CN115261260A - Fermentation method for improving spore number and stability of bacillus coagulans and preparation method of bacterial powder - Google Patents
Fermentation method for improving spore number and stability of bacillus coagulans and preparation method of bacterial powder Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims abstract description 169
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- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 67
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- 238000002360 preparation method Methods 0.000 title abstract description 18
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 32
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- 238000011081 inoculation Methods 0.000 claims description 8
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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Abstract
The invention discloses a fermentation method for improving the spore number and stability of bacillus coagulans and a bacterium powder preparation method, and belongs to the technical field of microbial fermentation engineering. The fermentation method for improving the spore number and stability of the bacillus coagulans comprises the following steps: fermenting in a seeding tank and feeding in batches; it is characterized in that DO in the fermentation process of the seeding tank is maintained to be more than 30 percent; the fed-batch fermentation comprises a first stage fermentation and a second stage fermentation; the DO in the first stage fermentation process is maintained to be more than 30 percent, and the feeding is started when the DO rises back; the DO is reduced and maintained by 10-20% in the second stage of fermentation; the DO rise-back means that DO rises by 10% or more within 1 min. The invention also provides a preparation method of the bacillus coagulans powder with high spore number based on the fermentation method.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a fermentation method for improving the spore number and stability of bacillus coagulans and a bacterium powder preparation method thereof.
Background
Probiotics are active microorganisms beneficial to the human body. Lactic acid bacteria, as the most important probiotic, have the effects of improving intestinal function, regulating the balance of intestinal flora and improving the health level of organisms, and are widely applied to the industries of food, medical treatment, health care, animal husbandry, aquatic products and the like. However, the stress resistance of the lactic acid bacteria to the environment is weak, and the defects of easy inactivation, short shelf life and the like of the live lactic acid bacteria preparation generally exist, so that the industrial development of the live lactic acid bacteria preparation is greatly restricted.
The bacillus coagulans is a special probiotic bacterium capable of forming spores, and is the only bacillus lactis in edible strain list. The lactobacillus milk powder has the beneficial functions of maintaining intestinal microecological balance, stimulating immunity, improving the health level of organisms, promoting digestion and absorption and the like of lactobacillus, and has the biological characteristics of strong environmental stress resistance, high temperature and high pressure resistance, acid and alkali resistance and drying resistance due to the formation of spores. This makes it an object of the probiotic market, particularly for applications in the food field.
The research on the bacillus coagulans in China is relatively late, and after the bacillus coagulans is listed in the edible strain list in 2016, the industrialization process of the bacillus coagulans in the food field is formally started in China. Especially in the food field, until now, the raw materials imported from foreign countries are mainly relied on. The dependence on import is not only limited by a plurality of factors and high in production cost, but also is not beneficial to the rapid development of related industries. And the bacillus coagulans products on the current market generally have the problems of low spore number, poor stability and the like. Therefore, the low-cost preparation process of the bacillus coagulans is a precondition and key for large-scale application of the bacillus coagulans. Currently, the research of bacillus coagulans mainly comprises a solid fermentation process and a liquid fermentation process. The solid fermentation process is easy to pollute, has considerable restriction on large-scale production, and is difficult to meet the requirements of food production. The method for preparing the bacillus coagulans by the liquid fermentation process has simple process, but low spore formation rate, high cost and short storage period.
Disclosure of Invention
Aiming at the problems in the project technology, the invention provides a bacillus coagulans and a preparation method of high spore count bacterial powder thereof. The bacillus coagulans is newly developed, has the characteristics of high density and high yield, is favorable for reducing the product cost, has high spore number and high stability, is favorable for product application, and has very important significance for promoting the application of the bacillus coagulans in the field of food.
The technical scheme of the invention is as follows:
a fermentation method for improving the spore number and stability of Bacillus coagulans comprises the following steps: fermenting in a seeding tank and fermenting in fed batch; it is characterized in that DO in the fermentation process of the seeding tank is maintained to be more than 30 percent; the fed-batch fermentation comprises a first stage fermentation and a second stage fermentation; the DO in the first stage fermentation process is maintained to be more than 30 percent, and the feeding is started when the DO rises back; the DO is reduced and maintained by 10-20% in the second stage of fermentation; the DO rise-back means that DO rises by 10% or more within 1 min.
Fermentation products (e.g., fungal meal) of bacterial species with high spore count and high survival rate can be obtained using a fermentation process characterized by the control of DO levels in each of the fermentation stages described above, wherein reduction of DO levels by the second stage fermentation to 10-20% is effective in promoting sporulation.
