CN103289935A - Compound strain microecological preparation and preparation method thereof - Google Patents
Compound strain microecological preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a compound strain microecological preparation and a preparation method thereof. The method comprises the following steps: respectively using brewer yeast, bacillus subtilis, bacillus licheniformis and bacillus coagulans as fermenting strains; in an optimized solid culture medium, cultivating in appropriate temperature conditions; and performing large-scale fermentation production through three-level amplification, wherein the weight ratio of four strain powders in the preparation is sequentially (40-60):(10-30):(10-30):(10-30). The preparation disclosed by the invention has the beneficial effects that the production process is simple and stable, and the strain density is high. Fermentation products can be directly used without separation and purification, and is easy to popularize. Four strain fermentation products are organically combined to adequately exert flora advantages of various strains so as to realize the purpose of compounding synergy. The preparation not only prevents diseases of digestive tract, but also promotes growth and weight increase of livestock, so that the preparation can be used as a feed additive for livestock.
Description
Technical field
The present invention relates to solid fermentation engineering field, relate in particular to a kind of compound strain probiotics that behind solid fermentation, is made up according to a certain percentage by yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and Bacillus coagulans.
Background technology
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) claim bread yeast or budding yeast again, is a kind of generally regarded as safe microorganism (GRAS), has higher protein content and other nutritive ingredient and be widely used in food and the feed.Yeast saccharomyces cerevisiae is and mankind's relation primary yeast the most widely, not only because it is used for making food and wine brewing such as bread and steamed bun traditionally, be used as the eucaryon model animals in modern molecule and cytobiology, the present invention is to use the S. cervisiae of Guangdong Microbes Inst DSMZ as fermented bacterium.In medicine industry, yeast and goods thereof are used for the treatment of some indigestion, and can improve and adjust the metabolic function of human body.In livestock industry, yeast is widely used as concentrated feed to increase the protein content in the feed, to improving dressing percentage, laying rate and the galactopoiesis rate of fowl poultry, the improvement of meat and the raising of pelage quality is all had obvious effects.
Subtilis (
Bacillus subtilis) subtilyne, polymyxin, nystatin, the linear gramicidins isoreactivity material that produce in the process of growth, these active substances have the obvious suppression effect to the conditioned pathogen of pathogenic bacterium or autogenous infection; Subtilis consumes the free oxygen in the environment rapidly, causes the enteron aisle hypoxemia, promotes the growth of useful anerobe, and produces organic acid such as lactic acid, reduces the enteron aisle pH value, suppresses other pathogenic bacterium growth indirectly; Stimulate growing of animal immune organ, activate T, bone-marrow-derived lymphocyte, improve immunoglobulin (Ig) and antibody horizontal, strengthen cellular immunization and humoral immune function, improve herd immunity; Subtilis thalline self synthesizes enzymes such as α-Dian Fenmei, proteolytic enzyme, lipase, cellulase, in digestive tube with animal body in digestive enzymes together play a role; Multiple vitamin B group such as energy synthesise vitamins B1, B2, B6, nicotinic acid, the activity of raising animal body internal interference element and scavenger cell.
Bacillus licheniformis (
Bacillus Subtilis) be a kind of of bacillus, cell size 0.8 μ m * (1.5～3.5) μ m, cellular form and arrangement are shaft-like, Dan Sheng, no Poly-salt particle in the cell, be a kind of Gram-positive bacillus, bacterium colony be flat, the edge is irregular, white, be facultative anaerobe.Bacillus licheniformis cellular form and arrangement are shaft-like, Dan Sheng, can adjust flora imbalance and reach therapeutic purpose, can impel body to produce antibacterial substance, kill pathogenic bacterium, after it enters animal intestinal, take the oxygen mechanism of action by force by its unique biological, pathogenic bacterium such as staphylococcus, yeast-like fungus in the enteron aisle there is antagonistic action, and probioticss such as bifidus bacillus, lactobacillus, bacterioide, digestibility suis are had the growth of promotion effect, oneself obtains the national Ministry of Agriculture and allows to add use in feed, is very outstanding fodder industry thalline additive.
