Culture method of bacillus coagulans high-bacterial-quantity fermentation liquor

Abstract

The invention provides a culture method of a bacillus coagulans high-bacterial-quantity fermentation broth, which comprises the following steps: (1) strain activation: inoculating a bacillus coagulans strain to a selective culture medium, picking a single colony, transferring the single colony to a test tube slant culture medium, then scribing, transferring the single colony to an eggplant bottle slant culture medium, carrying out microscopic examination, and carrying out freeze-drying on the single colony to obtain the bacillus coagulans high-bacterial-quantity fermentation broth; when the spore rate is more than 90%, storing in a refrigerator for later use. (2) expanding culture in a first-stage seed tank: scraping and washing mature bacterial sludge on the eggplant bottle slant strain activated in the step (1) by using sterile water, loading into a sterile triangular flask, oscillating and dispersing the bacterial sludge to obtain a uniform bacterial suspension, heating the bacterial suspension in a water bath at 80 DEG C for 20 minutes, and inoculating the bacterial suspension into the first-stage seed tank; the problems of low thallus concentration, low spore formation rate, high production cost and the like are solved, and the product competitiveness is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation engineering, in particular to a method for culturing a high-bacteria-quantity fermentation liquid of Bacillus coagulans. Background Art

[0002] Bacillus coagulans is a Gram-positive bacterium belonging to the Firmicutes phylum. Bacillus coagulans belongs to the genus Bacillus, and its cells are rod-shaped and Gram-positive. It is a facultative anaerobic bacterium that decomposes sugars to produce L-lactic acid and is a homolactic fermentation bacterium.

[0003] The characteristics of Bacillus coagulans are as follows: 1. Bacillus coagulans is a facultative anaerobic bacterium with an optimum growth temperature of 35 degrees Celsius to 50 degrees Celsius and an optimum growth pH of 5.5 to 6.5, and has high survival and reproduction capabilities; 2. It can produce lactic acid and antibacterial coagulants to inhibit the growth and reproduction of pathogenic microorganisms, which is beneficial to the health of the host; 3. It can exist in the form of spores, is resistant to high temperature and pressure, stomach acid, choline, has strong environmental resistance and a high resurrection rate; 4. It can secrete a variety of digestive enzymes and enzymatic factors to promote the digestion and absorption of feed and reduce the feed-to-weight ratio; 5. It has no toxic side effects, is safe to use, and improves the breeding environment.

[0004] It is precisely because Bacillus coagulans has the characteristics of both Bacillus and lactic acid bacteria and has good feeding effects and processing performance that it greatly makes up for the shortcomings of traditional lactic acid bacteria such as heat intolerance, poor preservation, and easy inactivation.

[0005] However, the traditional fermentation method has problems such as low bacterial concentration, low spore formation rate, and high production cost, which limit its industrial application. Therefore, it is of great significance to develop an efficient and economical method for culturing high-bacterial-quantity fermentation broth of Bacillus coagulans. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans, which solves the problems of low bacterial concentration, low spore formation rate, high production cost, etc. in the prior art.

[0007] According to an embodiment of the present invention, a method for culturing a high-bacterial-count fermentation broth of Bacillus coagulans comprises the following steps by weight:

[0008] (1) Activation of bacterial strains: Inoculate Bacillus coagulans onto a selective medium, pick a single colony and transfer it onto a test tube slant medium, then streak it onto an eggplant bottle slant medium, examine under a microscope, and store in a refrigerator when the spore rate is above 90% for future use;

[0009] (2) Primary seed tank expansion: scrape the mature bacterial sludge on the eggplant bottle slope surface activated in step (1) with sterile water, put it into a sterile triangular flask, shake and disperse the bacterial sludge to obtain a uniform bacterial suspension, heat the bacterial suspension in an 80° C. water bath for 20 minutes, and then inoculate it into the primary seed tank;

[0010] (3) Secondary seed tank expansion: The seed solution obtained in step (2) is inoculated into a secondary seed tank at an inoculation rate of 0.1% to 10%, and the strain is expanded for later fermentation;

[0011] (4) Fermentation in a fermenter: The seed solution obtained in step (3) is inoculated into a fermenter at an inoculation rate of 0.1% to 10% for fed-batch fermentation to obtain a high-bacteria fermentation solution.