The starting material supplement refers to adding a supplement culture medium;
preferably, the feed medium comprises the following components in percentage by mass: 5-6% of glucose, 1.5-2% of yeast powder, 0.5-1% of ammonium sulfate and the balance of water.
The conditions for fermentation in the seed tank comprise: the temperature is 30-45 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5-7, and the time is 6-12h;
preferably, the conditions for the fermentation in the seed tank include: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5.5-6.0, and the time is 6-8h;
the culture medium for fermentation in the seeding tank comprises: 5-25g/L of peptone, 5-25g/L of beef extract powder, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium hydrogen phosphate and 0.2-1g/L of magnesium sulfate;
preferably, the medium of the seedpot fermentation comprises: 10g/L of peptone, 5g/L of beef extract powder, 3g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium hydrogen phosphate and 0.5g/L of magnesium sulfate;
the conditions of the first stage fermentation of the fed-batch fermentation include: the inoculum size of the seeding tank fermentation product is 2-10%, the culture temperature is 30-45 deg.C, pH is 5-7, the rotation speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, and the tank pressure is 0.03-0.05Mpa;
preferably, the conditions of the first stage fermentation include: the inoculum size of the seeding tank fermentation product is 5%, the culture temperature is 37-42 ℃, the pH is 5.5-6.0, the rotation speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, and the tank pressure is 0.05Mpa;
the culture medium for the first stage fermentation comprises: 5-25g/L of peptone, 5-25g/L of beef extract, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium phosphate, 0.2-1g/L of magnesium sulfate, 0.2-1g/L of manganese sulfate, 0.2-1g/L of calcium chloride and 0.05-0.2% of defoamer;
preferably, the medium of the first stage fermentation comprises: 15g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 4g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 0.3g/L of calcium chloride and 0.05% of defoaming agent.
The conditions of the second stage fermentation of the fed-batch fermentation include: culturing at 30-45 deg.C, pH5.5-8.5, rotation speed of 50-100rpm, ventilation amount of 0.3-0.6vvm, tank pressure of 0.03-0.05Mpa, DO maintaining 10-20%, and time of 12-24 hr;
preferably, the conditions of the second stage fermentation of the fed-batch fermentation comprise: culturing at 37-42 deg.C, pH7.5-8.5, rotation speed of 50-100rpm, ventilation volume of 0.3-0.6vvm, pot pressure of 0.05Mpa, DO maintaining 10-20%, and time of 16h;
preferably, the temperature is raised to 60-75 ℃ after the end of the second stage of the fed-batch fermentation, maintained for 10-30min and then lowered to 25 ℃.
The function of this step is: the temperature is raised for a short time to accelerate the maturation of spores and enhance the heat resistance of the spores; simultaneously, the thallus which does not form spores is inactivated, and the number of the spores of the bacterial powder is further increased.
The conditions of pH and DO level reduction in the second stage fermentation are maintained at 10-20% to maximize spore formation.
The fermentation method for improving the spore number and the survival rate of the bacillus coagulans further comprises the following steps: rejuvenating and activating the strain before fermenting in a seeding tank;
preferably, the strain rejuvenation comprises: coating the frozen strain preservation solution on a solid culture medium after heat treatment;
preferably, the strain rejuvenation comprises: selecting a single colony on the solid culture medium coated with the strain preservation solution, carrying out heat treatment on the single colony, and coating the solid culture medium for the second time;
preferably, the solid medium refers to nutrient agar medium;
preferably, the strain activation comprises: inoculating single colony of the rejuvenated strain to nutrient agar culture medium, and culturing at 30-45 deg.C for 18-36 hr;
preferably, the nutrient agar medium comprises: 2g/L of glucose, 5g/L of yeast extract powder, 10g/L of peptone and 15-20g/L of agar, and the pH value is 7.0-7.2.
The fermentation method for improving the spore number and the survival rate of the bacillus coagulans further comprises the following steps: performing shake flask culture after rejuvenation and activation of the strain and before seed tank fermentation;
preferably, the shake flask culture comprises: eluting and inoculating the activated strain on the nutrient agar culture medium into a shake flask culture medium for shake culture;
preferably, the shake flask culture medium comprises: 10g/L of peptone, 5g/L of beef extract, 5g/L of yeast powder, 10g/L of glucose and 5g/L of sodium chloride;
preferably, the conditions of the shaking culture include: shaking at a rotation speed of 200r/min at 30-45 deg.C for 8-12h;
preferably, the strain is a bacillus coagulans strain; the bacillus coagulans strain is selected from the group consisting of: bacillus coagulans strain BC99, BC10, BC 3702.
