AU2016297402B2 - Bacillus coagulans, high cell density fermentation method thereof, and method for preparing spray-dried powder thereof - Google Patents
Bacillus coagulans, high cell density fermentation method thereof, and method for preparing spray-dried powder thereof Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims description 46
- 238000000855 fermentation Methods 0.000 title claims description 45
- 241000193749 Bacillus coagulans Species 0.000 title claims description 39
- 229940054340 bacillus coagulans Drugs 0.000 title claims description 39
- 239000000843 powder Substances 0.000 title claims description 25
- 238000000034 method Methods 0.000 title claims description 15
- 239000007788 liquid Substances 0.000 claims description 44
- 239000001963 growth medium Substances 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000001888 Peptone Substances 0.000 claims description 19
- 108010080698 Peptones Proteins 0.000 claims description 19
- 235000019319 peptone Nutrition 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 18
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 235000015278 beef Nutrition 0.000 claims description 13
- 239000006916 nutrient agar Substances 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000012807 shake-flask culturing Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- 238000009423 ventilation Methods 0.000 claims description 9
- 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 8
- 239000008103 glucose Substances 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 244000061458 Solanum melongena Species 0.000 claims description 6
- 235000002597 Solanum melongena Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 238000009472 formulation Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 239000010802 sludge Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 2
- 238000001694 spray drying Methods 0.000 claims description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 230000000813 microbial effect Effects 0.000 description 13
- 239000003795 chemical substances by application Substances 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 8
- 229960000448 lactic acid Drugs 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 7
- 230000009286 beneficial effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 241000186660 Lactobacillus Species 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
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- 230000000968 intestinal effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000186000 Bifidobacterium Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000003031 feeding effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
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- 238000010563 solid-state fermentation Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
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- C12R2001/07—Bacillus
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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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Abstract
Provided is a
Description
Description
Title of the Invention: Bacillus Coagulans, High Cell Density Fermentation
Method thereof, and Method for Preparing Spray-dried Powder thereof
Technical Field [0001] The present invention relates to the field of microbial technology, in particular to a Bacillus coagulans, a high cell density fermentation method thereof, and the prepared spray-dried powder.
Description of the Related Art [0002] Microbial ecological agent is a microbial bacterial preparation made by special processes, and contains microorganisms beneficial to the host. The microbial ecological agent has the effects of being natural and non-toxic and promoting host health, and is widely applied to the fields of human healthcare and animal health.
[0003] Lactobacillus and bacillus microbial ecological agents are two major varieties which are most widely used in the feed industry. Lactobacillus species mainly includes lactic acid bacteria, bifidobacteria and etc., while bacillus species mainly includes Bacillus subtilis, Bacillus licheniformis and etc.
[0004] The lactobacillus microbial ecological agent plays roles by producing lactic acid, changing pH values in the intestinal tract, and conditioning intestinal microenvironment. This type of microbial ecological agent has poor stress resistance and is easy to lose activity during processing. The bacillus microbial ecological agent is capable of producing spores and tolerating unfavorable environment, such as high temperature, stomach acid and choline, and has strong stress resistance, but doesn’t possess the intestinal conditioning function of the lactobacillus.
[0005] Bacillus Coagulans, is a Gram-positive bacterium, and a lactic-acid-producing Bacillus. The characteristics of the Bacillus Coagulans are: 1. Bacillus Coagulans can produce lactic acid under an anaerobic environment of the intestinal tract to form an acidic environment and inhibit the growth of a pathogenic microorganism, which is beneficial to host health; 2. Bacillus Coagulans can exist in a spore state, tolerate high temperature, stomach acid and choline, and has strong environmental stress resistance. It can be said that Bacillus Coagulans has both the advantages of lactobacillus and bacillus microbial ecological agents.
[0006] Due to its good feeding effect and processing performance, Bacillus Coagulans has been included in a directory of feed additives, and becomes an important member of the feed microbial ecological agent industry.
