CN110511885B - Composite nitrogen source suitable for bacillus coagulans fermentation and use method thereof - Google Patents
Composite nitrogen source suitable for bacillus coagulans fermentation and use method thereof Download PDFInfo
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Abstract
The invention discloses a composite nitrogen source suitable for bacillus coagulans fermentation and a using method thereof, belonging to the field of microbial fermentation. The composite nitrogen source is formed by mixing sesame seed meal hydrolyzed protein, corn gluten hydrolyzed protein and shrimp shell hydrolyzed protein, is applied to the multistage fermentation of the bacillus coagulans in a multistage variable speed feeding mode, realizes the high-efficiency and high-speed fermentation of the bacillus coagulans, has easily obtained raw materials and low price, greatly reduces the fermentation cost of the bacillus coagulans, and is more suitable for the industrial production of the bacillus coagulans.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a composite nitrogen source capable of rapidly and efficiently fermenting bacillus coagulans and a using method thereof.
Background
Bacillus coagulans (Bacillus coagulans) is facultative anaerobe, can grow in aerobic and anaerobic environments, can adapt to hypoxic intestinal environment, has high tolerance to acid and bile, and can ferment to produce lactic acid. Therefore, the bacillus coagulans has wide application prospect in the aspects of food, medical treatment, livestock and poultry and the like, particularly in the aspect of improving intestinal balance.
A large amount of carbon-nitrogen sources and inorganic salts are needed for the growth of the bacillus coagulans, and common culture medium components comprise glucose, yeast extract powder, peptone, sodium chloride and the like, such as: patent CN104017751A, which uses peptone as main nitrogen source, and jointly cultures with lactobacillus plantarum and lactobacillus casei, the fermentation time of the bacterial liquid is 36h, and the viable count is 2.98 x 109CFU/g; patent CN109207406A, culturing 70h with peptone, yeast extract and beef extract as main nitrogen source, viable count 5 x 109CFU/mL. In patent CN103160455A, yeast extract and tryptone are used as main nitrogen sources, the fermentation time of a bacterial liquid is 32 hours, the semi-closed fermentation of a solid fermentation medium is 72 hours, and the viable count is 1 x 1010CFU/g. Patent CN101390571A, using fish meal, soybean peptone, etc. as main nitrogen source, fermenting for 36h, viable count 1 x 1010CFU/mL. Patent CN109609432A, using soybean peptone and yeast extract as main nitrogen source, fermenting in 50L fermentation tank by feeding glucose, the fermentation time of bacterial liquid is 44h, and viable count is 4 x 1010CFU/mL. CN108085265A, the fermentation medium is glucose, corn flour, soybean meal, sodium chloride, magnesium sulfate at 0.5m3Fermenting for 18h in the fermentation tank, the viable count is 4 x 1010CFU/mL. Patent CN104974966A, peptone and yeast powder are used as main nitrogen sources, and the nitrogen source is 50m3Fermenting in a fermentation tank for 48h, the viable count is 1.5 multiplied by 1010CFU/mL. Therefore, the cost of the fermentation culture medium in the existing fermentation of the bacillus coagulans is high, the fermentation period is long, the number of live bacteria is low, and the industrial fermentation and the popularization and application of the bacillus coagulans are restricted.
The sesame seed meal is a byproduct of oil extraction of sesame, and a product obtained by degreasing the oil by a solvent leaching method is called defatted sesame seed meal powder, and the main component of the defatted sesame seed meal is sesame protein. The sesame protein mainly comprises alpha-globulin, accounting for about 80 percent, and is dissolved by dilute alkali solution, and then the pH value is adjusted to precipitate so as to obtain sesame protein isolate; hydrolyzing the sesame protein isolate into soluble protein by using protease, concentrating the soluble protein in vacuum, and performing spray drying to prepare the sesame seed meal protein hydrolysate.
Corn gluten meal, also known as corn gluten meal, corn protein meal or zein, is a main byproduct of corn starch produced by a wet milling process, contains about 50-60% of protein, 15-20% of starch, 10% of moisture, 6-8% of lipid, a small amount of cellulose and other trace components, but the corn gluten meal contains about 40% of impurities, so the starch and cellulose impurities are removed by treatment of alpha-amylase and cellulose, then the protein is subjected to thermal denaturation treatment by heat denaturation and sulfite, the protein is hydrolyzed into soluble protein by protease, and the soluble protein is subjected to vacuum concentration and then spray drying to prepare the corn gluten hydrolyzed protein.