The fermentation of the seed tank comprises primary seed tank fermentation and/or secondary seed tank fermentation;
preferably, the inoculation amount of the primary seed tank fermentation is 5%, and the fermentation conditions comprise: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 8h;
preferably, the conditions for the secondary seedtank fermentation include: the temperature is 37-42 ℃, the rotating speed is 150-300rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 6h;
preferably, after the fermentation in the primary seed tank is finished, the fermentation liquid in the primary seed tank is transferred to the secondary seed tank for fermentation in the secondary seed tank.
A method for preparing bacterial powder for improving the spore number and survival rate of Bacillus coagulans, which comprises fermenting the Bacillus coagulans by the bacterial fermentation method of any one of claims 1 to 7.
The preparation method of the high-survival-rate bacterial powder further comprises the following steps: and (3) separating fermentation liquor after the fed-batch fermentation is finished, collecting bacterial sludge, and obtaining bacterial powder by adopting a spray drying mode.
Preferably, the isolated fermentation broth refers to: separating the fermentation liquor by a butterfly centrifuge;
preferably, the air inlet temperature of the spray drying is 180-220 ℃, and the air outlet temperature is 90-100 ℃;
preferably, the bacillus coagulans is selected from the group consisting of: bacillus coagulans strain BC99, BC10, BC 3702.
The bacillus coagulans BC99 is preserved in the China general microbiological culture Collection center (CGMCC), and the microorganism preservation number is CGMCC No.21801; the classification is named as: bacillus coagulans Bacillus coagulons; preservation time: year 2021, month 02, day 01.
The invention has the beneficial technical effects that:
1. different control modes are set according to different environmental requirements of the growth stage and the spore formation stage of the bacillus coagulans BC 99. In the growth stage of the thalli, the logarithmic growth phase is prolonged by a feeding mode, the inhibition of a high-concentration substrate on the growth of the thalli is removed, the density of the thalli can be rapidly improved in a short period, and meanwhile, the feeding time is judged by DO, so that the method is convenient and accurate, and meanwhile, the proper pH and DO are controlled, the optimal growth condition is provided for the growth of the thalli, and the multiplication of the thalli is not influenced. In a spore formation stage, the pH is increased, the DO is reduced, a severe environment is provided, the spore formation is promoted, the spore formation rate is obviously improved, the heat resistance of spores is enhanced in a heating and heat shock mode after the fermentation is finished, and the stability of bacterial powder is improved.
2. The invention obtains the preparation method of the bacillus coagulans powder with high spore number by strain rejuvenation, culture medium, fermentation process and post-treatment optimization. The preparation process is simple, the number of spores is high, the bacillus coagulans can be stored for a long time, the survival rate can still be kept high, the stability is good, the production cost is obviously reduced, and the popularization of bacillus coagulans products is facilitated.
Detailed Description
The following detailed description of the present invention is provided in connection with specific examples, which should not be construed as limiting the scope of the present invention.
Sources of biological materials
The bacillus coagulans BC99 strain is from Wuhan Weikang probiotic research institute Limited company, is the strain described in the patent application 202110285874.6, and has the preservation number of CGMCC No.21801;
the BC10 strain is the BC99 strain described in the invention patent application 202010538017.8;
the BC3702 strain is 3702 strain described in patent application 202110285874.6;
group 1 example, fermentation Process of the invention
The present group of embodiments provides a fermentation method for increasing the number of spores and the survival rate of bacillus coagulans. All embodiments of this group share the following common features: the fermentation method for improving the spore number and the survival rate of the bacillus coagulans comprises the following steps: fermenting in a seeding tank and fermenting in fed batch; it is characterized in that DO in the fermentation process of the seeding tank is maintained to be more than 30 percent; the fed-batch fermentation comprises a first stage fermentation and a second stage fermentation; the DO in the first stage fermentation process is maintained to be more than 30 percent, and the feeding is started when the DO rises back; the DO is reduced and maintained by 10-20% in the second stage of fermentation; the DO rise-back means that DO rises by 10% or more within 1 min.