[0012] Preferably, the composition of the culture medium selected in step (1) is: peptone 0.5%-2%, beef extract 0.5%-2%, yeast extract 0.2%-0.5%, dipotassium hydrogen phosphate 0.1%-0.5%, triammonium citrate 0.1%-0.5%, sodium acetate 0.3%-0.5%, glucose 1%-2%, 0.05%-0.1% Tween 80, magnesium sulfate 0.01%-0.05%, manganese sulfate 0.001%-0.005%, calcium carbonate 0.2%-0.5%, agar 1%-2%, pH 6.2%-6.5%, and the balance is water, and sterilized at 115°C for 20 min for standby use;

[0013] The composition of the slant culture medium: peptone 0.5%-2%, beef extract 0.5%-2%, yeast extract 0.2%-0.5%, dipotassium hydrogen phosphate 0.1%-0.5%, triammonium citrate 0.1%-0.5%, sodium acetate 0.3%-0.5%, glucose 1%-2%, 0.05%-0.1% Tween 80, magnesium sulfate 0.01%-0.05%, manganese sulfate 0.001%-0.005%, agar 1%-2%, pH 6.2%-6.5%, the balance is water, sterilize at 115°C for 20 min for use.

[0014] Preferably, in step (1), the culture medium culture conditions are selected to be cultured at 35-45°C for 36-72 hours; the test tube slant culture medium and eggplant bottle slant culture conditions are cultured at 35-45°C for 24-48 hours.

[0015] Preferably, the culture medium of the primary seed tank in step (2) comprises: 0.5%-1.5% peptone, 0.2%-1% yeast extract powder, 0.5%-1.5% sodium chloride, PH 6.0%-7.5%, and the balance is water, and is sterilized at 118°C for 30 min.

[0016] Preferably, in step (2), the volume of the primary seed tank is 50-300 L, the liquid filling volume is 50-70%, the culture conditions are stirring speed 150-250 r / min, ventilation volume 0.5-1.0 vvm, temperature 35-45° C., and culture time 15-25 h.

[0017] Preferably, the secondary seed tank culture medium in step (3) is composed of: 1.0%-2.0% bean cake powder, 0.2%-1% corn flour, 0.1%-0.5% corn steep liquor powder, 0.1%-1% sodium chloride, 0.05%-0.5% magnesium sulfate, 0.1%-1% ammonium sulfate, pH 6.0%-7.5%, and the balance is water, and is sterilized at 118°C for 30 minutes.

[0018] Preferably, in step (3), the volume of the secondary seed tank is 500-1000 L, the liquid filling volume is 60-80%, the culture conditions are stirring speed 180-250 r / min, ventilation volume 0.5-1.0 vvm, temperature 35-45°C, and culture time 8-20 h.

[0019] Preferably, the fermentation medium in the fermenter in step (4) comprises: 2.0%-5.0% bean cake powder, 0.5%-2% corn flour, 1%-5% glucose, 0.1%-0.5% corn steep liquor powder, 0.1%-1% sodium chloride, 0.05%-0.5% magnesium sulfate, 0.01%-0.05% manganese sulfate, 0.1%-1% ammonium sulfate, 0.1%-0.5% potassium dihydrogen phosphate, 0.1%-1% sodium acetate, pH 6.0%-7.5%, and the balance is water, and sterilized at 118°C for 30 min.

[0020] Preferably, the feed medium in step (4) comprises 2%-6% glucose, 1%-3% yeast extract, and the remainder is water, and is sterilized at 118° C. for 30 min.

[0021] Preferably, the fermentation tank in step (4) has a volume of 5-30 m 3 , liquid volume 50-80%, culture conditions, stirring speed 100-200r / min, ventilation 0.5-1.0vvm, temperature 35-45℃, from the beginning of fermentation to the time when the pH first drops and then rebounds at 6.0-7.0, and the dissolved oxygen is maintained at 10%-30%, start feeding, the feeding cycle is 12-16h, during the feeding period, use 10%-30% sodium hydroxide solution to maintain the pH at 6.6-7.2, and the culture time is 36-72h.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The method for culturing the high-bacterial-count fermentation broth of Bacillus coagulans provided by the present invention is to inoculate the Bacillus coagulans strain on a selective culture medium supplemented with calcium carbonate, and the activity of a single colony can be preliminarily judged by the size of the transparent circle, which is conducive to picking a single colony with good performance for expanded culture.