The shrimp shell hydrolyzed protein is prepared by using the shrimp shell as a raw material, hydrolyzing protein in the shrimp shell by adopting alkaline protease to obtain soluble protein, and performing vacuum concentration and spray drying on the soluble protein.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a composite nitrogen source suitable for bacillus coagulans fermentation and a using method thereof.
Through a great deal of experiments and continuous efforts, the inventor finally obtains a composite nitrogen source suitable for bacillus coagulans fermentation, wherein the composite nitrogen source is formed by mixing sesame seed meal hydrolyzed protein, corn gluten hydrolyzed protein and shrimp shell hydrolyzed protein, and the mass ratio of the sesame seed meal hydrolyzed protein to the corn gluten hydrolyzed protein to the shrimp shell hydrolyzed protein is 2:2: 1.
Preferably, the composite nitrogen source suitable for the fermentation of the bacillus coagulans is added in an amount of 3-5% of the tank volume.
The application method of the composite nitrogen source suitable for the fermentation of the bacillus coagulans is characterized in that the composite nitrogen source is applied to the fermentation of the bacillus coagulans by adopting a variable-speed feeding method.
Preferably, the method for using the composite nitrogen source suitable for the fermentation of bacillus coagulans as described above comprises the following steps: inoculating a seed solution of bacillus coagulans into a fermentation tank filled with bottom water, fermenting for 10-18h at the fermentation temperature of 45-55 ℃, and adding a carbon source, a nitrogen source and a sodium hydroxide solution in batches during fermentation, wherein the specific feeding method comprises the following steps:
fermenting for 0-4h, wherein the flow rate of the composite nitrogen source is 0.5-2% of the total flow rate per hour, and the rotating speed is 100-; fermenting for 4-10h, wherein the flow rate of the composite nitrogen source is 2-15% of the total flow rate per hour, and the rotation speed is 150-; fermenting for 10-16h, wherein the feeding speed of the composite nitrogen source is 0.5-2% of the total feeding volume per hour, the rotating speed is 150-; stopping feeding the nitrogen source in the final fermentation stage 16-18;
fermenting for 0-6h, wherein the feeding speed of the feeding carbon source is 2.5-6.25% of the total feeding volume per hour; fermenting for 6-10h, wherein the feeding speed of the feeding carbon source is 3.75-6.25% of the total feeding volume per hour; fermenting for 10-16h, wherein the feeding speed of the carbon source is 3.75-6.25% of the total volume per hour, 1-2mol/L sodium hydroxide solution is fed in the whole fermentation process, the flow acceleration is controlled, and the pH value is maintained at 5.5-6.5.
Preferably, the method for using the composite nitrogen source suitable for bacillus coagulans fermentation is as described above, and the preparation medium of the bacillus coagulans seed solution is as follows: 20g/L of glucose, 2.5g/L of yeast extract powder, 5g/L of peptone, 5g/L of beef extract, 12.8g/L of sesame seed meal hydrolyzed protein powder, 8.5g/L of corn gluten hydrolyzed protein powder, 3.7g/L of shrimp shell hydrolyzed protein powder, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and pH of 5.0-6.0.
Preferably, the method for using the composite nitrogen source suitable for bacillus coagulans fermentation is as described above, the bottom water in the fermentation tank consists of water, yeast powder, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate and manganese sulfate, and the pH is adjusted to 5.0-6.0.
Preferably, the method for using the composite nitrogen source suitable for Bacillus coagulans is as described above, the concentration of the fed composite nitrogen source is 90-150g/L, the yeast extract powder solution is 20-40g/L, and the carbon source is 400-500g/L glucose solution.
Compared with the prior art, the invention has the advantages that:
1. the composite nitrogen source consisting of the sesame seed meal hydrolyzed protein powder, the corn gluten hydrolyzed protein powder and the shrimp shell hydrolyzed protein powder is applied to the fermentation of the bacillus coagulans, the variable-speed fed-batch method is adopted, the fermentation period is obviously shortened, the fermentation period is only 10-18h in a 10L fermentation tank, and the number of the viable bacteria of the bacillus coagulans is as high as 1.5 x 1011CFU/mL; fermenting in 500L fermentation tank for 16-18h, and the viable count of Bacillus coagulans is 2.4 x 1011CFU/mL, the bacillus coagulans is rapidly and efficiently fermented.