Fermentation products (e.g., fungal meal) of the bacterial species with high spore count and high survival rate can be obtained by a fermentation process characterized by the control of DO levels at each of the fermentation stages described above, wherein the reduction of DO by the second stage fermentation to 10-20% is effective in promoting sporulation.
In some embodiments, the start-up feeding refers to the addition of a feed medium;
preferably, the feed medium comprises the following components in percentage by mass: 5-6% of glucose, 1.5-2% of yeast powder, 0.5-1% of ammonium sulfate and the balance of water.
In other embodiments, the conditions of the seedtank fermentation include: the temperature is 30-45 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5-7, and the time is 6-12h;
preferably, the conditions for the fermentation in the seed tank include: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5.5-6.0, and the time is 6-8h;
the culture medium for fermentation in the seeding tank comprises: 5-25g/L of peptone, 5-25g/L of beef extract, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium phosphate and 0.2-1g/L of magnesium sulfate;
preferably, the medium for the fermentation in the seed tank comprises: 10g/L of peptone, 5g/L of beef extract powder, 3g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium hydrogen phosphate and 0.5g/L of magnesium sulfate;
in specific embodiments, the conditions of the first stage fermentation of the fed-batch fermentation include: the inoculum size of the seeding tank fermentation product is 2-10%, the culture temperature is 30-45 deg.C, pH is 5-7, the rotation speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, and the tank pressure is 0.03-0.05Mpa;
preferably, the conditions of the first stage fermentation include: the inoculum size of the seeding tank fermentation product is 5%, the culture temperature is 37-42 ℃, the pH is 5.5-6.0, the rotation speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, and the tank pressure is 0.05Mpa;
the culture medium for the first stage fermentation comprises: 5-25g/L of peptone, 5-25g/L of beef extract, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium phosphate, 0.2-1g/L of magnesium sulfate, 0.2-1g/L of manganese sulfate, 0.2-1g/L of calcium chloride and 0.05-0.2% of defoamer;
preferably, the medium of the first stage fermentation comprises: 15g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 4g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 0.3g/L of calcium chloride and 0.05% of defoaming agent.
In some embodiments, the conditions of the second stage fermentation of the fed-batch fermentation comprise: culturing at 30-45 deg.C, pH5.5-8.5, rotation speed of 50-100rpm, ventilation amount of 0.3-0.6vvm, tank pressure of 0.03-0.05Mpa, DO maintaining 10-20%, and time of 12-24 hr;
preferably, the conditions of the second stage fermentation of the fed-batch fermentation comprise: culturing at 37-42 deg.C, pH7.5-8.5, rotation speed of 50-100rpm, ventilation amount of 0.3-0.6vvm, tank pressure of 0.05Mpa, DO maintenance of 10-20%, and time of 16h;
preferably, the temperature is raised to 60-75 ℃ after the second stage fermentation of the fed-batch fermentation is finished, maintained for 10-30min and then cooled to 25 ℃.
The function of this step is: the temperature is raised for a short time to accelerate the maturation of spores and enhance the heat resistance of the spores; simultaneously, the thallus which does not form spores is inactivated, and the number of the spores of the bacterial powder is further increased.
The conditions of pH and DO level reduction in the second stage fermentation are maintained at 10-20% to maximize spore formation.
In a further embodiment, the fermentation method for increasing the spore number and survival rate of bacillus coagulans further comprises: rejuvenating and activating the strain before fermenting in a seeding tank;
preferably, the strain rejuvenation comprises: coating the frozen strain preservation solution on a solid culture medium after heat treatment;
preferably, the strain rejuvenation comprises: picking the single colony on the solid culture medium coated with the strain preservation solution, performing heat treatment again, and coating the solid culture medium for the second time;
preferably, the solid medium refers to a nutrient agar medium;
preferably, the strain activation comprises: inoculating single colony of the rejuvenated strain to nutrient agar culture medium, and culturing at 30-45 deg.C for 18-36 hr;
preferably, the nutrient agar medium comprises: 2g/L of glucose, 5g/L of yeast extract powder, 10g/L of peptone and 15-20g/L of agar, and the pH value is 7.0-7.2.