[0024] 2. The method for culturing the high-bacterial-count fermentation broth of Bacillus coagulans provided by the present invention heats the bacterial suspension of the slant bacteria entering the tank in a water bath at 80° C. for 20 minutes, which is beneficial to the consistency of bacterial growth.

[0025] 3. The method for culturing the high-bacterial-count fermentation broth of Bacillus coagulans provided by the present invention adopts three-stage fermentation and feed control in the fermentation stage to maintain the pH at 6.6-7.2, so that Bacillus coagulans is in the most suitable growth environment, which greatly improves the bacterial mass and spore rate of Bacillus coagulans.

[0026] 4. In the method for culturing the high-bacterial-count fermentation broth of Bacillus coagulans provided by the present invention, a high-bacterial-count fermentation broth is obtained by optimizing the culture medium and the fermentation process, and a high-bacterial-count bacterial powder preparation can be provided, thereby reducing production costs and improving product quality. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with embodiments.

[0028] This embodiment 1

[0029] A method for culturing a high-bacterial-count fermentation broth of Bacillus coagulans is provided, which comprises the following steps, measured by weight:

[0030] (1) Bacterial Activation

[0031] Inoculate the Bacillus coagulans strain onto the selective medium and culture it at 40°C for 72 hours; pick a single colony and transfer it to the test tube slant medium and culture it at 40°C for 48 hours; then streak it onto the eggplant bottle slant medium and culture it at 40°C for 48 hours. Examine under a microscope and store it in a refrigerator when the spore rate is above 90% for later use.

[0032] The composition of the selection medium: 0.5% peptone, 0.5% beef extract, 0.2% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.1% triammonium citrate, 0.3% sodium acetate, 1% glucose, 0.05% Tween 80, 0.01% magnesium sulfate, 0.001% manganese sulfate, 0.2% calcium carbonate, 1% agar, pH 6.2, the balance is water, sterilize at 115°C for 20 min for use.

[0033] The composition of the slant culture medium: 0.5% peptone, 0.5% beef extract, 0.2% yeast extract, 0.1% dipotassium hydrogen phosphate, 0.1% triammonium citrate, 0.3% sodium acetate, 1% glucose, 0.05% Tween 80, 0.01% magnesium sulfate, 0.001% manganese sulfate, 1% agar, pH 6.2, the balance is water, sterilize at 115°C for 20 min for use.

[0034] (2) Primary seed tank expansion

[0035] Use sterile water to scrape off the mature bacterial mud on the slope of the eggplant bottle with the activated eggplant bottle slope, put it into a sterile triangular bottle, oscillate to disperse the bacterial mud to obtain a uniform bacterial suspension, heat the bacterial suspension in an 80℃ water bath for 20 minutes, and access the first-level seed tank.

[0036] The medium composition of the first-stage seed tank is: peptone 0.5%, yeast extract powder 0.2%, sodium chloride 0.5%, pH 6.0, and the balance is water. The volume of the seed tank is 200L, and the liquid volume is 50%.

[0037] Culture conditions: stirring speed 200r / min, ventilation volume 0.6vvm, temperature 40℃, culture time 18h.

[0038] (3) Secondary seed tank expansion

[0039] The seed liquid is inoculated into the secondary seed tank at a rate of 0.1% to expand the strain for later fermentation. The secondary seed tank culture medium composition: 1% bean cake powder, 0.2% corn powder, 0.1% corn steep liquor powder, 0.1% sodium chloride, 0.05% magnesium sulfate, 0.05% ammonium sulfate, pH 6.0, and the balance is water. The seed tank volume is 500L, and the liquid volume is 60%.

[0040] Culture conditions: stirring speed 180r / min, ventilation volume 0.5vvm, temperature 35℃, culture time 8h.

[0041] (4) Fermentation in a fermenter

[0042] The seed solution was inoculated into the fermentation tank at an inoculation rate of 0.1%.

[0043] Fermentation medium composition: soybean meal powder 2%, corn flour 0.5%, glucose 1%, corn steep liquor powder 0.1%, sodium chloride 0.1%, magnesium sulfate 0.05%, manganese sulfate 0.01%, ammonium sulfate 0.1%, dipotassium hydrogen phosphate 0.1%, sodium acetate 0.1%, pH 6.0. Fermentation tank volume 5m 3 , liquid volume 50%.