[0007] More and more attentions have been paid to Bacillus Coagulans, and a great deal of researches have been conducted. ZL200710122111 .X discloses preparation of Bacillus Coagulans feed additives, and the viable count in the fermentation liquid reaches up to 5.0 x 109 cfu/mL. ZL 200810235512.0 discloses a liquid state fermentation process for Bacillus Coagulans CGMCC No.2602, in a 1T fermentor, the total bacteria count in the fermentation liquid is 6.0 X 109 cfu/mL, the spore count is 5.8 X109 cfu/mL, and the spore rate is 96.7%. ZL 201110428027.7 discloses a solid state fermentation process for Bacillus Coagulans CICC No.20138, the number of cells is 1.16X 101° cfu/g and the spore rate is 77.2%. ZL 201210464404.7 discloses a feedback feed-batch culture process for Bacillus Coagulans CGMCC No.6681, and the viable count in the fermentation liquid reaches up to 5.8 X109 cfu/mL.
[0008] Although a great deal of researches have been conducted in the industry, the selling price of Bacillus Coagulans in the market remains at a high level, which makes the product difficult to be popularized. This is because the industrial fermentation level of Bacillus Coagulans is low, cell loss during processing is high and the cell content in the finished product is low. These unfavorable factors directly result in a high product price and influence popularity and application of Bacillus Coagulans.
Technical Problems [0009] In view of the above problems in the prior art, the applicant provides a Bacillus coagulans, a high cell density fermentation method thereof, and a method for preparing a spray-dried powder. The newly-developed Bacillus coagulans according to the present invention has the characteristics of high cell density fermentation and high cell content, therefore, it is beneficial to reducing the product cost, is convenient for market popularization of products, and has a positive significance in promoting healthy livestock and poultry cultivation.
Solution
Technical Solution [0010] The technical solution of the present invention is as follows:
[0011] A Bacillus coagulans (Bacillus coagulans) provided by the present invention has been deposited at the China Center for Type Culture Collection with a deposit number of CCTCC M 2015273. Particularly, the present invention is implemented in the following steps:
[0012] 1. Screening of Bacillus coagulans [0013] 2g of soil sample near a dairy farm in Mashan, Wuxi, Jiangsu is weighed. 98 mL of sterile physiological saline is added. After heat treatment for 10 min at the temperature of 80°C, the mixture is oscillated on a shaker at a temperature of 37°C and at a speed of 120 rpm for 20 min, 1 mL of the supernatant is pipetted into an LB culture medium for dilution and coating, so as to isolate bacterial strains. The genus of the bacterial species is effectively and accurately determined through such means for the strain as morphological observation, physiological & biochemical experiments, and 16S rDNA analysis.
[0014] Finally, a strain with a serial number of ZS-007 is obtained, and identified as a Bacillus coagulans.
[0015] ZS-007 is deposited in the China Center for Type Culture Collection on May 3, 2015 with a deposit number of CCTCC M 2015273.
[0016] 2. Testing of Bacillus coagulans lactic-acid-producing capability [0017] Bacillus coagulans ZS-007 is inoculated into an acid-producing capability test culture medium, and cultured under static conditions at a temperature of 37 °C to conduct an acid-producing capability test. Compositions of the acid-producing capability test culture medium: 20 g/L glucose, 10 g/L peptone, 5 g/L yeast powder, 2 g/L potassium dihydrogen phosphate, 1 g/L magnesium sulfate and 0.5 g/L manganese sulfate. 10% NaOH is added to the culture medium to adjust pH to 7.0, and then the culture medium is sterilized at a temperature of 121 °C for 20 min.
[0018] The lactic acid content in the fermentation liquid is determined by HPLC, and the results are shown in FIG. 1. It can be seen from FIG. 1 that the lactic acid content is 10 g/L or so.
[0019] 3. High cell density fermentation of the Bacillus coagulans [0020] 1) Strain activation [0021] The Bacillus coagulans strain is inoculated into a nutrient agar slant culture medium, and incubated at a temperature of 25-37 °C for 24-48 hr; then streaked on a nutrient agar eggplant bottle slant, and incubated at a temperature of 25-37 °C for 24-48 hr.
[0022] Compositions of the nutrient agar slant culture medium and the nutrient agar eggplant bottle are both: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, 15-20g/L agar, with a pH of 7.2-7.4.
[0023] 2) Shake-flask culture [0024] The activated strain is inoculated into a shake-flask, and incubated with shaking for 18-30 hr.
[0025] Formulation of shake-flask culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. 20 mL of liquid volume in 250mL shake-flask, and 50 mL of liquid volume in 500mL shake-flask.
[0026] Culture conditions: the shake-flask is rotated at a speed of 200 r/min and a temperature of 25-37 °C.