Bacillus coagulans (
Bacillus coagulans), gram-positive microorganism not only belongs to hard (or thick) wall bacterium door, and belongs to the enteron aisle milk-acid bacteria, is called " the spore milk-acid bacteria is arranged " again.Be the bacillus class milk-acid bacteria through a kind of " generally believing safety " of drugs approved by FDA, oral after, Bacillus coagulans is in blind, knot and the rectum field planting of animal, fermentation produces a large amount of antibiotic coagulins, lactic acid, amino acid, VITAMIN and multiple digestive ferment.The survival rate of Bacillus coagulans after simulated gastric fluid (pH1.4) is handled 2 hours is 95.29%, and the bifidus bacillus survival rate is 5.20%, and the survival rate of lactobacillus is 5.50%.Studies show that Bacillus coagulans is extremely strong to the acidic conditions tolerance of simulated gastric fluid, its survival is unaffected, obviously is better than other probiotics, can enter enteron aisle by stomach smoothly, thereby guarantee the normal performance of drug effect, Bacillus coagulans is sealed in the Plastic Bottle, places and handle in 55 ℃ of loft drier after 60 hours, the result who measures viable count shows, still be not less than 100,000,000 cfu/g, moisture content does not have obvious change, and the Bacillus coagulans preparation can be in room temperature preservation, and validity period can reach 24 months.
The functional characteristics of Bacillus coagulans: (1) effectively suppresses the growth of harmful bacterium, and improvement intestinal microecology environment promotes intestinal growth, strengthens intestinal function; (2) improve feed quality, promote digesting and assimilating of feed, reduce feedstuff-meat ratio; (3) effectively improve weanling pig because the flora imbalance diarrhea that stress cause with anaphylaxis; (4) without any side effects, use safety, and can reduce obnoxious flavoures such as ammonia in the environment, hydrogen sulfide, improve breeding environment.
Yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and four kinds of bacterial strains of Bacillus coagulans are carried out organic assembling, give full play to the flora advantage of each bacterial classification, not only can prevent digestive tract diseases, and can promote growth of animals or poultry and put on weight, reach the purpose of compound synergic.
Summary of the invention
The purpose of this invention is to provide a kind of compound strain probiotics and preparation method thereof, by organic assembling and the collocation of different efficacies bacterial strain, improve the action effect of probiotics.
The technical scheme that realizes the object of the invention is as follows:
A kind of compound strain probiotics and preparation method thereof may further comprise the steps:
(1) yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) spawn culture:
1. actication of culture: the present invention adopt yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) as fermented bacterium, yeast saccharomyces cerevisiae claims bread yeast or budding yeast again, purchases in Guangdong Microbes Inst DSMZ preserving number: CICC1795.Yeast saccharomyces cerevisiae is and mankind's relation primary yeast the most widely, it is used for making food and wine brewing such as bread and steamed bun traditionally, containing elements such as the high-quality protein, food fibre, vitamin complex and the organoselenium that meet the body demand, chromium, zinc, iron, is the health-nutrition source of a kind of pure natural, pollution-free and environmental protection; Its protein content is higher, and amino acid balance is good, and the digestibility height is rule the yeast saccharomyces cerevisiae bacterial classification of-80 ℃ of preservations at the YPD solid plate, cultivates 16 ~ 24 hours for 30 ℃;
2. prepare first order seed: with the yeast saccharomyces cerevisiae bacterial classification of activation, being inoculated in the 200 mL YPD liquid nutrient mediums with transfering loop picking one ring, triangular flask is placed shaking table, is under the condition of 30 ℃ and 250rpm in temperature, after the shaking culture 16 ~ 24 hours, make the first order seed bacterium;
3. prepare the solid fermentation substratum: the solid butt: be that basestocks (contains Semen Maydis powder 40 ~ 60wt%), glucose 2wt%, urea 0.5wt%, K with Semen Maydis powder and wheat-flour
2HPO
41.0wt% and vitamin H 0.01wt%; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is to stir under the natural condition in pH value; Be under 121 ℃ with solid medium in temperature, autoclave sterilization 20min namely can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation: will insert the first order seed bacterium according to 5 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, and keep that humidity will be 50 ~ 70%, temperature is under 25 ~ 30 ℃ the condition, to cultivate about 20 hours, makes secondary seed;
5. with the secondary seed that obtains, according to step 4. further solid fermentation obtain three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to 4. further solid fermentation enlarged culturing of step; Yeast culture is carried out enumeration, require viable count to reach 1.0 * 10
8More than the bright song of cfu/g;