[0044] The composition of the feed medium was: 2% glucose, 1% yeast extract, and the balance was water.

[0045] Culture conditions: stirring speed 100r / min, ventilation volume 0.5vvm, temperature 35℃, culture time 36h.

[0046] Fed-batch culture: From the beginning of fermentation to when the pH drops and rebounds to 6.0, and the dissolved oxygen is maintained at 10%, start feeding, and the feeding cycle is 12 hours. During the feeding period, use 10% sodium hydroxide solution to maintain the pH at 6.6.

[0047] During the culture process, defoaming agent is added according to the increase of foam to prevent contamination by bacteria caused by foam escaping from the fermentation tank.

[0048] When the fermentation liquid is taken for microscopic examination, when more than 90% of the bacteria have formed spores, the fermentation ends and the final fermentation liquid is obtained.

[0049] The plate coating method was used to determine the number of live bacteria and spores in the final fermentation liquid, and the spore rate was calculated. Among them, the spore rate is the ratio of the number of spores to the number of live bacteria. The culture medium used was a slant culture medium. When determining the number of live bacteria in the final fermentation liquid, the gradient dilution was used for determination; when determining the number of spores, the final fermentation liquid was heat-treated at 80℃ for 20 minutes and then gradiently diluted for determination. The number of live bacteria in the final fermentation liquid was 1.26×10 10 cfu / ml, spore count 1.20×10 10 cfu / ml, spore rate 95.2%.

[0050] (5) Preparation of bacterial powder

[0051] Finally, the fermentation liquid was added with starch and spray-dried to obtain bacterial powder. The operating conditions were: inlet air temperature 180°C, outlet air temperature 80°C. The number of viable bacteria in the dry bacterial powder was 2.25×1011 cfu / g.

[0052] Embodiment 2:

[0053] This embodiment provides a method for culturing a high-bacterial-count fermentation broth of Bacillus coagulans, which comprises the following steps, measured by weight:

[0054] (I) Bacterial Activation

[0055] Inoculate the Bacillus coagulans strain onto the selective medium and culture it at 37°C for 72 hours; pick a single colony and transfer it to the test tube slant medium and culture it at 37°C for 48 hours; then streak it onto the eggplant bottle slant medium and culture it at 37°C for 48 hours. Examine under a microscope and store it in a refrigerator when the spore rate is above 90% for later use.

[0056] The composition of the selection medium is as follows: 2% peptone, 2% beef extract, 0.5% yeast extract, 0.5% dipotassium hydrogen phosphate, 0.5% triammonium citrate, 0.5% sodium acetate, 2% glucose, 0.1% Tween 80, 0.05% magnesium sulfate, 0.005% manganese sulfate, 0.5% calcium carbonate, 2% agar, pH 6.5, the balance is water, sterilize at 115°C for 20 min for use.

[0057] The composition of the slant culture medium: 2% peptone, 2% beef extract, 2% yeast extract, 0.5% dipotassium hydrogen phosphate, 0.5% triammonium citrate, 0.5% sodium acetate, 2% glucose, 0.1% Tween 80, 0.05% magnesium sulfate, 0.005% manganese sulfate, 2% agar, pH 6.5, the balance is water, sterilize at 115°C for 20 min and set aside.

[0058] ⑵First-level seed tank expansion

[0059] Use sterile water to scrape off the mature bacterial mud on the slope of the eggplant bottle with the activated eggplant bottle slope, put it into a sterile triangular bottle, oscillate to disperse the bacterial mud to obtain a uniform bacterial suspension, heat the bacterial suspension in an 80℃ water bath for 20 minutes, and access the first-level seed tank.

[0060] The first-level seed tank culture medium consists of: 1.5% peptone, 1% yeast extract powder, 1.5% sodium chloride, pH 7.5, and the balance is water. The seed tank volume is 300L, and the liquid volume is 70%.

[0061] Culture conditions: stirring speed 250r / min, ventilation volume 1.0vvm, temperature 45℃, culture time 25h.

[0062] (3) Secondary seed tank expansion

[0063] The seed liquid is inoculated into the secondary seed tank at a 10% inoculation rate to expand the strain for later fermentation. The secondary seed tank culture medium composition: 2% bean cake powder, 2% corn powder, 5% glucose, 0.5% corn steep liquor powder, 1% sodium chloride, 0.5% magnesium sulfate, 1% ammonium sulfate, pH 7.5, and the balance is water. The seed tank volume is 1000L, and the liquid volume is 80%.