2. The sesame seed meal hydrolyzed protein powder, the corn gluten hydrolyzed protein powder and the shrimp shell hydrolyzed protein powder used in the invention have low price, greatly reduce the fermentation cost of the bacillus coagulans, have easily available raw materials, and are more suitable for the industrial production of the bacillus coagulans.
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FIG. 1 is a graph showing the optimum amount of a complex nitrogen source to be added in fermentation of Bacillus coagulans.
Detailed Description
The foregoing aspects of the present invention are described in further detail below by way of examples, but it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above aspects of the present invention are within the scope of the present invention.
Example 1: preparation of bacillus coagulans by using optimal addition amount of composite nitrogen source
(1) Bacterial strains
Bacillus coagulans (Bacillus coagulans) IDCC1201, deposited in the laboratory of fermentation engineering A613 of the university of Industrial North Hubei.
(2) Preparation of the starting materials
Sesame seed meal: the method comprises the steps of firstly utilizing 1% of Tween 80 surfactant to carry out degreasing pretreatment on sesame seed meal for 1h, washing the sesame seed meal for 3 times at normal temperature, centrifuging to obtain precipitate for proteolysis, utilizing alkaline protease to hydrolyze the pretreated sesame seed meal, hydrolyzing for 4h at 55 ℃ in a material-water ratio of 1:10, carrying out vacuum concentration on soluble protein obtained by hydrolysis, and then carrying out spray drying to prepare sesame seed meal hydrolyzed protein powder, wherein the protein content is 82-87%, and the total nitrogen recovery rate of the sesame seed meal is more than 87%.
Corn gluten meal: the protein content in the raw material is 58.9 percent, the corn gluten meal is firstly crushed by a jet mill and then sieved by a 60-mesh sieve, the crushed and sieved corn gluten meal is added with separated water to be regulated into a certain concentration, and the corn gluten meal is firstly treated by cellulase: the temperature is 50 ℃, the pH value is 5.0, the enzyme concentration is 0.6 percent, the time is 3.0h, and the ratio of material to water is 1: 3. And then, treating by adopting alpha-amylase: the temperature is 65 ℃, the pH value is 6.5, the enzyme concentration is 0.8%, the time is 1.0h, the material-water ratio is 1:4, sodium sulfite is added for denaturation, the temperature is 45 ℃, the material-water ratio is 1:4, the time is 30min, the adding amount is 6%, after the concentration and the pH value are adjusted, papain is added for hydrolysis, the temperature is 60 ℃, the pH value is 6.5, the material-water ratio is 1:6, the time is 5h, the enzyme amount is 3%, then the hydrolysate is centrifuged, washed, the supernatant is collected, after vacuum concentration, spray drying is carried out, and the maize yellow powder hydrolyzed protein powder is prepared, wherein the protein content is 81-84%. The total nitrogen recovery rate of the corn gluten meal is 95.1 percent.
Shrimp shell hydrolyzed protein: the protein content of the shrimp shell raw material is 25-30%. Crushing the shrimp shell, adding water into the crushed shrimp shell, heating to 50 ℃, preheating for 30min, adding alkaline protease for hydrolysis at the temperature of 50 ℃ for 5h, wherein the ratio of the water to the material is 1:3, the pH value is 8.0, filtering after enzyme deactivation to obtain filtrate again, concentrating in vacuum, and performing spray drying to prepare the hydrolyzed protein powder of the shrimp shell, wherein the protein content of the hydrolyzed protein powder is 82-84%. The total nitrogen recovery of the crayfish shells was 66.92%.
(3) Preparation of the Medium
The strain activation liquid culture medium contains: 2% of glucose, 1% of yeast extract powder, 2% of peptone and 6.0-7.0 of pHs.
The liquid seed culture medium contains: 20g/L of glucose, 2.5g/L of yeast extract powder, 5g/L of peptone, 5g/L of beef extract, 12.8g/L of sesame seed meal hydrolyzed protein powder, 8.5g/L of corn gluten hydrolyzed protein powder, 3.7g/L of shrimp shell hydrolyzed protein powder, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 2g/L of dipotassium phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and pH 6.0-7.0.