6. the Yeast protein culture with solid fermentation carries out bake drying, pulverizes then and packages spare;
(2) subtilis (
Bacillus subtilis) spawn culture:
1. actication of culture:
The present invention adopt subtilis (
Bacillus subtilis) as fermented bacterium, purchase in microbial strains preservation center, Guangdong Province, preserving number, GIM 1.131, with the Bacillus subtilis strain of-80 ℃ of preservations at seed culture medium (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g adds water to 1 liter, and autoclave sterilization is standby) solid rules on the inclined-plane, cultivates about 16 hours activation for 37 ℃;
2. prepare first order seed:
With the subtilis of activation, be inoculated in (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO in the 300 mL liquid seed culture mediums with transfering loop picking one ring
4Be added into 0.2%, add water to 1 liter, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 16 ~ 20 hours;
3. prepare the solid fermentation substratum:
The solid butt: be basestocks (ratio of quality is 7:2:1) with Semen Maydis powder, wheat-flour and soybean cake powder, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs; Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation:
To insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made secondary seed; Secondary seed according to carrying out enlarged culturing in 8 ~ 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 8 ~ 10% inoculum size; The subtilis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
9More than the bright song of CFU/g, the gemma rate of formation is more than 90%;
5. dry:
Handle 60min with inner drying with heating under the oven dry of subtilis solid fermentation culture, airing or 65 ℃, preserve standby;
(3) Bacillus licheniformis (
Bacillus licheniformis) spawn culture:
1. actication of culture:
The present invention adopt Bacillus licheniformis (
Bacillus licheniformis) as fermented bacterium, with the Bacillus licheniformis bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g, autoclave sterilization is standby) rule on the solid plate, cultivated 18 ~ 20 hours for 37 ℃;
2. prepare first order seed:
With the yeast saccharomyces cerevisiae bacterial classification of activation, be inoculated in seed culture medium in the 200 mL liquid seed culture mediums (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO with transfering loop picking one ring
4Be added into 0.2%, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 18 ~ 20 hours;
3. prepare the solid fermentation substratum: the solid butt: be basestocks (Semen Maydis powder is 9:1 with the ratio of the quality of soybean cake powder) with Semen Maydis powder and soybean cake powder, glucose 1.0%, urea 0.3 ~ 0.5%, NaCl 1.0%.It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is natural condition, stirs; Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation: will insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made secondary seed; Secondary seed according to carrying out enlarged culturing in 8 ~ 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 8 ~ 10% inoculum size; The Bacillus licheniformis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
9CFU/g is bright bent, and the gemma rate of formation is more than 80%;
(4) Bacillus coagulans (
Bacillus coagulans) spawn culture:
1. actication of culture: the present invention adopt Bacillus coagulans (
Bacillus coagulans) as fermented bacterium, preserving number: GIM 1.421 purchases in microbial strains preservation center, Guangdong Province; With the Bacillus coagulans bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2% and agar powder 15g, through standby behind the autoclave sterilization) rule on the solid plate, cultivated 20 ~ 24 hours for 37 ~ 40 ℃;
2. prepare first order seed: with the Bacillus coagulans of activation, being inoculated in the 200 mL liquid seed culture mediums to the five rings with transfering loop picking three, triangular flask is placed shaking table, is under the condition of 37 ~ 40 ℃ and 250rpm in temperature, after the shaking culture 20 ~ 24 hours, make the first order seed bacterium;
3. prepare the solid fermentation substratum: the solid butt: be basestocks (Semen Maydis powder is 2:1 with the ratio of the quality of soybean cake powder), KH with Semen Maydis powder and soybean cake powder
2PO
40.3 ~ 0.5wt%, MnSO
4H
2O 0.02wt% and MgSO
47H
2O 0.125wt ‰; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is natural condition in pH value, stirs, and is that autoclave sterilization 20min namely can be used as the use of solid fermentation substratum after cooling under 121 ℃ the condition in temperature with solid medium;
4. solid fermentation: will insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, stir, keep humidity be 50 ~ 70% and temperature be under 37 ~ 40 ℃ the condition, to cultivate 20 ~ 24 hours, make secondary seed; With the secondary seed that obtains, obtain three grades of seeds according to the further solid fermentation of 8 ~ 10% inoculum size; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further solid fermentation enlarged culturing of 8 ~ 10% inoculum size; The Bacillus coagulans culture is carried out enumeration, and the viable bacteria bulk concentration can reach 1.0 * 10
9More than the bright song of CFU/g, the gemma rate of formation is more than 80%.