In a further embodiment, the fermentation method for increasing the number of spores and the survival rate of bacillus coagulans is characterized by further comprising: performing shake flask culture after rejuvenation and activation of the strain and before seed tank fermentation;
preferably, the shake flask culture comprises: eluting and inoculating the activated strain on the nutrient agar culture medium into a shake flask culture medium for shake culture;
preferably, the shake flask culture medium comprises: 10g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 10g/L of glucose and 5g/L of sodium chloride;
preferably, the conditions of the shaking culture include: shaking at a rotation speed of 200r/min at 30-45 deg.C for 8-12h;
preferably, the strain is a bacillus coagulans strain; the bacillus coagulans strain is selected from the group consisting of: bacillus coagulans BC99 strain, BC10 strain, BC3702 strain.
In some embodiments, the seedtank fermentation comprises a primary seedtank fermentation and a secondary seedtank fermentation;
preferably, the inoculation amount of the primary seed tank fermentation is 5%, and the fermentation conditions comprise: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 8h;
preferably, the conditions for the secondary seedtank fermentation include: the temperature is 37-42 ℃, the rotating speed is 150-300rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 6h;
preferably, after the fermentation in the primary seed tank is finished, the fermentation liquid in the primary seed tank is transferred to the secondary seed tank for fermentation in the secondary seed tank.
Group 2 example, preparation of fungal powder of the invention
The embodiment of the group provides a preparation method of bacterial powder for improving the spore number and the survival rate of bacillus coagulans. All embodiments of this group share the following common features: the preparation method of the bacterial powder for improving the spore number and the survival rate of the bacillus coagulans comprises the step of fermenting the bacillus coagulans by using the bacterial fermentation method provided by any one of group 1 embodiments.
In a further embodiment, the method for preparing the bacterial powder with high survival rate further comprises the following steps: and (3) separating fermentation liquor after the fed-batch fermentation is finished, collecting bacterial sludge, and obtaining bacterial powder by adopting a spray drying mode.
Preferably, the isolated fermentation broth refers to: separating the fermentation liquor by a butterfly centrifuge;
preferably, the air inlet temperature of the spray drying is 180-220 ℃, and the air outlet temperature is 90-100 ℃;
preferably, the target strain is bacillus coagulans BC99 strain.
Experimental example 1 rejuvenation of Bacillus coagulans BC99
(1) Preparing bacillus coagulans BC99 into bacterial suspension, performing gradient dilution, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 48h at 37-42 ℃;
(2) Selecting single colony forming spore to prepare bacterial suspension, and performing heat treatment in hot water of 80-95 deg.C for 15-30min;
(3) Carrying out gradient dilution on the bacterial suspension after heat treatment, selecting bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 36h at 37-42 ℃;
(4) Selecting single colony forming spore to make into bacterial suspension, and performing secondary heat treatment in hot water of 80-95 deg.C for 15-30min;
(5) And (3) carrying out gradient dilution on the bacterial suspension subjected to heat treatment, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing at 37-42 ℃ for 24h to obtain a single colony of spores, namely the bacillus coagulans capable of producing spores rapidly.
Experimental example 2 preparation of fungal powder in 100L fermenter
The experimental example takes a 100L fermentation tank as an example, and provides a method for fermenting and preparing bacterial powder by bacillus coagulans with high spore number, which comprises the following steps:
(1) Inoculating the single colony of the rejuvenated bacillus coagulans to a nutrient agar slant, and culturing for 24-36h at 37 ℃. The nutrient agar culture medium comprises: 2g/L glucose, 5g/L yeast extract, 10g/L peptone, 15-20g/L agar, pH7.0-7.2, and sterilizing at 121 deg.C for 15min.
(2) And eluting the activated strains, inoculating the strains into a shake flask, and carrying out shake culture for 8-12h. The formula of the shake flask culture medium is as follows: 10g/L of peptone, 5g/L of beef extract, 5g/L of yeast powder, 10g/L of glucose and 5g/L of sodium chloride, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 30min. The liquid loading of the 500mL shake flask was 100mL. The culture conditions are as follows: the shaking speed is 200r/min, and the temperature is 37 ℃.
(3) Inoculating the shake flask culture seeds into a seeding tank, wherein the inoculation amount is 5 percent, the volume of the seeding tank is 5L, and the liquid filling is 60 percent. The culture medium of the seeding tank comprises: 10g/L of peptone, 5g/L of beef extract powder, 3g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate and sterilization at 121 ℃ for 30min. The culture conditions are as follows: the temperature is 37 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the DO is maintained to be more than 30%, the pH value is controlled to be 5.5-6.0, and the culture time is 8h.
