CN101715876B - Preparation method of high-efficiency compound microorganism soybean meal leaven - Google Patents

Preparation method of high-efficiency compound microorganism soybean meal leaven Download PDF

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CN101715876B
CN101715876B CN2009100589142A CN200910058914A CN101715876B CN 101715876 B CN101715876 B CN 101715876B CN 2009100589142 A CN2009100589142 A CN 2009100589142A CN 200910058914 A CN200910058914 A CN 200910058914A CN 101715876 B CN101715876 B CN 101715876B
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million cfu
culture medium
soybean meal
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CN101715876A (en
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冯建国
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Sichuan Kewang Biotechnology Co Ltd
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Abstract

The invention relates to a preparation method of a high-efficiency compound microorganism soybean meal leaven, belonging to the technical field of biological enzymolysis and fermentation. The preparation method comprises the following steps of: respectively carrying out optimal screening on lactobacillus acidophilus, lactobacillus lactis, saccharomyces cerevisiae, Candida utilis, bacillus subtilis and bacillus licheniformis; mixing and culturing by shaking a bottle; culturing by using a 10L fermentation tank; manufacturing a microorganism compound protective agent; spray drying in low temperature; preparing the soybean meal leaven and the like. The high-efficiency compound microorganism soybean meal leaven can efficiently inhibit the growth of mixed bacterium and eliminate or remarkably lower anti-nutritional factors and flatulent factors in soybean meal, enhance the protein utilization ratio and strengthen the immunity and has the excellent characteristics of acid resistance, salt resistance, temperature resistance and bile resistance. The invention has the advantages of simple preparation method, low cost, stable and reliable produced product quality and wide application range.

Description

The preparation method of high-efficiency compound microorganism soybean meal leaven
Technical field
The present invention relates to a kind of preparation method of microbe leaven, especially relate to the preparation method of high-efficiency compound microorganism soybean meal leaven, belong to the biological enzymolysis fermentation technical field.
Background technology
Dregs of beans is the accessory substance behind the soybean extract oil, and its crude protein content is up to 40%-50%, and is nutritious, with low cost, and protein and amino acid whose composition are reasonable, is widely used as the feed of animal.But contain a large amount of ANFs in the dregs of beans, not only influenced the utilization ratio of protein, and can cause the intestinal tract injury of growing animal, cause growing animal wean back diarrhoea, the health of appreciable impact animal and surviving.The way that solves is to add soybean meal leaven in dregs of beans, in order to the ANFs in elimination or the reduction dregs of beans.The soybean meal leaven that adopts mainly contains three kinds at present: the one, and the bean-dregs feed of last batch fermentation; The 2nd, before mould residue in the covering that uses during bean pulp fermentation; The 3rd, the fungal mycelium of high hot undue growth.The vigor inequality of these leavenings, poor growth, and often suffer other microbial contaminations the fermentation that is difficult to predict occurs, even causes all fermentation failures.Therefore, before fermentation, need dregs of beans is carried out high-temperature sterilization, use the lactic acid acidified substrate again, in case microbiological contamination; Usually also need bubbling air in the fermentation, cost is very high.
Summary of the invention
The objective of the invention is to overcome above-mentioned defective, a kind of preparation method of high-efficiency compound microorganism soybean meal leaven is provided.