[0027] 3) Seed tank enlarged culture [0028] The strain after enlarged culture is used for later fermentation. Compositions of seed tank culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4; the seed tank is 10-1000L in volume, with 50-80% of liquid volume.
[0029] The culture is conducted at a stirring speed of 180-250 r/min, a ventilation amount of 0.5-1.0 vvm, and a temperature of 25-37 °C for 18-30hr.
[0030] 4) Fermentor fermentation [0031] The seed liquid with an inoculum size of 0.1%-10% is inoculated into the fermentor, the fermentation culture medium is composed of 25 g/L glucose, 15 g/L peptone, 10 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate, with an initial pH of 7.0. The fermentor is 1-50m3 in volume, with 50-80% of liquid volume.
[0032] The culture is conducted at a stirring speed of 100-200 r/min, a ventilation amount of 0.5-0.8 vvm, and a temperature of 25-37 °C for 16-48hr.
[0033] The fermentation liquid is taken for microscopic examination, when more than 90% of bacterial bodies form spores, i.e., a fermentation end-point is arrived, the final fermentation liquid is obtained.
[0034] The viable count and the spore count in the final fermentation liquid are determined with a spread plate method, and the spore rate is calculated. In particular, the spore rate is the ratio of the spore count and the viable count. The culture medium used is a nutrient agar culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, 15-20g/L agar, with a pH of 7.2-7.4. The viable count in the final fermentation liquid is determined after gradient dilution; after the final fermentation liquid is taken for heat treatment for 10 min at the temperature of 80°C and then gradient dilution, the spore count is determined. The viable count in the final fermentation liquid is 1.5*1Ο10 cfu/mL or above, the spore count is 1.4x1O10 cfu/mL or above, and the spore rate is 90.8% or above.
[0035] 4. Preparation of spray-dried powder [0036] Wet bacterial sludge is collected after the final fermentation liquid is centrifuged, and then spray-dried to obtain the dry bacterial powder. Operating conditions are: an air inlet temperature of 210 °C and an air outlet temperature of 100 °C. The viable count in the spray-dried powder is 8.0x1011 cfu/g or above.
[0037] The obtained dry bacterial powder can be mixed with starch, glucose or any other filler, such as a carrier conforming to standards to obtain a Bacillus coagulans microbial ecological agent. Beneficial Effects of the Present Invention.
Beneficial Effects [0038] The beneficial technical effects of the prevent invention lie in that:
[0039] By adopting the liquid fermentation process, the Bacillus coagulans viable count in the fermentation liquid is 1.52*101° cfu/mL or above, the spore count is 1.38*101° cfu/mL or above, and the spore rate is 90.8% or above. In the dry bacterial powder obtained by spray drying, the viable count is 8.0*1011 cfu/g or above. The spray-dried powder can be mixed with starch, glucose or any other filler to obtain a Bacillus coagulans microbial ecological agent.
[0040] By adopting the present invention, the production process is simple and stable, the viable count in the fermentation liquid is high, so that application cost can be directly reduced, and the product can be easily popularized, and the present invention has a significance in promoting a wide application of the Bacillus coagulans microbial ecological agent.
Brief Description of the Drawings Description of the Drawings [0041] FIG. 1 shows a lactic acid content of Bacillus coagulans in a fermentation liquid by an HPLC determination according to the present invention.
Embodiment
Embodiment of the Present Invention [0042] Embodiment 1: Screening of Bacillus coagulans [0043] 2g of soil sample near a dairy farm in Mashan, Wuxi, Jiangsu is weighed. 98 mL of sterile physiological saline is added. After heat treatment for 10 min at the temperature of 80°C, the mixture is oscillated on a shaker at a temperature of 37°C and at a speed of 120 rpm for 20 min, 1 mL of the supernatant is pipetted into an LB culture medium for dilution and coating, so as to isolate bacterial strains. The genus of the bacterial species is effectively and accurately determined through such means for the strain as morphological observation, physiological & biochemical experiments, and 16S rDNA analysis.
[0044] Finally, a strain with a serial number of ZS-007 is obtained, and identified as a Bacillus coagulans.
[0045] ZS-007 is deposited in the China Center for Type Culture Collection on May 3, 2015 with a deposit number of CCTCC M 2015273.