[0064] Culture conditions: stirring speed 250r / min, ventilation volume 1vvm, temperature 45℃, culture time 20h.

[0065] (4) Fermentation in a fermenter

[0066] The seed liquid was inoculated into the fermentation tank at a 10% inoculation rate.

[0067] Fermentation medium composition: soybean meal 5%, corn meal 2%, glucose 5%, corn steep liquor 0.5%, sodium chloride 1%, magnesium sulfate 0.5%, manganese sulfate 0.05%, ammonium sulfate 1%, dipotassium hydrogen phosphate 0.5%, sodium acetate 1%, pH 7.5, the balance is water. Fermentation tank volume 30m 3 , liquid filling volume 80%.

[0068] The composition of the feed medium was glucose 6%, yeast extract 3%, and the balance was water.

[0069] Culture conditions: stirring speed 200r / min, ventilation volume 1.0vvm, temperature 45℃, culture time 72h.

[0070] Fed-batch culture: From the beginning of fermentation to the time when the pH drops and rebounds to 7.0, and the dissolved oxygen is maintained at 30%, start feeding, and the feeding cycle is 16 hours. During the feeding period, use 30% sodium hydroxide solution to maintain the pH at 7.2.

[0071] During the culture process, defoaming agent is added according to the increase of foam to prevent contamination by bacteria caused by foam escaping from the fermentation tank.

[0072] When the fermentation liquid is taken for microscopic examination, when more than 90% of the bacteria have formed spores, the fermentation ends and the final fermentation liquid is obtained.

[0073] The plate coating method was used to determine the number of live bacteria and spores in the final fermentation liquid, and the spore rate was calculated. Among them, the spore rate is the ratio of the number of spores to the number of live bacteria. The culture medium used was a slant culture medium. When determining the number of live bacteria in the final fermentation liquid, the gradient dilution was used for determination; when determining the number of spores, the final fermentation liquid was heat-treated at 80℃ for 20 minutes and then gradiently diluted for determination. The number of live bacteria in the final fermentation liquid was 7.80×10 9 cfu / ml, spore count 7.03×10 9 cfu / ml, spore rate 90.0%.

[0074] (5) Preparation of bacterial powder

[0075] Finally, the fermentation liquid was added with starch and spray dried to obtain bacterial powder. The operating conditions were: inlet air temperature 200°C, outlet air temperature 80°C. The number of live bacteria in the dry bacterial powder was 1.63×10 11 cfu / g.

[0076] Embodiment 3:

[0077] This embodiment provides a method for culturing a high-bacterial-count fermentation broth of Bacillus coagulans. The method comprises the following steps:

[0078] (1) Bacterial Activation

[0079] Inoculate the Bacillus coagulans strain onto the selective medium and culture it at 42°C for 72 hours; pick a single colony and transfer it to the test tube slant medium and culture it at 42°C for 48 hours; then streak it onto the eggplant bottle slant medium and culture it at 42°C for 48 hours. Examine under a microscope and store it in a refrigerator when the spore rate is above 90% for later use.

[0080] The composition of the selection medium: peptone 1.25%, beef extract 1.25%, yeast extract 0.3%, dipotassium hydrogen phosphate 0.3%, triammonium citrate 0.3%, sodium acetate 0.4%, glucose 1.5%, 0.075% Tween 80, magnesium sulfate 0.03%, manganese sulfate 0.003%, calcium carbonate 0.3%, agar 1.5%, pH 6.3, the balance is water, sterilize at 115°C for 20 min for use.

[0081] The composition of the slant culture medium: peptone 1.25%, beef extract 1.25%, yeast extract 0.3%, dipotassium hydrogen phosphate 0.3%, triammonium citrate 0.3%, sodium acetate 0.4%, glucose 1.5%, 0.075% Tween 80, magnesium sulfate 0.03%, manganese sulfate 0.003%, agar 1.5%, pH 6.3, the balance is water, sterilize at 115°C for 20 min for use.

[0082] (2) Primary seed tank expansion

[0083] Use sterile water to scrape off the mature bacterial mud on the slope of the eggplant bottle with the activated eggplant bottle slope, put it into a sterile triangular bottle, oscillate to disperse the bacterial mud to obtain a uniform bacterial suspension, heat the bacterial suspension in an 80℃ water bath for 20 minutes, and access the first-level seed tank.