2. accurately take by weighing the culture of yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and Bacillus coagulans respectively, the mass ratio of four kinds of bacterium powder is followed successively by in the preparation: 40-60:10-30:10-30:10-30, mix, be the compound strain probiotics.
Beneficial effect of the present invention: with Semen Maydis powder, wheat-flour and soybean cake powder as basestocks, add an amount of carbon source, nitrogenous source, inorganic salt and somatomedin and carry out solid fermentation, reduce culture medium cost, improved probiotics viable bacteria concentration, for its application in aquaculture provides the basis.The probiotics viable bacteria that not only contains high density through the compound strain probiotics of this method fermentative production, and contain rich in protein, amino acid, VITAMIN and trace element, and contain digestive ferment and some unknown growth-stimulating factors, have higher biological activity, therefore, can promote animal metabolism, whet the appetite, be conducive to digesting and assimilating of animal, accelerate growing and the performance of production performance of animal, can also improve the defending and fighting against diseases ability of animal.Production technique simple and stable of the present invention, cell density height, tunning need not separation and purification and just can directly use, and are easy to promote; The compound strain culture is added in the pig feed, not only can prevent digestive tract diseases, and can promote poultry growth and put on weight.
Embodiment
Further specify the present invention by the following examples, but not as restriction of the present invention.
Embodiment 1 solid fermentation is produced the technical process of Yeast protein feed, may further comprise the steps:
Yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) bacterium:
(1) actication of culture:
The present invention adopt yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) as fermented bacterium, yeast saccharomyces cerevisiae claims bread yeast or budding yeast again, purchases in Guangdong Microbes Inst DSMZ preserving number: CICC1795.Yeast saccharomyces cerevisiae is and mankind's relation primary yeast the most widely, it is used for making food and wine brewing such as bread and steamed bun traditionally, contain the high-quality protein, the food fibre that meet the body demand, meet VITAMIN and organoselenium, elements such as chromium, zinc, iron, be the health-nutrition source of a kind of pure natural, pollution-free and environmental protection.Its protein content is higher, and amino acid balance is good, the digestibility height.The yeast saccharomyces cerevisiae bacterial classification of-80 ℃ of preservations is rule at the YPD solid plate, cultivated 16 hours for 30 ℃;
(2) preparation first order seed:
With the yeast saccharomyces cerevisiae bacterial classification of activation, be inoculated in the 200 mL YPD liquid nutrient mediums with transfering loop picking one ring, triangular flask is placed shaking table, be that shaking culture made primary seed solution after 16 hours under 30 ℃ of conditions with 250rpm in temperature;
(3) preparation solid fermentation substratum:
Solid butt: be that basestocks (contains Semen Maydis powder 40 ~ 60wt%), glucose 2wt%, urea 0.5wt%, K with Semen Maydis powder and wheat-flour
2HPO
41.0wt% and vitamin H 0.01wt%.It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is to stir under the natural condition in pH value; Be under 121 ℃ with solid medium in temperature, autoclave sterilization 20min namely can be used as the solid fermentation substratum after cooling and uses;
(4) solid fermentation: will insert the first order seed bacterium according to 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, and keep that humidity will be 50 ~ 70%, temperature is under 25 ~ 30 ℃ the condition, to cultivate 20 hours, makes secondary seed; Secondary seed according to carrying out enlarged culturing in 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 25 ~ 30 ℃ cultivated about 20 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 10% inoculum size; Yeast culture is carried out enumeration, and viable count reaches 2.6 * 10
8Cfu/g;
(5) the Yeast protein culture with solid fermentation carries out bake drying, pulverizes then and packages spare.