(4) Inoculating the seed solution obtained in the step 3 into a 100L tank for fed batch fermentation, wherein a fermentation medium comprises: 10g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 0.3g/L of calcium chloride, 0.05% of defoaming agent and sterilization at 121 ℃ for 30min. The liquid loading amount was 60%. The first stage fermentation control conditions: the inoculation amount is 5 percent, the culture temperature is 37 ℃, the pH is controlled to be 5.5-6.0, the rotating speed is 100-250rpm, the ventilation amount is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the DO control is more than 30 percent, when the DO rises, the material is supplemented, and the composition of a supplemented culture medium is as follows: 5% of glucose, 2% of yeast powder and 0.5% of ammonium sulfate, sterilizing at 115 ℃ for 30min, and feeding period is 12h. And the second stage fermentation control conditions are as follows: the culture temperature is 37 ℃, the pH is controlled to be 7.5-8.5, the rotating speed is 50-100rpm, the ventilation volume is 0.3-0.6vvm, the tank pressure is 0.03-0.05Mpa, the DO is maintained for 10-20%, and the culture time at the stage is 16h. When the fermentation is finished, the temperature is increased to 75 ℃ and maintained for 30min. The DO rebound indicates: when the rotation speed, the ventilation volume and the tank pressure are not changed, DO rises by 10% or more in 1 min.
(5) And (3) separating the fermentation liquor by a butterfly centrifuge, collecting bacterial sludge, and obtaining bacillus coagulans powder by adopting a spray drying mode. The air inlet temperature of the spray drying tower is 200 ℃, and the air outlet temperature is 90 ℃. The detected spore number of the bacterial powder is 9.5 multiplied by 1011cfu/g. Spore number detection culture composition: 5.0g of glucose, 10.0g of peptone, 5.0g of yeast powder, 5.0g of beef extract, 0.25g of sodium chloride, 0.15g of anhydrous calcium chloride, 0.1g of manganese sulfate monohydrate, 0.5g of L-cysteine hydrochloride and 15.0g of agar powder, metering the volume to 1000mL, and adjusting the pH value to 5.0-5.5.
(6) And sealing 200g of the bacterial powder by using an aluminum foil bag, placing the bacterial powder in an incubator at 37 ℃ for storage, detecting the number of spores of the bacterial powder every 6 months, and calculating the survival rate of the bacterial powder stored at different time. The spore number and survival rate data of different strains are respectively shown in the following tables 1 and 2:
TABLE 1 spore count of fungal powder prepared from different Bacillus coagulans strains
TABLE 2 storage stability of fungal powders prepared with different BC strains
(7) The survival rate of BC99 bacterial powder still reaches 90% after being stored for 3 years, the high survival rate is not only related to the characteristics of the bacterial strain, but also mainly caused by the preparation process of the bacterial powder.
Experimental examples 3, preparation of fungal powder in 20T fermenter
In this experimental example, a 20T fermentation tank is taken as an example, and a method for fermenting and preparing bacterial powder from bacillus coagulans with high spore number is provided, which comprises the following steps:
(1) Inoculating the single colony of the rejuvenated bacillus coagulans to a nutrient agar slant, and culturing for 24-36h at 37 ℃. The nutrient agar culture medium comprises the following components: 2g/L glucose, 5g/L yeast extract powder, 10g/L peptone, 15-20g/L agar, pH7.0-7.2, and sterilizing at 121 deg.C for 15min.
(2) And eluting the activated strains, inoculating the strains into a shake flask, and performing shake culture for 8-12h. The formula of the shake flask culture medium is as follows: 10g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 10g/L of glucose and 5g/L of sodium chloride, adjusting the pH value to 7.0-7.2, and sterilizing at 121 ℃ for 30min. The liquid loading of the 500mL shake flask was 150mL. The culture conditions are as follows: the shaking speed is 200r/min, and the temperature is 37 ℃.
(3) The seed cultured in the shake flask is inoculated into a seeding tank, the inoculation amount is 5 percent, the volume of a first-stage seeding tank is 50L, and the liquid loading amount is 60 percent. The culture conditions are as follows: the temperature is 37 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the DO is maintained to be more than 30 percent, the pH is controlled to be 5.5-6.0, and the culture time is 8h. Transferring the cultured primary seed liquid to a secondary seed tank, wherein the volume of the secondary seed tank is 1000L, the liquid loading amount is 60%, and the culture conditions are as follows: the temperature is 37 ℃, the rotating speed is 150-300rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the DO is maintained to be more than 30 percent, the pH is controlled to be 5.5-6.0, and the culture time is 6h. The culture medium of the seeding tank comprises: 10g/L of peptone, 5g/L of beef extract powder, 3g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate and sterilization at 121 ℃ for 30min. The seed transferring refers to transferring all the primary seed liquid to a secondary seed tank.