The objective of the invention is to be achieved by following technical proposals:
A kind of preparation method of high-efficiency compound microorganism soybean meal leaven comprises the steps:
1) bacterial classification optimization screening:
Respectively lactobacillus acidophilus, lactobacillus lactis, saccharomyces cerevisiae, candida utili, Bacillus subtillis, bacillus licheniformis are carried out acidproof, the anti-bile salt of bacterial strain, anti-high-copper and heatproof domestication;
1. the acidproof domestication of bacterial strain: pH 3.0-3.5 filters out acidproof bacterial strain;
2. anti-cholic acid domestication: in culture medium, add the cholate of variable concentrations, bacterial strain is tamed;
3. anti-high-copper seed selection: in containing the MRS broth bouillon of 200-250mg/kg copper, grow;
4. heatproof domestication: the thalline culture after will activating is seeded in LB, the TB fluid nutrient medium, heats certain hour behind the cultivation certain hour in 40-60 ℃ of water-bath, continues subsequently culture is placed 37 ℃ of cultivations;
2) shake a bottle Mixed culture:
The culture medium that strain culturing adopts glucose or cornstarch, fish meal or dusty yeast, inorganic salts etc. to form carries out in shaking bottle;
1. lactobacillus acidophilus is cultivated: the MTB culture medium consist of glucose 80-90g/L, sucrose 8-10g/L, peptone 3-5g/L, dusty yeast 2-25g/L, natrium citricum 10-12g/L, KH 2PO 42.2-2.4g/L, K 2HPO 410-12g/L carries out deep fermentation under 26-37 ℃;
2. lactobacillus lactis is cultivated: the GTB culture medium consist of glucose 60-70g/L, beef extract 8-10g/L, dusty yeast 10-15g/L, MgSO 41.2-1.6g/L, NaCl 2.2-2.5g/L, KH 2PO 42.2-2.4g/L, K 2HPO 410-12g/L, initial pH value is 6-6.5, and cultivation temperature is 26-35 ℃, and inoculum concentration is 3%;
3. the Mixed culture of Bacillus acidi lactici: lactobacillus acidophilus after will cultivating and lactobacillus lactis are put into MTB culture medium Mixed culture, by regulating the proportioning of bacterial classification in the fermentation, adopt 35,37 ℃ of two sections cultivation temperature, incubation time 18-24 hour;
4. saccharomyces cerevisiae is cultivated: culture medium consists of casein peptone 12-15g/L, dusty yeast 12-15g/L, glucose 25-30g/L, KH 2PO 42.2-2.4g/L, K 2HPO 43H 2O 15-16.34g/L, cultivation temperature is 26-30 ℃;
5. candida utili is cultivated: culture medium consists of glucose 20-25g/L, dusty yeast 10-12g/L, (NH 4) 2HPO 42.5-3g/L, KH 2PO 42.2-2.4g/L, K 2HPO 43H 2O 15-16.34g/L;
6. Bacillus subtillis is cultivated: culture medium consists of dregs of beans 10-12g/L, corn flour 18-21g/L, fish meal 7-7.3g/L, MgSO 418-2.1g/L pH is 5.5-6.1, temperature is 26-35 ℃;
7. bacillus licheniformis is cultivated: culture medium consists of dregs of beans 8-10g/L, corn flour 20-25g/L, fish meal 6.5-7.0g/L, MgSO 42.2-2.5g/L pH is 4.6-5.1, temperature 26-33.5 ℃;
3) 10L fermentation tank culture:
The result who shakes the bottle cultivation is put into the 10L fermentation tank respectively amplify cultivation;
4) make the microorganism composite protectant:
Protective agent is made up of skimmed milk powder, cysteine, sodium glutamate, and its concentration is skimmed milk powder 80-85g/L, cysteine 0.04-0.05mol/L, sodium glutamate 25-30g/L; Protective agent is added in lactobacillus acidophilus and the lactobacillus lactis, after vacuum freeze drying, it is preserved under 4 ℃;
5) low temperature spray drying:
The low temperature spray drying temperature is 20-38 ℃;
6) preparation soybean meal leaven:
Lactobacillus acidophilus 3.0-4.0 hundred million CFU/g, lactobacillus lactis 2.0-3.0 hundred million CFU/g, bacillus subtilis 10.0-12.0 hundred million CFU/g, bacillus licheniformis 5.0-6.0 hundred million CFU/g, saccharomyces cerevisiae 3.0-3.5 hundred million CFU/g, candida utili 2.0-3.0 hundred million CFU/g are mixed and get final product, efficient soybean meal leaven total bacteria count 〉=2,500,000,000 CFU/g;
Be preferably: lactobacillus acidophilus 3.5 hundred million CFU/g, lactobacillus lactis 2.5 hundred million CFU/g, bacillus subtilis 11.0 hundred million CFU/g, bacillus licheniformis 5.5 hundred million CFU/g, saccharomyces cerevisiae 3.0 hundred million CFU/g, candida utili 2.5 hundred million CFU/g;
The preparation method of high-efficiency compound microorganism soybean meal leaven of the present invention compared with prior art has the following advantages:
1) science is chosen bacillus subtilis, bacillus licheniformis, lactobacillus acidophilus, lactobacillus lactis, six kinds of microorganisms of saccharomyces cerevisiae and candida utili, suppress varied bacteria growing when utilizing their comprehensive function to reach bean pulp fermentation, eliminate the dregs of beans ANFs, the flatulence factor, improve protein utilization rate, improve palatability, microorganism is synthesized multivitamin and the enzyme that helps growth of animal simultaneously, secondary metabolite etc., while is with the effect of probiotics, has the animal intestinal of improvement microecological balance, the stimulating immune system reaction, improve immunity, reduce cholesterol levels, reduce acidulant in the feed, effects such as antibiotic use;
2) every kind of microorganism is optimized screening and domestication, makes it to have the good characteristic of acidproof, salt tolerant, heatproof and anti-bile;
3) culture medium, the condition of culture to selected microorganism is optimized control, guarantees the high-density growth of each bacterial classification, reduced fermentation costs, and the vigor of leavening is even, and growth does not subject to other microbial contaminations fast;
4) in lactobacillus acidophilus and lactobacillus lactis, added protective agent; but and under the tolerable temperature of thalline, adopt spray technique that thalline is carried out drying; greatly reduce cost; verified the influence of condition of culture, harvest time, freezing rate, residual moisture and rehydration condition, guaranteed the activity of thalline in the product dry thalline survival rate.The bacterium powder of being grown is easy to packed and transported, is the developing direction of microorganism formulation.
The specific embodiment
Embodiment 1
The preparation method of high-efficiency compound microorganism soybean meal leaven of the present invention comprises the steps:
1) bacterial classification optimization screening:
Respectively lactobacillus acidophilus, lactobacillus lactis, saccharomyces cerevisiae, candida utili, Bacillus subtillis, bacillus licheniformis are carried out acidproof, the anti-bile salt of bacterial strain, anti-high-copper and heatproof domestication;
1. the acidproof domestication of bacterial strain: pH 3.5, filter out acidproof bacterial strain;
2. anti-cholic acid domestication: in culture medium, add the cholate of variable concentrations, bacterial strain is tamed;
3. anti-high-copper seed selection: in containing the MRS broth bouillon of 250mg/kg copper, grow;
4. heatproof domestication: the thalline culture after will activating is seeded in LB, the TB fluid nutrient medium, heats certain hour behind the cultivation certain hour in 40,45,50,55,60 ℃ of water-baths, continues subsequently culture is placed 37 ℃ of cultivations;
2) shake a bottle Mixed culture:
The culture medium that strain culturing adopts glucose or cornstarch, fish meal or dusty yeast, inorganic salts etc. to form carries out in shaking bottle;