(4) Optimum addition amount of composite nitrogen source
The composite nitrogen source is added into a 500mL triangular flask containing 100mL of liquid containing 2% (W/V) glucose according to the proportion of 1%, 2%, 3%, 4%, 5% and 6% (W/V), 1% (V/V) of activated bacillus coagulans seed bacteria liquid is inoculated, the pH value is adjusted to be 5.0-6.0, and after the bacillus coagulans seed bacteria liquid is cultured for 16-18h at the temperature of 45-55 ℃, the viable count of the bacillus coagulans is measured by a dilution coating plate method. The results are shown in FIG. 1. As can be seen from FIG. 1, when the addition amount is 3-5% (W/V), the viable count of Bacillus coagulans is high, and when the composite nitrogen source of 4% (W/V) is added, the viable count of Bacillus coagulans is highest and can reach 2.14 x 1010CFU/mL。
Example 2: bacillus coagulans is fermented on a 10L fermentation tank in a fed-batch mode
(1) The strain is as follows: the same as in example 1.
(2) Culture medium
The strain activation liquid culture was the same as in example 1.
The liquid seed medium was the same as in example 1.
The secondary seed culture medium contains: 2% of glucose, 5% of yeast extract powder, 1% of peptone, 1% of beef extract, 1.28% of sesame seed meal hydrolyzed protein powder, 0.85% of corn gluten hydrolyzed protein powder, 0.37% of shrimp shell hydrolyzed protein powder, 0.2% of diammonium hydrogen citrate, 0.5% of sodium acetate, 0.2% of dipotassium hydrogen phosphate, 0.058% of magnesium sulfate, 0.025% of manganese sulfate and pH 5.0-6.0.
The bottom water in the horizontal fermentation of 10L fermentation tank contains: 1.5L of water, 15g of yeast powder, 1.2g of dipotassium phosphate, 0.2g of magnesium sulfate heptahydrate and 0.375g of manganese sulfate, and adjusting the pH value to 5.0-6.0.
Preparation of fed-batch nitrogen source: the volume of the nitrogen source added in the flow is 3L, and the nitrogen source comprises 2L of 250g of compound nitrogen source and 1L of 30g of yeast extract, and the mixture is sterilized for 20min at 115 ℃.
Preparation of a fed-batch carbon source: the volume of the carbon source fed-batch was 1.6L, the component was 750g glucose, and the mixture was sterilized at 115 ℃ for 20 min.
Preparation of fed-batch alkali solution: the volume of the fed-batch alkaline solution is 500mL, the component is sodium hydroxide with the concentration of 1.5mol/L, and the solution is sterilized for 20min at 115 ℃.
(3) Fermentation culture
Multi-stage fed-batch fermentation: inoculating the Bacillus coagulans seed liquid prepared from the secondary seed culture medium into a 10L full-automatic fermentation tank filled with 1.5L bottom water according to 3% (V/V) of the tank volume, wherein the fermentation temperature is 45-55 ℃, and the fermentation period is 14-18 h. In the early stage of fermentation, the composite nitrogen source flow is accelerated for 0-4 h: 10mL/h-40mL/h, and the rotation speed is 100-150 r/min; in the middle stage of fermentation for 4-10h, the compound nitrogen source flow addition rate is as follows: 40mL/h-300mL/h, and the rotation speed is 150-200 r/min; in the later stage of fermentation for 10-16h, the composite nitrogen source flow acceleration rate is as follows: 40-100 mL/h, and simultaneously feeding yeast extract powder: 80mL/h-160mL/h, the rotating speed is 150-; stopping feeding the nitrogen source in the last fermentation stage for 16-18 h.
Fermenting for 0-6h, wherein the carbon source flow acceleration rate is 40mL/h-100 mL/h; the fermentation medium stage is 6-10h, and the carbon source flow acceleration rate is 60mL/h-100 mL/h; the later stage of fermentation is 10-16h, and the carbon source flow acceleration rate is 60mL/h-100 mL/h. Adding 1-2mol/L sodium hydroxide solution during the whole fermentation process, controlling the flow acceleration, and maintaining the pH at 5.0-7.0.
(4) And (3) measuring the viable count: the dilution coating plate method is used.
(5) As a result:
fermentation time: for 18 hours.
Viable count: 1.5*1011CFU/mL。
Example 3: bacillus coagulans is fermented on a 500L fermentation tank in a fed-batch mode
(1) The strain is as follows: the same as in example 1.
(2) Culture medium
The strain activating liquid medium was the same as in example 1.
The liquid seed medium was the same as in example 1.
The secondary seed medium was the same as in example 1.
Preparation of fed-batch nitrogen source: the volume of the nitrogen source fed-batch was 150L, including 100L of a nitrogen source containing 12kg of a complex nitrogen source and 50L of a nitrogen source containing 2kg of a yeast extract, and sterilized at 115 ℃ for 20 min.