Subtilis (
Bacillus subtilis) spawn culture:
(1) actication of culture:
The present invention adopt subtilis (
Bacillus subtilis) as fermented bacterium, purchase in microbial strains preservation center, Guangdong Province, preserving number, GIM 1.131, with the Bacillus subtilis strain of-80 ℃ of preservations at seed culture medium (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g adds water to 1 liter, and autoclave sterilization is standby) solid rules on the inclined-plane, cultivates about 16 hours activation for 37 ℃;
(2) preparation first order seed:
With the subtilis of activation, be inoculated in (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO in the 300 mL liquid seed culture mediums with transfering loop picking one ring
4Be added into 0.2%, add water to 1 liter, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 16 ~ 20 hours;
(3) preparation solid fermentation substratum:
The solid butt: be basestocks (ratio of quality is 3:1:1) with soybean cake powder, Semen Maydis powder and wheat-flour, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs, and solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
(4) solid fermentation:
To insert the first order seed bacterium according to 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 18 hours, made secondary seed.Secondary seed according to carrying out enlarged culturing in 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 18 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 10% inoculum size.The subtilis culture is carried out enumeration, and the viable bacteria bulk concentration reaches 1.8 * 10
9CFU/g is bright bent, and the gemma rate of formation is 92%;
(5) drying:
Handle 60min with inner drying with heating under the oven dry of subtilis solid fermentation culture, airing or 65 ℃, preserve standby;
Bacillus licheniformis (
Bacillus licheniformis) spawn culture:
(1) actication of culture:
The present invention adopt Bacillus licheniformis (
Bacillus licheniformis) as fermented bacterium, with the Bacillus licheniformis bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g, autoclave sterilization is standby) rule on the solid plate, cultivated 18 hours for 37 ℃;
(2) preparation first order seed:
With the yeast saccharomyces cerevisiae bacterial classification of activation, be inoculated in seed culture medium in the 200 mL liquid seed culture mediums (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO with transfering loop picking one ring
4Be added into 0.2%, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 18 hours;
(3) preparation solid fermentation substratum: solid butt: solid butt: be basestocks (Semen Maydis powder is 9:1 with the ratio of the quality of soybean cake powder) with Semen Maydis powder and soybean cake powder, glucose 1.0%, urea 0.3 ~ 0.5%, NaCl 1.0%, it is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is natural condition, stirs, solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
(4) solid fermentation: will insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made secondary seed.Secondary seed according to carrying out enlarged culturing in 8 ~ 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made three grades of seeds.Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 8 ~ 10% inoculum size.The Bacillus licheniformis culture is carried out enumeration, and the viable bacteria bulk concentration reaches 9.15 * 10
9CFU/g is bright bent, and the gemma rate of formation is 89%.
Bacillus coagulans (
Bacillus coagulans) spawn culture:
(1) actication of culture: the present invention adopt Bacillus coagulans (
Bacillus coagulans) as fermented bacterium, preserving number: GIM 1.421 purchases in microbial strains preservation center, Guangdong Province.With the Bacillus coagulans bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2% and agar powder 15g, through standby behind the autoclave sterilization) rule on the solid plate, cultivated 24 hours for 37 ~ 40 ℃;
(2) preparation first order seed: with the Bacillus coagulans of activation, being inoculated in the 200 mL liquid seed culture mediums to the five rings with transfering loop picking three, triangular flask is placed shaking table, is under the condition of 37 ℃ and 250rpm in temperature, after the shaking culture 24 hours, make the first order seed bacterium;
(3) preparation solid fermentation substratum: solid butt: be basestocks (Semen Maydis powder is 2:1 with the ratio of the quality of soybean cake powder), KH with Semen Maydis powder and soybean cake powder
2PO
40.3 ~ 0.5wt%, MnSO
4H
2O 0.02wt% and MgSO
47H
2O 0.125wt ‰; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is natural condition in pH value, stirs, and is that autoclave sterilization 20min namely can be used as the use of solid fermentation substratum after cooling under 121 ℃ the condition in temperature with solid medium;
(4) solid fermentation: will insert the first order seed bacterium according to 8% inoculum size through the solid fermentation substratum of sterilising treatment, stir, keep humidity be 50 ~ 70% and temperature be under 37 ℃ the condition, to cultivate 24 hours, make secondary seed;
(5) with the secondary seed that obtains, obtain three grades of seeds according to the further solid fermentation of step (4);
(6) can be according to concrete cultivation consumption, further with three grades of seeds according to the further solid fermentation enlarged culturing of step (4); The Bacillus coagulans culture is carried out enumeration, and the viable bacteria bulk concentration can reach 1.61 * 10
9CFU/g is bright bent, and the gemma rate of formation is 83%.