(4) Inoculating the seed solution obtained in the step 3 into a 20L tank for fed-batch and batch fermentation, wherein a fermentation medium comprises: 15g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 4g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 0.3g/L of calcium chloride, 0.05% of defoaming agent and sterilization at 121 ℃ for 30min. The first stage fermentation control conditions: the inoculation amount is 5 percent, the culture temperature is 37 ℃, the pH is controlled to be 5.5-6.0, the rotating speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, the tank pressure is 0.05Mpa, the DO control is more than 30 percent, the feeding operation is carried out in time when the DO begins to rise, and the feeding culture medium comprises: glucose 6%, yeast powder 1.5%, ammonium sulfate 1%, sterilizing at 115 deg.C for 30min, and supplementing for 12 hr. And the second stage fermentation control conditions are as follows: the culture temperature is 37 deg.C, pH is controlled at 7.5-8.5, rotation speed is 50-100rpm, ventilation amount is 0.3-0.6vvm, tank pressure is 0.05Mpa, DO is maintained at 10-20%, and the culture time is 16h. When the fermentation is finished, the temperature is increased to 75 ℃ and maintained for 30min.
(5) And separating the fermentation liquor by a butterfly centrifuge, collecting bacterial sludge, and obtaining bacillus coagulans powder by adopting a spray drying mode. The air inlet temperature of the spray drying tower is 200 ℃, and the air outlet temperature is 90 ℃. The number of spores of the detected bacterial powder is 8.2 multiplied by 1011cfu/g。
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A fermentation method for improving the spore number and stability of Bacillus coagulans comprises the following steps: fermenting in a seeding tank and fermenting in fed batch; it is characterized in that DO in the fermentation process of the seeding tank is maintained to be more than 30 percent; the fed-batch fermentation comprises a first stage fermentation and a second stage fermentation; the DO in the first stage fermentation process is maintained to be more than 30 percent, and the feeding is started when the DO rises back; the DO is reduced and maintained by 10-20% in the second stage of fermentation; the DO rise-back means that DO rises by 10% or more within 1 min.
2. The fermentation method for improving the spore number and stability of bacillus coagulans according to claim 1, wherein the start feeding refers to adding a feeding medium;
and/or the feed medium comprises the following components in percentage by mass: 5-6% of glucose, 1.5-2% of yeast powder, 0.5-1% of ammonium sulfate and the balance of water.
3. The method of claim 1, wherein the conditions of the seed tank fermentation include: the temperature is 30-45 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5-7, and the time is 6-12h;
and/or, the conditions of the seed tank fermentation include: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.03-0.05Mpa, the pH value is 5.5-6.0, and the time is 6-8h;
and/or, the culture medium for the fermentation of the seeding tank comprises: 5-25g/L of peptone, 5-25g/L of beef extract, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium phosphate and 0.2-1g/L of magnesium sulfate;
and/or, the culture medium for the fermentation of the seeding tank comprises: 10g/L of peptone, 5g/L of beef extract powder, 3g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 2g/L of dipotassium phosphate and 0.5g/L of magnesium sulfate.
4. The method of claim 1, wherein the conditions of the first stage fermentation of the fed-batch fermentation comprise: the inoculum size of the seeding tank fermentation product is 2-10%, the culture temperature is 30-45 deg.C, pH is 5-7, rotation speed is 100-200rpm, ventilation volume is 0.6-1.2vvm, and tank pressure is 0.03-0.05Mpa;
and/or, the conditions of the first stage fermentation include: the inoculation amount of the fermentation product in the seeding tank is 5 percent, the culture temperature is 37-42 ℃, the pH value is 5.5-6.0, the rotating speed is 100-200rpm, the ventilation volume is 0.6-1.2vvm, and the tank pressure is 0.05Mpa;
and/or, the medium of the first stage fermentation comprises: 5-25g/L of peptone, 5-25g/L of beef extract powder, 3-15g/L of yeast powder, 10-30g/L of glucose, 5-20g/L of sodium chloride, 1-5g/L of dipotassium phosphate, 0.2-1g/L of magnesium sulfate, 0.2-1g/L of manganese sulfate, 0.2-1g/L of calcium chloride and 0.05-0.2% of defoaming agent;
and/or the culture medium of the first stage fermentation comprises: 15g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 25g/L of glucose, 5g/L of sodium chloride, 4g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of manganese sulfate, 0.3g/L of calcium chloride and 0.05% of defoaming agent.