Preparation of a fed-batch carbon source: the volume of the carbon source added is 80L, the component is glucose 40kg, and the mixture is sterilized at 115 ℃ for 20 min.
Preparation of fed-batch alkali solution: adding aqueous alkali 300L, sodium hydroxide as component, 2mol/L, sterilizing at 115 deg.C for 20 min.
(3) Fermentation culture
Multi-stage fed-batch fermentation: inoculating the Bacillus coagulans prepared in the secondary seed culture medium into a 500L full-automatic fermentation tank filled with 75L bottom water according to 3% (V/V) of the tank volume, wherein the fermentation temperature is 45-55 ℃, and the fermentation period is 14-18 h. In the early stage of fermentation, the composite nitrogen source flow is accelerated for 0-4 h: 0.5L-2L/h, the rotation speed is 100-; in the middle stage of fermentation for 4-10h, the composite nitrogen source flow acceleration rate is as follows: 2L-15L/h, the rotation speed is 150-; in the later stage of fermentation for 10-16h, the composite nitrogen source flow acceleration rate is as follows: 2L-5L/h, simultaneously adding yeast extract powder: 4L-8L/h, the rotating speed is 150 plus of 200r/min, and the dissolved oxygen content is controlled at 30 percent; stopping feeding the nitrogen source in the last fermentation stage for 16-18 h.
Fermenting for 0-6h, wherein the carbon source flow acceleration rate is 2L-5L/h; the fermentation medium stage is 6-10h, and the carbon source flow acceleration rate is 3L-5L/h; the fermentation later stage is 10-16h, and the carbon source flow acceleration rate is 3L-5L/h. 2mol/L sodium hydroxide solution is added in the whole fermentation process, the flow acceleration is controlled, and the pH value is maintained at 5.0-7.0.
(4) And (3) measuring the viable count: the dilution coating plate method is used.
(5) As a result:
fermentation time: for 18 hours.
Viable count: 2.4*1011CFU/mL。
Comparative example 1
The method is the same as example 2, but the complex nitrogen source fed during the fermentation process is only lack of shrimp shell hydrolyzed protein compared with example 2, and the number of viable bacteria is measured after the fermentation is finished. The result is:
fermentation time: for 44 hours.
Viable count: 2.1*108CFU/mL。
Comparative example 2
The implementation method is the same as that of example 2, but compared with example 2, the complex nitrogen source fed during the fermentation process only lacks the sesame seed meal hydrolyzed protein, and the number of viable bacteria is measured after the fermentation is finished. The result is:
fermentation time: for 36 hours.
Viable count: 4.5*108CFU/mL。
Comparative example 3
The implementation method is the same as that of example 2, but the composite nitrogen source fed-batch in the fermentation process is formed by mixing sesame seed meal hydrolyzed protein, corn gluten hydrolyzed protein and shrimp shell hydrolyzed protein, the mass ratio of the sesame seed meal hydrolyzed protein, the corn gluten hydrolyzed protein and the shrimp shell hydrolyzed protein is 1:1:1, and the number of live bacteria is measured after the fermentation is finished. The result is:
fermentation time: for 44 hours.
Viable count: 3.4*109CFU/mL。
Comparative example 4
The implementation method is the same as that of example 2, but the compound nitrogen source fed-batch in the fermentation process is formed by mixing sesame seed meal hydrolyzed protein, corn gluten hydrolyzed protein and yeast extract, the mass ratio of the sesame seed meal hydrolyzed protein, the corn gluten hydrolyzed protein and the yeast extract is 2:2:1, and the number of viable bacteria is measured after the fermentation is finished. The result is:
fermentation time: for 28 hours.