1. lactobacillus acidophilus is cultivated: the MTB culture medium consist of glucose 90g/L, sucrose 10g/L, peptone 5g/L, dusty yeast 25g/L, natrium citricum 12g/L, KH 2PO 42.4g/L, K 2HPO 412g/L carries out deep fermentation under 37 ℃;
2. lactobacillus lactis is cultivated: the GTB culture medium consist of glucose 70g/L, beef extract 10g/L, dusty yeast 15g/L, MgSO 41.6g/L, NaCl 2.5g/L, KH 2PO 42.4g/L, K 2HPO 412g/L, initial pH value is 6.5, and cultivation temperature is 35 ℃, and inoculum concentration is 3%;
3. the Mixed culture of Bacillus acidi lactici: lactobacillus acidophilus after will cultivating and lactobacillus lactis are put into MTB culture medium Mixed culture, by regulating the proportioning of bacterial classification in the fermentation, adopt 35,37 ℃ of two sections cultivation temperature, incubation time 24 hours;
4. saccharomyces cerevisiae is cultivated: culture medium consists of casein peptone 15g/L, dusty yeast 15g/L, glucose 30g/L, KH 2PO 42.4g/L, K 2HPO 43H 2O 16.34g/L, cultivation temperature is 30 ℃;
5. candida utili is cultivated: culture medium consists of glucose 25g/L, dusty yeast 12g/L, (NH 4) 2HPO 43g/L, KH 2PO 42.4g/L, K 2HPO 43H 2O 16.34g/L;
6. Bacillus subtillis is cultivated: culture medium consists of dregs of beans 12g/L, corn flour 21g/L, fish meal 7.3g/L, MgSO 42.1g/L pH is 6.1, temperature is 35 ℃;
7. bacillus licheniformis is cultivated: culture medium consists of dregs of beans 10g/L, corn flour 25g/L, fish meal 7.0g/L, MgSO 42.5g/L pH is 5.1, temperature is 33.5 ℃;
3) 10L fermentation tank culture:
The result who shakes the bottle cultivation is put into the 10L fermentation tank respectively amplify cultivation;
4) make the microorganism composite protectant:
Protective agent is made up of skimmed milk powder, cysteine, sodium glutamate, and its concentration is skimmed milk powder 85g/L, cysteine 0.05mol/L, sodium glutamate 30g/L; Protective agent is added in lactobacillus acidophilus and the lactobacillus lactis, after vacuum freeze drying, it is preserved under 4 ℃;
5) low temperature spray drying:
The low temperature spray drying temperature is 26 ℃;
6) preparation soybean meal leaven:
Lactobacillus acidophilus 3.5 hundred million CFU/g, lactobacillus lactis 2.5 hundred million CFU/g, bacillus subtilis 11.0 hundred million CFU/g, bacillus licheniformis 5.5 hundred million CFU/g, saccharomyces cerevisiae 3.0 hundred million CFU/g, candida utili 2.50 hundred million CFU/g are mixed and get final product, and efficient soybean meal leaven total bacteria count is 2,800,000,000 CFU/g.

Claims (2)

1. the preparation method of a high-efficiency compound microorganism soybean meal leaven is characterized in that: comprise the steps:
1) bacterial classification optimization screening:
Respectively lactobacillus acidophilus, lactobacillus lactis, saccharomyces cerevisiae, candida utili, Bacillus subtillis, bacillus licheniformis are carried out acidproof, the anti-bile salt of bacterial strain, anti-high-copper and heatproof domestication;
1. the acidproof domestication of bacterial strain: pH3.0-3.5 filters out acidproof bacterial strain;
2. anti-cholic acid domestication: in culture medium, add the cholate of variable concentrations, bacterial strain is tamed;
3. anti-high-copper seed selection: in containing the MRS broth bouillon of 200-250mg/kg copper, grow;
4. heatproof domestication: the thalline culture after will activating is seeded in LB, the TB fluid nutrient medium, heats certain hour behind the cultivation certain hour in 40-60 ℃ of water-bath, continues subsequently culture is placed 37 ℃ of cultivations;
2) shake a bottle Mixed culture:
Strain culturing adopts and to mainly contain the culture medium that glucose or cornstarch, fish meal or dusty yeast, inorganic salts are formed, and carries out in shaking bottle;
1. lactobacillus acidophilus is cultivated: the MTB culture medium consist of glucose 80-90g/L, sucrose 8-10g/L, peptone 3-5g/L, dusty yeast 2-25g/L, natrium citricum 10-12g/L, KH 2PO 42.2-2.4g/L, K 2HPO 410-12g/L carries out deep fermentation under 26-37 ℃;
2. lactobacillus lactis is cultivated: the GTB culture medium consist of glucose 60-70g/L, beef extract 8-10g/L, dusty yeast 10-15g/L, MgSO 41.2-1.6g/L, NaCl 2.2-2.5g/L, KH 2PO 42.2-2.4g/L, K 2HPO 410-12g/L, initial pH value is 6-6.5, and cultivation temperature is 26-35 ℃, and inoculum concentration is 3%;
3. the Mixed culture of Bacillus acidi lactici: lactobacillus acidophilus after will cultivating and lactobacillus lactis are put into MTB culture medium Mixed culture, by regulating the proportioning of bacterial classification in the fermentation, adopt 35,37 ℃ of two sections cultivation temperature, incubation time 18-24 hour;
4. saccharomyces cerevisiae is cultivated: culture medium consists of casein peptone 12-15g/L, dusty yeast 12-15g/L, glucose 25-30g/L, KH 2PO 42.2-2.4g/L, K 2HPO 43H 2O 15-16.34g/L, cultivation temperature is 26-30 ℃;
5. candida utili is cultivated: culture medium consists of glucose 20-25g/L, dusty yeast 10-12g/L, (NH 4) 2HPO 42.5-3g/L, KH 2PO 42.2-2.4g/L, K 2HPO 43H 2O 15-16.34g/L;
6. Bacillus subtillis is cultivated: culture medium consists of dregs of beans 10-12g/L, corn flour 18-21g/L, fish meal 7-7.3g/L, MgSO 418-2.1g/L pH is 5.5-6.1, temperature is 26-35 ℃;
7. bacillus licheniformis is cultivated: culture medium consists of dregs of beans 8-10g/L, corn flour 20-25g/L, fish meal 6.5-7.0g/L, MgSO 42.2-2.5g/L pH is 4.6-5.1, temperature 26-33.5 ℃;
3) 10L fermentation tank culture:
The result who shakes the bottle cultivation is put into the 10L fermentation tank respectively amplify cultivation;
4) make the microorganism composite protectant:
Protective agent is made up of skimmed milk powder, cysteine, sodium glutamate, its concentration is skimmed milk powder 80-85g/L, cysteine 0.04-0.05mol/L, sodium glutamate 25-30g/L, protective agent is added in lactobacillus acidophilus and the lactobacillus lactis, after vacuum freeze drying, it is preserved under 4 ℃;
5) low temperature spray drying:
The low temperature spray drying temperature is 20-38 ℃;
6) preparation soybean meal leaven:
Lactobacillus acidophilus 3.0-4.0 hundred million CFU/g, lactobacillus lactis 2.0-3.0 hundred million CFU/g, bacillus subtilis 10.0-12.0 hundred million CFU/g, bacillus licheniformis 5.0-6.0 hundred million CFU/g, saccharomyces cerevisiae 3.0-3.5 hundred million CFU/g, candida utili 2.0-3.0 hundred million CFU/g are mixed and get final product; Efficient soybean meal leaven total bacteria count 〉=2,500,000,000 CFU/g;
2. the preparation method of a high-efficiency compound microorganism soybean meal leaven as claimed in claim 1 is characterized in that:
Described lactobacillus acidophilus is that 3.5 hundred million CFU/g, lactobacillus lactis are that 2.5 hundred million CFU/g, bacillus subtilis are that 11.0 hundred million CFU/g, bacillus licheniformis are that 5.5 hundred million CFU/g, saccharomyces cerevisiae are that 3.0 hundred million CFU/g, candida utili are 2.5 hundred million CFU/g.
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CN103202393A (en) * 2012-01-11 2013-07-17 四川省环亚生物科技有限公司 Preparation method of efficient composite micro-ecologic premix for animals
CN104970201A (en) * 2015-07-16 2015-10-14 宁波市江东农茹生物科技有限公司 Production method for fermented soybean meal rich in arginine and lysine

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