[0046] Embodiment 2: 1 m3 fermentor preparation [0047] Strain activation: the strain obtained in Embodiment 1 is inoculated into a nutrient agar slant culture medium, and incubated at a temperature of 30 °C for 24 hr; then streaked on a nutrient agar eggplant bottle slant, and incubated at a temperature of 30 °C for 24 hr. Compositions of the nutrient agar slant culture medium and the nutrient agar eggplant bottle are both: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, 15-20g/L agar, with a pH of 7.2-7.4.
[0048] Shake-flask culture: the activated strain is inoculated into a shake-flask, and incubated with shaking for 20 hr. Formulation of shake-flask culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. 50 mL of liquid volume in 500mL conical flask. Culture conditions: the shake-flask is rotated at a speed of 200 r/min and a temperature of 30 °C.
[0049] Fermentor fermentation: the liquid in twenty conical flasks is collected as seed for inoculation in 1 m3 fermentor. The liquid volume is 65% of the 1 m3 fermentor. Compositions of culture medium: 25 g/L glucose, 15 g/L peptone, 10 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate, with an initial pH of 7.0. The cultured seed is inoculated into the fermentor for fermentation. The culture is conducted at a stirring speed of 200 r/min, a ventilation amount of 0.8 vvm, and a temperature of 30 °C for 40-44 hr.
[0050] The viable count in the fermentation liquid is 1.52*101° cfu/g, the spore count is 1.38*101° cfu/g, and the spore rate is 90.8 %.
[0051] Embodiment 3: 3 m3 fermentor preparation [0052] Strain activation is the same as that in Embodiment 2.
[0053] Shake-flask culture: the activated strain is inoculated into a shake-flask, and incubated with shaking for 20 hr. Formulation of shake-flask culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. 20 mL of liquid volume in 250mL conical flask. Culture conditions: the shake-flask is rotated at a speed of 200 r/min and a temperature of 30 °C.
[0054] Seed tank enlarged culture: the seed liquid in ten 250mL shake-flasks is collected for seed tank inoculation. Compositions of seed tank culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. The seed tank is 30 L in volume, with 80% of liquid volume. The culture is conducted at a stirring speed of 200 r/min, a ventilation amount of 0.8 vvm, and a temperature of 30 °C for 20 hr.
[0055] Fermentor fermentation: 3 m3 fermentor, with 70% of liquid volume. Compositions of the culture medium: 25 g/L glucose, 15 g/L peptone, 10 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate, with an initial pH of 7.0. The cultured seed is inoculated into the fermentor for fermentation. The culture is conducted at a stirring speed of 150 r/min, a ventilation amount of 0.7 vvm, and a temperature of 35 °C for 36-42 hr.
[0056] The viable count in the fermentation liquid is 1.79x101° cfu/g, the spore count is 1.72x101° cfu/g, and the spore rate is 96.1 %.
[0057] Embodiment 4: 30 m3 fermentor and preparation of spray-dried powder [0058] Strain activation is the same as that in Embodiment 2.
[0059] Shake-flask culture: the activated strain is inoculated into a shake-flask, and incubated with shaking for 20 hr. Formulation of shake-flask culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. 50 mL of liquid volume in 500mL conical flask. Culture conditions: the shake-flask is rotated at a speed of 200 r/min and a temperature of 30 °C.
[0060] Seed tank enlarged culture: the seed liquid in twenty 500mL shake-flasks is collected for seed tank inoculation. Compositions of seed tank culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4. The seed tank is 500 L in volume, with 75% of liquid volume. The culture is conducted at a stirring speed of 200 r/min, a ventilation amount of 0.8 vvm, and a temperature of 30 °C for 20 hr.
[0061] Fermentor fermentation: 30 m3 fermentor, with 70% of liquid volume. Compositions of the culture medium: 25 g/L glucose, 15 g/L peptone, 10 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate, with an initial pH of 7.0. The cultured seed is inoculated into the fermentor for fermentation. The culture is conducted at a stirring speed of 100 r/min, a ventilation amount of 0.6 vvm, and a temperature of 35 °C for 40-45 hr.
[0062] The viable count in the fermentation liquid is 1.99x101° cfu/g, the spore count is 1.92x101° cfu/g, and the spore rate is 96.5 %.