Viable count: 1.2*1010CFU/mL。
Claims (7)
1. A composite nitrogen source suitable for bacillus coagulans fermentation is characterized in that: the composite nitrogen source is formed by mixing sesame seed meal hydrolyzed protein, corn gluten hydrolyzed protein and shrimp shell hydrolyzed protein, wherein the mass ratio of the sesame seed meal hydrolyzed protein to the corn gluten hydrolyzed protein to the shrimp shell hydrolyzed protein is 2:2: 1; the preparation method of the sesame seed meal hydrolyzed protein comprises the following steps: degreasing and pretreating sesame seed meal by using 1% of tween 80, washing the sesame seed meal for 3 times after pretreating for 1h, centrifuging the sesame seed meal, taking precipitate, adding alkaline protease to hydrolyze the precipitate for 4h at the temperature of 55 ℃ and the material-water ratio of 1:10, concentrating soluble protein obtained by hydrolysis in vacuum, and performing spray drying to prepare sesame seed meal protein hydrolysate powder; the preparation method of the maize yellow powder hydrolyzed protein comprises the following steps: 1) adding deionized water into pulverized and sieved zeaxanthin powder, and treating with cellulase at 50 deg.C, pH5.0, enzyme concentration 0.6% for 3.0h, with a material-water ratio of 1: 3; 2) adding alpha-amylase into the solution treated in the step 1), treating at 65 ℃, pH6.5, enzyme concentration 0.8% for 1.0h and material-water ratio of 1:4, adding sodium sulfite for denaturation after enzymolysis, at 45 ℃, material-water ratio of 1:4, time 30min and adding amount 6%; 3) adding papain into the solution treated in the step 2) for hydrolysis, at the temperature of 60 ℃, with the pH value of 6.5 and the material-water ratio of 1:6, for 5 hours, adding 3 percent of enzyme, then centrifuging the hydrolysate, washing, collecting supernatant, concentrating in vacuum, and then adopting spray drying to prepare maize yellow powder hydrolyzed protein powder; the preparation method of the shrimp shell hydrolyzed protein comprises the following steps: crushing shrimp shells, adding water, heating to 50 ℃, preheating for 30min, adding alkaline protease for hydrolysis at 50 ℃ for 5 hours at a material-water ratio of 1:3 and a pH value of 8.0, inactivating enzyme, filtering to obtain filtrate, concentrating in vacuum, and spray-drying.
2. The complex nitrogen source suitable for fermentation by bacillus coagulans as claimed in claim 1, wherein: the addition amount of the composite nitrogen source is 3-5% of the volume of the tank.
3. The method of claim 1, wherein the method comprises the steps of: the composite nitrogen source is applied to the multistage fermentation of the bacillus coagulans by adopting a variable-speed feeding method.
4. The method of claim 3, wherein the method comprises the steps of: inoculating a seed solution of bacillus coagulans into a fermentation tank filled with bottom water, fermenting for 10-18h at the fermentation temperature of 45-55 ℃, and adding a carbon source, a nitrogen source and a sodium hydroxide solution in batches during fermentation, wherein the specific feeding method comprises the following steps: fermenting for 0-4h, wherein the flow rate of the composite nitrogen source is 0.5-2% of the total flow rate per hour, and the rotating speed is 100-; fermenting for 4-10h, wherein the flow rate of the composite nitrogen source is 2-15% of the total flow rate per hour, and the rotation speed is 150-; fermenting for 10-16h, wherein the feeding speed of the composite nitrogen source is 2.0-5% of the total feeding volume per hour, the rotating speed is 150-; stopping feeding the nitrogen source in the last fermentation stage for 16-18 h; fermenting for 0-6h, wherein the feeding speed of the feeding carbon source is 2.5-6.25% of the total feeding volume per hour; fermenting for 6-10h, wherein the feeding speed of the feeding carbon source is 3.75-6.25% of the total feeding volume per hour; fermenting for 10-16h, wherein the feeding speed of the feeding carbon source is 3.75-6.25% of the total volume of the feeding carbon source per hour, and 1-2mol/L sodium hydroxide solution is fed during the whole fermentation process, controlling the flow acceleration, and maintaining the pH value at 5.5-6.5.
5. The method of claim 4, wherein the method comprises the steps of: the preparation culture medium of the bacillus coagulans seed solution comprises the following components: 20g/L of glucose, 2.5g/L of yeast extract powder, 5g/L of peptone, 5g/L of beef extract, 12.8g/L of sesame seed meal hydrolyzed protein powder, 8.5g/L of corn gluten hydrolyzed protein powder, 3.7g/L of shrimp shell hydrolyzed protein powder, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 2g/L of dipotassium hydrogen phosphate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate and pH of 5.0-6.0.
6. The method of claim 4, wherein the method comprises the steps of: the bottom water of the fermentation tank consists of water, yeast powder, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate and manganese sulfate, and the pH value is adjusted to 5.0-6.0.
7. The method of claim 4, wherein the method comprises the steps of: the concentration of the fed-batch composite nitrogen source is 90-150g/L, the concentration of the yeast extract powder solution is 20-40g/L, and the concentration of the fed-batch carbon source is 400-500g/L glucose solution.
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| CN110511885A (en) | 2019-11-29 |
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