5. accurately take by weighing the culture of yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and Bacillus coagulans respectively, the mass ratio of four kinds of bacterium powder is followed successively by in the preparation: 55:15:15:15, mix, and be the compound strain probiotics.
Embodiment 2-3
Each stage of yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and Bacillus coagulans cultivates operation and is equal to embodiment 1, and above-mentioned four inoculating proportions see the following form respectively in the final probiotics:
Four kinds of bacterium powder mass ratioes in table 1 probiotics of the present invention
| ? | Yeast saccharomyces cerevisiae | Subtilis | Bacillus licheniformis | Bacillus coagulans |
| Embodiment 2 | 60 | 10 | 15 | 15 |
| Embodiment 3 | 50 | 15 | 15 | 20 |
Claims (2)
1. compound strain probiotics and preparation method thereof is characterized in that, may further comprise the steps:
(1) yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) spawn culture:
1. actication of culture: the present invention adopt yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae) as fermented bacterium, yeast saccharomyces cerevisiae claims bread yeast or budding yeast again, purchases in Guangdong Microbes Inst DSMZ preserving number: CICC1795; Yeast saccharomyces cerevisiae is and mankind's relation primary yeast the most widely, it is used for making food and wine brewing such as bread and steamed bun traditionally, containing elements such as the high-quality protein, food fibre, vitamin complex and the organoselenium that meet the body demand, chromium, zinc, iron, is the health-nutrition source of a kind of pure natural, pollution-free and environmental protection; Its protein content is higher, and amino acid balance is good, and the digestibility height is rule the yeast saccharomyces cerevisiae bacterial classification of-80 ℃ of preservations at the YPD solid plate, cultivates 16 ~ 24 hours for 30 ℃;
2. prepare first order seed: with the yeast saccharomyces cerevisiae bacterial classification of activation, being inoculated in the 200 mL YPD liquid nutrient mediums with transfering loop picking one ring, triangular flask is placed shaking table, is under the condition of 30 ℃ and 250rpm in temperature, after the shaking culture 16 ~ 24 hours, make the first order seed bacterium;
3. prepare the solid fermentation substratum: the solid butt: be that basestocks (contains Semen Maydis powder 40 ~ 60wt%), glucose 2wt%, urea 0.5wt%, K with Semen Maydis powder and wheat-flour
2HPO
41.0wt% and vitamin H 0.01wt%; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is to stir under the natural condition in pH value; Be under 121 ℃ with solid medium in temperature, autoclave sterilization 20min namely can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation: will insert the first order seed bacterium according to 5 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, and keep that humidity will be 50 ~ 70%, temperature is under 25 ~ 30 ℃ the condition, to cultivate about 20 hours, makes secondary seed;
5. with the secondary seed that obtains, according to step 4. further solid fermentation obtain three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to 4. further solid fermentation enlarged culturing of step; Yeast culture is carried out enumeration, require viable count to reach 1.0 * 10
8More than the bright song of cfu/g;
6. the Yeast protein culture with solid fermentation carries out bake drying, pulverizes then and packages spare;
(2) subtilis (
Bacillus subtilis) spawn culture:
1. actication of culture:
The present invention adopt subtilis (
Bacillus subtilis) as fermented bacterium, purchase in microbial strains preservation center, Guangdong Province, preserving number, GIM 1.131, with the Bacillus subtilis strain of-80 ℃ of preservations at seed culture medium (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g adds water to 1 liter, and autoclave sterilization is standby) solid rules on the inclined-plane, cultivates about 16 hours activation for 37 ℃;
2. prepare first order seed:
With the subtilis of activation, be inoculated in (peptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO in the 300 mL liquid seed culture mediums with transfering loop picking one ring
4Be added into 0.2%, add water to 1 liter, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 16 ~ 20 hours;
3. prepare the solid fermentation substratum:
The solid butt: be basestocks (ratio of quality is 7:2:1) with Semen Maydis powder, wheat-flour and soybean cake powder, glucose 5.0%, CaCO
31.5%, MnSO
4H
2O 0.04%; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and the pH value is adjusted to 6.5, stirs; Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation:
To insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made secondary seed; Secondary seed according to carrying out enlarged culturing in 8 ~ 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37 ℃ cultivated 16 ~ 20 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 8 ~ 10% inoculum size; The subtilis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
9More than the bright song of CFU/g, the gemma rate of formation is more than 90%;
5. dry:
Handle 60min with inner drying with heating under the oven dry of subtilis solid fermentation culture, airing or 65 ℃, preserve standby;
(3) Bacillus licheniformis (
Bacillus licheniformis) spawn culture:
1. actication of culture:
The present invention adopt Bacillus licheniformis (
Bacillus licheniformis) as fermented bacterium, with the Bacillus licheniformis bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2%, agar powder 15g, autoclave sterilization is standby) rule on the solid plate, cultivated 18 ~ 20 hours for 37 ℃;
2. prepare first order seed:
With the yeast saccharomyces cerevisiae bacterial classification of activation, be inoculated in seed culture medium in the 200 mL liquid seed culture mediums (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO with transfering loop picking one ring
4Be added into 0.2%, autoclave sterilization is standby), triangular flask is placed shaking table, 37 ℃, 250rpm, shaking culture made primary seed solution after 18 ~ 20 hours;
3. prepare the solid fermentation substratum: the solid butt: be basestocks (Semen Maydis powder is 9:1 with the ratio of the quality of soybean cake powder) with Semen Maydis powder and soybean cake powder, glucose 1.0%, urea 0.3 ~ 0.5%, NaCl 1.0%, it is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, the pH value is natural condition, stirs; Solid medium through 121 ℃, 20min autoclave sterilization, namely be can be used as the solid fermentation substratum after cooling and uses;
4. solid fermentation: will insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, and stir, keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made secondary seed; Secondary seed according to carrying out enlarged culturing in 8 ~ 10% the inoculum size adding sterilization solid fermentation substratum, is stirred, and keeping humidity is 50 ~ 70%, and temperature is 35 ~ 37.5 ℃ cultivated 18 ~ 20 hours, made three grades of seeds; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further enlarged culturing of 8 ~ 10% inoculum size; The Bacillus licheniformis culture is carried out enumeration, require the viable bacteria bulk concentration can reach 1.0 * 10
9CFU/g is bright bent, and the gemma rate of formation is more than 80%;
(4) Bacillus coagulans (
Bacillus coagulans) spawn culture:
1. actication of culture: the present invention adopt Bacillus coagulans (
Bacillus coagulans) as fermented bacterium, preserving number: GIM 1.421 purchases in microbial strains preservation center, Guangdong Province; With the Bacillus coagulans bacterial classification of-80 ℃ of preservations at seed culture medium (tryptone 10g, yeast extract 5g, NaCl 10g, 25% concentration MnSO
4Be added into 0.2% and agar powder 15g, through standby behind the autoclave sterilization) rule on the solid plate, cultivated 20 ~ 24 hours for 37 ~ 40 ℃;
2. prepare first order seed: with the Bacillus coagulans of activation, being inoculated in the 200 mL liquid seed culture mediums to the five rings with transfering loop picking three, triangular flask is placed shaking table, is under the condition of 37 ~ 40 ℃ and 250rpm in temperature, after the shaking culture 20 ~ 24 hours, make the first order seed bacterium;
3. prepare the solid fermentation substratum: the solid butt: be basestocks (Semen Maydis powder is 2:1 with the ratio of the quality of soybean cake powder), KH with Semen Maydis powder and soybean cake powder
2PO
40.3 ~ 0.5wt%, MnSO
4H
2O 0.02wt% and MgSO
47H
2O 0.125wt ‰; It is the sterilized water of 1:1 ~ 2 that the solid butt is added mass ratio, and is natural condition in pH value, stirs, and is that autoclave sterilization 20min namely can be used as the use of solid fermentation substratum after cooling under 121 ℃ the condition in temperature with solid medium;
4. solid fermentation: will insert the first order seed bacterium according to 8 ~ 10% inoculum size through the solid fermentation substratum of sterilising treatment, stir, keep humidity be 50 ~ 70% and temperature be under 37 ~ 40 ℃ the condition, to cultivate 20 ~ 24 hours, make secondary seed; With the secondary seed that obtains, obtain three grades of seeds according to the further solid fermentation of 8 ~ 10% inoculum size; Can be according to concrete cultivation consumption, further with three grades of seeds according to the further solid fermentation enlarged culturing of 8 ~ 10% inoculum size; The Bacillus coagulans culture is carried out enumeration, and the viable bacteria bulk concentration can reach 1.0 * 10
9More than the bright song of CFU/g, the gemma rate of formation is more than 80%.