[0063] Wet bacterial sludge is collected by centrifugation, and then spray-dried to obtain the dry bacterial powder. Operating conditions are: an air inlet temperature of 210 °C and an air outlet temperature of 100 °C. The viable count in the spray-dried powder is 8.32x1011 cfu/g.
[0064] Embodiment 5: Preparation of spray-dried powder [0065] The fermentation liquid of the Bacillus coagulans is obtained with the fermentation method in Embodiment 4, wet bacterial sludge is collected by centrifugation, and then spray-dried to obtain the dry bacterial powder. Operating conditions are: an air inlet temperature of 210 °C and an air outlet temperature of 100 °C. The viable count in the spray-dried powder is 6.54x1011 cfu/g.
[0066] Those skilled in the art can make various modifications and changes to the above description, and all of these modifications and changes belong to the protection scope defined by the appended claims of the present invention.
Claims (3)
1) strain activation the Bacillus coagulans strain is inoculated into a nutrient agar slant culture medium, and incubated at a temperature of 25-37 °C for 24-48 hr; then streaked on a nutrient agar eggplant bottle slant, and incubated at a temperature of 25-37 °C for 24-48 hr; compositions of the nutrient agar slant culture medium and the nutrient agar eggplant bottle are both: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, 15-20g/L agar, with a pH of 7.2-7.4;
1. A Bacillus coagulans strain ZS-007, deposited at the China Center for Type Culture Collection with a deposit number of CCTCC M 2015273.
2) shake-flask culture the strain obtained in step 1) is inoculated into a shake-flask, and incubated with shaking for 18-30 hr; formulation of shake-flask culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4; 20 mL of liquid volume in 250mL shake-flask, and 50 mL of liquid volume in 500mL shake-flask; culture conditions: the shake-flask is rotated at a speed of 200 r/min and a temperature of 25-37 °C;
3) seed tank enlarged culture compositions of seed tank culture medium: 10 g/L peptone, 3g/L beef extract, 5 g/L sodium chloride, with a pH of 7.2-7.4; the seed tank is 10-1000L in volume, with 50-80% of liquid volume; the culture is conducted at a stirring speed of 180-250 r/min, a ventilation amount of 0.5-1.0 vvm, and a temperature of 25-37 °C for 18-30hr;
4) fermentor fermentation the seed liquid with an inoculum size of 0.1 %-10% obtained in step 3) is inoculated into the fermentor, the fermentation culture medium is composed of 25 g/L glucose, 15 g/L peptone, 10 g/L yeast powder, 1 g/L potassium dihydrogen phosphate, and 0.5 g/L magnesium sulfate, with an initial pH of 7.0; the fermentor is 1-50m3 in volume, with 50-80% of liquid volume; the culture is conducted at a stirring speed of 100-200 r/min, a ventilation amount of 0.5-0.8 vvm, and a temperature of 25-37 °C for 16-48hr; the fermentation liquid is taken for microscopic examination, when more than 90% of cells form spores, i.e., a fermentation end-point is arrived, the fermentation liquid is obtained.
2. A high cell density fermentation method of the Bacillus coagulans according to claim 1, characterized in that, the method particularly comprises the following steps:
3. A method for preparing a spray-dried powder based on the high cell density fermentation method of the Bacillus coagulans according to claim 2, characterized in that, wet bacterial sludge is collected after the fermentation liquid obtained in step 4) is centrifuged, and then spray-dried to obtain the dry bacterial powder; conditions of spray-drying are: an air inlet temperature of 210 °C and an air outlet temperature of 100 °C; the viable count in the spray-dried powder is 8.0x1011 cfu/g or above.