[0084] The medium composition of the first-stage seed tank is: 1% peptone, 0.6% yeast extract powder, 1% sodium chloride, and the balance is water, pH 6.7. The volume of the seed tank is 175L, and the liquid volume is 60%.

[0085] Culture conditions: stirring speed 200r / min, ventilation volume 0.75vvm, temperature 40℃, culture time 20h.

[0086] (3) Secondary seed tank expansion

[0087] The seed liquid is inoculated into the secondary seed tank at a 5% inoculation rate to expand the strain for later fermentation. The secondary seed tank culture medium composition: soybean meal powder 1.5%, corn flour 0.6%, corn steep liquor dry powder 0.3%, sodium chloride 0.4%, magnesium sulfate 0.3%, ammonium sulfate 0.4%, pH 6.7. The seed tank volume is 750L, and the liquid volume is 70%.

[0088] Culture conditions: stirring speed 210r / min, ventilation volume 0.7vvm, temperature 42℃, culture time 14h.

[0089] (4) Fermentation in a fermenter

[0090] The seed liquid was inoculated into the fermentation tank at a 5% inoculation rate.

[0091] Fermentation medium composition: soybean meal powder 3%, corn meal 1.25%, glucose 3%, corn steep powder 0.3%, sodium chloride 0.5%, magnesium sulfate 0.3%, manganese sulfate 0.03%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.3%, sodium acetate 0.5%, pH 6.7. Fermentation tank volume 17m 3 , liquid volume 65%.

[0092] The composition of the feed medium was as follows: 4% glucose, 1.5% yeast extract, and the balance was water.

[0093] Culture conditions: stirring speed 150r / min, ventilation volume 0.8vvm, temperature 42℃, culture time 50h.

[0094] Fed-batch culture: From the beginning of fermentation to the time when the pH drops and rebounds to 6.5, and the dissolved oxygen is maintained at 12%, feed is started, and the feeding cycle is 14 hours. During the feeding period, 20% sodium hydroxide solution is used to maintain the pH at 6.9.

[0095] During the culture process, defoaming agent is added according to the increase of foam to prevent contamination by bacteria caused by foam escaping from the fermentation tank.

[0096] When the fermentation liquid is taken for microscopic examination, when more than 90% of the bacteria have formed spores, the fermentation ends and the final fermentation liquid is obtained.

[0097] The plate coating method was used to determine the number of live bacteria and spores in the final fermentation liquid, and the spore rate was calculated. Among them, the spore rate is the ratio of the number of spores to the number of live bacteria. The culture medium used was a slant culture medium. When determining the number of live bacteria in the final fermentation liquid, the gradient dilution was used for determination; when determining the number of spores, the final fermentation liquid was heat-treated at 80℃ for 20 minutes and then gradiently diluted for determination. The number of live bacteria in the final fermentation liquid was 1.03×10 10 cfu / ml, spore count 9.90×10 9 cfu / ml, spore rate 96.1%.

[0098] (5) Preparation of bacterial powder

[0099] Finally, the fermentation liquid was added with starch and spray dried to obtain bacterial powder. The operating conditions were: inlet air temperature 180°C, outlet air temperature 70°C. The number of live bacteria in the dry bacterial powder was 1.95×10 11 cfu / g.

[0100] Comparative Example 1

[0101] (1) Bacillus coagulans is inoculated into a selective culture medium and cultured in a constant temperature incubator at 40 to 45°C for 24 hours to obtain a single colony, and then a single colony is selected and cultured on a seed culture medium for seed culture and preservation.

[0102] Specifically, a culture medium is selected for the separation and detection of Bacillus coagulans, and its ingredients are as follows: 0.5% glucose, 1% peptone, 0.5% yeast powder, 0.5% beef extract, 0.015% anhydrous calcium chloride, 0.25% sodium chloride, 0.01% manganese sulfate monohydrate, 0.05% L-cysteamine hydrochloride, and the balance is water. Hydrochloric acid is used to adjust the pH to 5.0-5.5, and then 1.5% agar powder is added, and the medium is prepared by sterilization at 115°C for 30 minutes.