2. accurately take by weighing the culture of yeast saccharomyces cerevisiae, subtilis, Bacillus licheniformis and Bacillus coagulans respectively, the mass ratio of four kinds of bacterium powder is followed successively by in the preparation: 40-60:10-30:10-30:10-30, mix, be the compound strain probiotics.
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Cited By (7)
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|---|---|---|---|---|
| CN105076708A (en) * | 2015-09-22 | 2015-11-25 | 河南科技大学 | Poultry yeast culture and preparation method thereof |
| CN105192365A (en) * | 2015-09-25 | 2015-12-30 | 河南宏翔生物科技有限公司 | Preparation method of yeast culture for pigs |
| CN105777234A (en) * | 2016-05-31 | 2016-07-20 | 沈阳市现代三川生物技术研究所 | Method for preparing biological organic fertilizer through common fermentation of nitrogen-fixing bacteria, phosphate solubilizing bacteria and potassium bacteria |
| CN107043725A (en) * | 2017-04-25 | 2017-08-15 | 西安鑫汉宝生物科技有限公司 | Method of bacillus subtilis and bacillus coagulans mixed fermentation and application thereof |
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN111955622A (en) * | 2020-07-10 | 2020-11-20 | 湖北华大瑞尔科技有限公司 | Compound feed additive and preparation method thereof |
| CN113693159A (en) * | 2021-08-03 | 2021-11-26 | 江苏京牧生物技术有限公司 | Mixed type micro-ecological feed additive |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076708A (en) * | 2015-09-22 | 2015-11-25 | 河南科技大学 | Poultry yeast culture and preparation method thereof |
| CN105192365A (en) * | 2015-09-25 | 2015-12-30 | 河南宏翔生物科技有限公司 | Preparation method of yeast culture for pigs |
| CN105777234A (en) * | 2016-05-31 | 2016-07-20 | 沈阳市现代三川生物技术研究所 | Method for preparing biological organic fertilizer through common fermentation of nitrogen-fixing bacteria, phosphate solubilizing bacteria and potassium bacteria |
| CN107043725A (en) * | 2017-04-25 | 2017-08-15 | 西安鑫汉宝生物科技有限公司 | Method of bacillus subtilis and bacillus coagulans mixed fermentation and application thereof |
| CN109468259A (en) * | 2018-10-24 | 2019-03-15 | 亚太星原农牧科技海安有限公司 | A kind of culture medium for promoting gemma to generate |
| CN109468259B (en) * | 2018-10-24 | 2022-04-05 | 亚太星原农牧科技海安有限公司 | Culture medium for promoting spore generation |
| CN111955622A (en) * | 2020-07-10 | 2020-11-20 | 湖北华大瑞尔科技有限公司 | Compound feed additive and preparation method thereof |
| CN111955622B (en) * | 2020-07-10 | 2023-01-24 | 湖北华大瑞尔科技有限公司 | Compound feed additive and preparation method thereof |
| CN113693159A (en) * | 2021-08-03 | 2021-11-26 | 江苏京牧生物技术有限公司 | Mixed type micro-ecological feed additive |
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