5. The method as claimed in claim 1 or 4, wherein the conditions of the second stage fermentation of the fed-batch fermentation include: culturing at 30-45 deg.C, pH5.5-8.5, rotation speed of 50-100rpm, ventilation amount of 0.3-0.6vvm, tank pressure of 0.03-0.05Mpa, DO maintaining 10-20%, and time of 12-24 hr;
and/or, the conditions of the second stage fermentation of the fed-batch fermentation comprise: culturing at 37-42 deg.C, pH7.5-8.5, rotation speed of 50-100rpm, ventilation volume of 0.3-0.6vvm, pot pressure of 0.05Mpa, DO maintaining 10-20%, and time of 16h;
and/or after the second stage of fermentation of the fed-batch fermentation is finished, raising the temperature to 60-75 ℃, maintaining for 10-30min, and then cooling to 25 ℃.
6. The fermentation method for increasing the spore number and stability of bacillus coagulans according to claim 1, further comprising: rejuvenating and activating the strain before fermenting in a seeding tank;
and/or, the strain rejuvenation comprises: coating the frozen strain preservation solution on a solid culture medium after heat treatment;
and/or, the strain rejuvenation comprises: picking the single colony on the solid culture medium coated with the strain preservation solution, performing heat treatment again, and coating the solid culture medium for the second time;
and/or, the solid medium refers to nutrient agar medium;
and/or, the strain activation comprises: inoculating single colony of the rejuvenated strain to a nutrient agar culture medium, and culturing at 30-45 ℃ for 18-36h;
and/or, the nutrient agar medium comprises: 2g/L of glucose, 5g/L of yeast extract powder, 10g/L of peptone and 15-20g/L of agar, and the pH value is 7.0-7.2.
7. The method of claim 6, further comprising: performing shake flask culture after rejuvenation and activation of the strain and before seed tank fermentation;
and/or, the shake flask culture comprises: eluting and inoculating the activated strain on the nutrient agar culture medium into a shake flask culture medium for shake culture;
and/or, the shake flask culture medium comprises: 10g/L of peptone, 5g/L of beef extract powder, 5g/L of yeast powder, 10g/L of glucose and 5g/L of sodium chloride;
and/or, the conditions of the shaking culture comprise: shaking at a rotation speed of 200r/min at 30-45 deg.C for 8-12h;
and/or, the strain is a bacillus coagulans strain; the bacillus coagulans strain is selected from the group consisting of: bacillus coagulans BC99 strain, BC10 strain, BC3702 strain.
8. The fermentation method for improving spore number and stability of bacillus coagulans according to claim 3, wherein the seed tank fermentation comprises a primary seed tank fermentation and a secondary seed tank fermentation;
and/or the inoculation amount of the first-stage seed tank fermentation is 5%, and the fermentation conditions comprise: the temperature is 37-42 ℃, the rotating speed is 150-500rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 8h;
and/or, the conditions of the secondary seed tank fermentation comprise: the temperature is 37-42 ℃, the rotating speed is 150-300rpm, the ventilation volume is 0.6-1.0vvm, the tank pressure is 0.05Mpa, the pH value is 5.5-6.0, and the culture time is 6h;
and/or transferring the fermentation liquor of the first-stage seed tank to a second-stage seed tank for fermentation after the fermentation of the first-stage seed tank is finished.
9. A method for preparing bacterial powder for improving the spore number and stability of Bacillus coagulans, which is characterized by comprising the fermentation method for improving the spore number and stability of the Bacillus coagulans according to any one of claims 1-7.
10. The method for preparing bacterial powder for improving spore number and stability of bacillus coagulans according to claim 9, further comprising: and (3) separating fermentation liquor after the fed-batch fermentation is finished, collecting bacterial sludge, and obtaining bacterial powder by adopting a spray drying mode.
And/or, the separation of the fermentation broth refers to: separating the fermentation liquor by a butterfly centrifuge;
and/or the air inlet temperature of the spray drying is 180-220 ℃, and the air outlet temperature is 90-100 ℃;
and/or, the bacillus coagulans is selected from: bacillus coagulans strain BC99, BC10, BC 3702.
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