[0103] The composition of the seed culture medium is as follows: 2% glucose, 2% peptone, 1% yeast extract powder, and the balance is water, sterilized at 115°C for 30 minutes. A single colony is cultured on this seed culture medium at 40°C to 45°C and a shaking table at 150rpm to 200rpm for 8 to 12 hours.

[0104] After seed culture, add 10-30% glycerol to the seed culture solution, mix well and transfer into a centrifuge tube, 1.5 mL / tube, and store in a -80°C refrigerator.

[0105] (2) The preserved Bacillus coagulans was inoculated into a slant culture medium and cultured in a constant temperature incubator at 37°C for 2 to 3 days until spores were formed. The culture medium was then taken out and stored in a refrigerator at 4°C for future use. The shelf life is 1 to 3 months.

[0106] Specifically, the composition of the slant culture medium is as follows: weigh 5 g of bran, add 90 mL of water and boil over low heat for 30 min to 1 h, filter the juice with gauze, make the volume to 100 mL, add 1.5% agar powder, and sterilize at 115°C for 30 min.

[0107] (3) Pick a loop of bacterial slurry from the Bacillus coagulans cultured on the slant medium and inoculate it into the seed medium.

[0108] The culture was carried out at 200 rpm in a shaking incubator for 8 to 10 hours. The composition of the seed culture medium was as follows: 2% glucose, 2% peptone, 1% yeast extract powder, and the balance was water, and the culture medium was sterilized at 115°C for 30 minutes.

[0109] (4) The seed liquid after the expanded culture in step (3) is placed in a fermentation tank filled with fermentation medium for separation.

[0110] Batch-fed fermentation culture, wherein the fermentation medium has the following components: 1% glucose, 1.5% yeast extract, 0.5% corn steep liquor, 0.5% potassium dihydrogen phosphate, 0.12% manganese sulfate, 0.15% magnesium sulfate, 0.005% defoamer, and the balance is water, and is prepared by adjusting the pH to 6.8-7.5 using sodium hydroxide solution and sterilizing at 115°C for 30 minutes. The feed medium for feed-batch is 3% glucose, 2% yeast extract, and the balance is water, and is sterilized at 115°C for 30 minutes.

[0111] The specific process of fermentation culture is as follows: 18L fermentation medium is loaded into a 30L fermentation tank, sterilized at 115℃ for 30min, and then water is added to cool down to 40-45℃. Seed liquid is added at an inoculum rate of 1-10%, the initial stirring speed is 100rpm-600rpm, the initial ventilation volume is 0.5m3 / h-1.5m3 / h, and fermentation begins. The pH drops first and then rebounds. The dissolved oxygen begins to be maintained above 10%. When the pH rebounds to 5.5-6.5, feeding begins, and 0.35% feed medium is added each time until it is completed. During the feeding process, Bacillus coagulans will maintain the fermentation pH at 5.0-7.5 through the conversion of its own metabolic pathways. A large number of literature and studies have shown that Bacillus coagulans is conducive to its growth under this pH environment. The feeding is completed in about 24h, the glucose in the culture medium is completely exhausted in 27h, the vegetative body of Bacillus coagulans begins to form spores, and the spores completely fall off in 35h-45h.

[0112] The above selective medium was used to detect 70×10 vegetative cells in the fermentation broth. 8 cfu / mL, spores 63×10 8 cfu / mL, and the spore rate reached 90%.

[0113] The average spore rate of Examples 1-3 of the present invention reaches 93.7%, which is better than the spore rate of 90% in Comparative Example 1. Therefore, the spore rate of the present invention is better.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for culturing a high-bacterial-count fermentation broth of Bacillus coagulans, characterized in that, by weight, The steps include: (1) Activation of bacterial strains: Inoculate Bacillus coagulans onto a selective medium, pick a single colony and transfer it onto a test tube slant medium, then streak it onto an eggplant bottle slant medium, examine under a microscope, and store in a refrigerator when the spore rate is above 90% for future use; (2) Primary seed tank expansion: scrape the mature bacterial sludge on the eggplant bottle slope surface activated in step (1) with sterile water, put it into a sterile triangular flask, shake and disperse the bacterial sludge to obtain a uniform bacterial suspension, heat the bacterial suspension in an 80° C. water bath for 20 minutes, and then inoculate it into the primary seed tank; (3) Secondary seed tank expansion: The seed solution obtained in step (2) is inoculated into a secondary seed tank at an inoculation rate of 0.1% to 10%, and the strain is expanded for later fermentation; (4) Fermentation in a fermenter: The seed solution obtained in step (3) is inoculated into a fermenter at an inoculation rate of 0.1% to 10% for fed-batch fermentation to obtain a high-bacteria fermentation solution.

2. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The composition of the culture medium selected in step (1) is as follows: 0.5%-2% peptone, 0.5%-2% beef extract, 0.2%-0.5% yeast extract, 0.1%-0.5% dipotassium hydrogen phosphate, 0.1%-0.5% triammonium citrate, 0.3%-0.5% sodium acetate, 1%-2% glucose, 0.05%-0.1% Tween 80, 0.01%-0.05% magnesium sulfate, 0.001%-0.005% manganese sulfate, 0.2%-0.5% calcium carbonate, 1%-2% agar, pH 6.2-6.5, the balance being water, and sterilized at 115° C. for 20 min for later use; The composition of the slant culture medium: peptone 0.5%-2%, beef extract 0.5%-2%, yeast extract 0.2%-0.5%, dipotassium hydrogen phosphate 0.1%-0.5%, triammonium citrate 0.1%-0.5%, sodium acetate 0.3%-0.5%, glucose 1%-2%, 0.05%-0.1% Tween 80, magnesium sulfate 0.01%-0.05%, manganese sulfate 0.001%-0.005%, agar 1%-2%, pH 6.2-6.5, the balance is water, sterilized at 115°C for 20 min for use.

3. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: In the step (1), the culture medium culture conditions are selected to be cultured at 35-45° C. for 36-72 hours; the test tube slant culture medium and eggplant bottle slant culture conditions are cultured at 35-45° C. for 24-48 hours.

4. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The primary seed tank culture medium in step (2) is composed of: Peptone 0.5%-1.5%, yeast extract powder 0.2%-1%, sodium chloride 0.5%-1.5%, PH 6.0-7.5, the balance is water, sterilize at 118℃ for 30min.

5. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: In the step (2), the volume of the primary seed tank is 50-300 L, the liquid filling volume is 50-70%, the culture conditions are stirring speed 150-250 r / min, ventilation volume 0.5-1.0 vvm, temperature 35-45° C., and culture time 15-25 h.

6. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The secondary seed tank culture medium in step (3) is composed of: 1.0%-2.0% bean cake powder, 0.2%-1% corn powder, 0.1%-0.5% corn steep liquor powder, 0.1%-1% sodium chloride, 0.05%-0.5% magnesium sulfate, 0.1%-1% ammonium sulfate, pH 6.0-7.5, and the balance is water, and is sterilized at 118° C. for 30 minutes.

7. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: In the step (3), the volume of the secondary seed tank is 500-1000 L, the liquid filling volume is 60-80%, the culture conditions are stirring speed 180-250 r / min, ventilation volume 0.5-1.0 vvm, temperature 35-45° C., and culture time 8-20 h.

8. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The fermentation medium in the fermentation tank in step (4) comprises: 2.0%-5.0% bean cake powder, 0.5%-2% corn powder, 1%-5% glucose, 0.1%-0.5% corn steep liquor powder, 0.1%-1% sodium chloride, 0.05%-0.5% magnesium sulfate, 0.01%-0.05% manganese sulfate, 0.1%-1% ammonium sulfate, 0.1%-0.5% potassium dihydrogen phosphate, 0.1%-1% sodium acetate, pH 6.0-7.5, and the balance is water, and sterilized at 118° C. for 30 min.

9. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The feed medium in step (4) is composed of 2%-6% glucose, 1%-3% yeast extract, and the balance is water, and is sterilized at 118° C. for 30 minutes.

10. The method for culturing a high-bacterial-quantity fermentation broth of Bacillus coagulans according to claim 1, characterized in that: The fermentation tank volume in step (4) is 5-30m 3 , liquid volume 50-80%, culture conditions, stirring speed 100-200r / min, ventilation 0.5-1.0vvm, temperature 35-45℃, from the beginning of fermentation to the time when the pH first drops and then rebounds at 6.0-7.0, and the dissolved oxygen is maintained at 10%-30%, start feeding, the feeding cycle is 12-16h, during the feeding period, use 10%-30% sodium hydroxide solution to maintain the pH at 6.6-7.2, and the culture time is 36-72h.