1 age in days health broiler, is randomly divided into 3 groups, often group 1000.Matched group is without any additive in daily ration；Test group is for adding 0.1% microorganism formulation in omnidistance daily ration, feeding management is the most identical with feedstuff change.When 28 age in days, often group randomly draws the close chicken of body weight 8 (often group repeats 4), butchers, win thymus, spleen and fabricius bursa after claiming whose body weight on an empty stomach, claims fresh weight after rejecting fat.Calculating thymus index, index and spleen index and bursal index, this example that Immune Organs Index accounts for every l kg body weight with immune organ fresh weight (g) represents, it may be assumed that Immune Organs Index=immune organ quality ÷ body weight × 100%.Weigh in 42 ages in days, ramming material, calculate daily ingestion amount, daily gain and feed conversion rate；Record test of full phase chicken death condition.

As shown in Table 1 and Table 2, result of the test shows, the daily gain of test group broiler and daily ingestion amount are higher than matched group broiler, and feedstuff-meat ratio is relatively low；The broiler mortality rate of test group is also below matched group broiler；In terms of Immune Organs Index, the index and spleen index of matched group broiler, bursal index and thymus index are above matched group broiler.Illustrate that complex microorganism preparations can improve Intestine of Broiler microecological balance, promote the propagation of probiotics, enhance the resistance against diseases of broiler, improve efficiency of feed utilization, reduce feedstuff-meat ratio.

The impact on growth of meat chicken performance of table 1 complex microorganism preparations

The impact on broiler Immune Organs Index of table 2 complex microorganism preparations

Test example 2: complex microorganism preparations application in pig

The complex microorganism preparations of this test is the product of embodiment 25 preparation, selects Du-length-Dasanyuan cross-bred pig 100 that 50 age in days body weight are close, healthy, is randomly divided into 2 groups, often group 50.Matched group is without any additive in daily ration；Test group is the complex microorganism preparations of interpolation 0.1% in daily ration.Respectively 65,80,95 age in days time blood sampling, measure IgA, the IgG in serum and periphery lymphocyte situation of change.

As shown in table 3, test shows, in test group porcine blood serum, IgA, IgG and T lymphocyte OD value is all above matched group, illustrate that complex microorganism preparations can improve the immunity of pig, the probiotic bacteria that reason is in complex microorganism preparations is in the internal breeding of Intestinum Sus domestica road and produces the benefit materials such as bacteriocin, improve microecological balance in intestinal, it is suppressed that the growth of pathogen, thus strengthen the immunocompetence of pig.

The impact on pig immune energy of table 3 complex microorganism preparations

Above-described is only the preferred embodiment of the present invention, it is noted that for a person skilled in the art, without departing under general idea premise of the present invention, it is also possible to making some changes and improvements, these also should be considered as protection scope of the present invention.

Claims (3)

1. a complex microorganism preparations, it is characterised in that: include following active component: at least two in enterococcus faecalis, saccharomyces cerevisiae, bacillus subtilis, and zymosan and corn starch.

A kind of complex microorganism preparations the most according to claim 1, it is characterised in that: it is made up of the raw material of following weight portion: enterococcus faecalis mycopowder 0.1 ~ 15 part, saccharomyces cerevisiae mycopowder 0.1 ~ 15 part, bacillus subtilis mycopowder 0.1 ~ 10 part, zymosan 0.1 part, corn starch 59.9 ~ 74.9 parts.

The preparation method of a kind of complex microorganism preparations the most according to claim 1 and 2, it is characterised in that: specifically comprise the following steps that

(1) enterococcus faecalis spawn culture:

A. actication of culture: the strain that Freezing Glycerine preserves is activated on slant medium, cultivates 24 h at 37 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: peptone 20 g/L, yeast powder 5.0 g/L, glucose 20 g/L, tween 80 1.0 mL/L, dipotassium hydrogen phosphate 2.0 g/L, Triammonium citrate 2.0 g/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 g/L, sodium acetate 2.0 g/L；Adjust pH to 7.1 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. primary seed solution is prepared: being inoculated in seed culture medium with the strain of inoculating loop picking activation, at 37 DEG C, quiescent culture 18-20 h, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: peptone 30 ~ 34 G/L, yeast powder 9.0 ~ 11 g/L, glucose 26 ~ 30 g/L, tween 80 1.0 mL/L, Triammonium citrate 2.0 G/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 g/L, calcium carbonate 10 g/L；Regulate pH to 7.1 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, be liquid fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 2.5 ~ 5.0%, primary seed solution be seeded in liquid fermentation medium, is 34 ~ 37 DEG C in temperature, and hunting speed is fermentation 12 ~ 14 h under conditions of 120 ~ 150 r/min, i.e. obtains Enterococcus faecalis fermentation liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:20 ~ 40 with corn starch, obtain enterococcus faecalis mycopowder, its viable count is 2.0 × 10¹⁰More than cfu/g；

(2) saccharomyces cerevisiae strain is cultivated:

A. the strain that Freezing Glycerine preserves is activated on slant medium, cultivate 24 h for 30 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 g/L, peptone 5.0 g/L, dipotassium hydrogen phosphate 1.0 g/L, magnesium sulfate 0.5 g/L；Regulate pH to 6.0 ± 0.1,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 30 DEG C in temperature, hunting speed is cultivation 22 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. liquid fermentation medium is prepared: the component of culture medium includes: glucose 110 ~ 120 G/L, brown sugar (Saccharum Sinensis Roxb.) 20 g/L, peptone 28 ~ 32 g/L, yeast powder 10 g/L, KH₂PO₄ 1.0 g/L、K₂HPO₄ 10 g/L、MgSO₄ 1.0 g/L, NaCl 1.0 g/L, regulate pH to 6.0 ± 0.1, sterilizing 20 min at 121 DEG C with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, are fermentation medium；

E. liquid fermentation: according to the inoculum concentration of 5.0% ~ 10%, primary seed solution be seeded in liquid fermentation medium, is 27 ~ 30 DEG C in temperature, and hunting speed is 180 ~ 200 r/min bottom fermentation 16 ~ 18 h, i.e. obtains fermentation by saccharomyces cerevisiae liquid；

F. being centrifuged by fermentation liquid, abandon supernatant, precipitation mixed in the ratio of 1:5 ~ 10 with corn starch, obtain saccharomyces cerevisiae mycopowder, its viable count is 1.0 × 10¹⁰More than cfu/g；

(3) Bacillus subtilis strain is cultivated:

A. actication of culture: activated on slant medium by the strain that Freezing Glycerine preserves, cultivates 24 h for 34 DEG C；

B. seed culture medium is prepared: the component of culture medium includes: glucose 10 G/L, peptone 1.0 g/L, yeast powder 3.0 g/L, NaCl 5.0 g/L；Regulate pH to 7.0 ± 0.2,121 DEG C of sterilizing 20 min with sodium hydroxide solution or dilute hydrochloric acid, after culture medium cools down, can be used as primary-seed medium；

C. preparing primary seed solution: be inoculated in seed culture medium with the strain of inoculating loop picking activation, be 35 DEG C in temperature, hunting speed is cultivation 18 ~ 24 h under conditions of 150 r/min, obtains primary seed solution；

D. solid fermentation culture medium is prepared: the component of culture medium includes: 80.5 ~ 85.5 parts of wheat bran, Semen Maydis powder 10 ~ 15 parts, glucose 0.5 part, peptone 2 parts, NaCl 0.5 part, manganese sulfate 0.5 part, 0.5 part of magnesium sulfate, calcium carbonate 0.5 part, material-water ratio 1:0.8,121 DEG C of sterilizing 25 min, after culture medium cools down, can be used as solid fermentation and cultivate；

E. fermentation: with the inoculum concentration of 2.5% ~ 5%, primary seed solution be seeded in solid medium, stir, by 8 layers of gauze sealing, standing for fermentation 48 at 35 ~ 38 DEG C H, every 12 h turn over even once；

F. solid fermentation thing drying be dry, pulverize, obtain bacillus subtilis mycopowder, its viable count reaches 2.0 × 10¹⁰Cfu/g, spore number reaches 1.4 × 10¹⁰cfu/g；

(4) by enterococcus faecalis mycopowder, saccharomyces cerevisiae mycopowder, bacillus subtilis mycopowder, zymosan and corn starch mix homogeneously, it is complex microorganism preparations.