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| PCT/CN2016/090895 WO2017012570A1 (en) | 2015-07-22 | 2016-07-21 | Bacillus coagulans, high cell density fermentation method thereof, and method for preparing spray-dried powder thereof |
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| CN104974966B (en) * | 2015-07-22 | 2017-12-12 | 江南大学 | A kind of bacillus coagulans and its fermentation process in high density and dry bacterium powder preparation method |
| CN105441354B (en) * | 2015-10-28 | 2019-07-02 | 南京工业大学 | A kind of preparation method of high activity bacillus coagulans active bacteria formulation |
| CN106801013A (en) * | 2015-11-26 | 2017-06-06 | 瑞普（天津）生物药业有限公司 | One kind mixing probiotics dried object and preparation method |
| CN108179116B (en) * | 2016-12-09 | 2020-12-29 | 天津农学院 | High-density fermentation production method of mushrooms |
| CN109207406A (en) * | 2018-10-24 | 2019-01-15 | 广东绿百多生物开发有限公司 | A kind of method and culture medium for improving bacillus coagulans cell and forming gemma |
| CN109287890B (en) * | 2018-11-16 | 2022-04-19 | 青岛贝宝海洋科技有限公司 | Germination agent-containing bacillus coagulans feed additive for aquaculture and preparation method thereof |
| CN109593680B (en) * | 2018-12-26 | 2022-02-22 | 武汉科诺生物科技股份有限公司 | Bacillus thuringiensis liquid fermentation medium and bacterial powder and oil suspension agent thereof |
| CN109609407B (en) * | 2018-12-27 | 2022-03-04 | 黄河三角洲京博化工研究院有限公司 | Thermophilic microorganism strain for in-situ sludge reduction and application thereof |
| CN109943506A (en) * | 2019-03-25 | 2019-06-28 | 微来世界生物科技(苏州)有限公司 | A kind of culture medium and its fermentation process of suitable bacillus coagulans BC01 |
| CN110117561B (en) * | 2019-05-05 | 2020-07-28 | 中铁十八局集团有限公司 | Compound biological agent for increasing survival rate of new trees and preparation method |
| CN110129230B (en) * | 2019-05-20 | 2020-11-17 | 江南大学 | Microbial preparation and application thereof in domestic sewage treatment of expressway service area |
| CN110511884B (en) * | 2019-07-24 | 2022-09-20 | 盛锦合生物科技(江苏)有限公司 | Bacillus coagulans and application thereof in feed processing |
| CN110734873B (en) * | 2019-10-10 | 2022-04-22 | 华南理工大学 | Method for increasing number of bacillus coagulans spores and application |
| CN110804574A (en) * | 2019-12-06 | 2020-02-18 | 湖北华扬科技发展有限公司 | High-concentration bacillus coagulans liquid fermentation method |
| CN111296848A (en) * | 2020-03-26 | 2020-06-19 | 山东正信生物科技股份有限公司 | Edible bacillus coagulans powder and preparation process thereof |
| CN111603489A (en) * | 2020-05-28 | 2020-09-01 | 武汉微康益生菌研究院有限公司 | Microbial inoculum for improving constipation and preparation method thereof |
| CN111876345A (en) * | 2020-06-29 | 2020-11-03 | 武汉微康益生菌研究院有限公司 | High-density and high-spore conversion rate fermentation method for bacillus coagulans |
| CN112048450A (en) * | 2020-08-14 | 2020-12-08 | 润盈生物工程（上海）有限公司 | Method for repeatedly fermenting twice by using bacillus coagulans fermentation centrifugal supernatant |
| CN114645006B (en) * | 2020-12-21 | 2023-09-01 | 中国科学院微生物研究所 | High sugar-tolerant lactic acid production strain and application thereof in D-lactic acid production |
| CN114437984A (en) * | 2022-02-18 | 2022-05-06 | 鑫九通科技(武汉)有限公司 | Preparation method of lactic acid-producing bacillus coagulans microbial preparation |
| CN115261260A (en) * | 2022-06-16 | 2022-11-01 | 漯河微康生物科技有限公司 | Fermentation method for improving spore number and stability of bacillus coagulans and preparation method of bacterial powder |
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| WO2014081395A1 (en) * | 2012-11-23 | 2014-05-30 | Agency For Science, Technology And Research | Highly Efficient Production Of Lactic Acid From Hemicellulose Sugars By Newly Isolated Thermophilic Bacillus Coagulans Strains |
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| CN102943057B (en) * | 2012-11-16 | 2014-06-25 | 南京工业大学 | Bacillus coagulans and process for high-density fermentation of bacillus coagulans |
| CN104232525A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus coagulans preparation |
| CN104974966B (en) * | 2015-07-22 | 2017-12-12 | 江南大学 | A kind of bacillus coagulans and its fermentation process in high density and dry bacterium powder preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001083704A1 (en) * | 2000-05-02 | 2001-11-08 | Biofermin Pharmaceutical Co., Ltd. | Spray-dried microbial cells |
| WO2014081395A1 (en) * | 2012-11-23 | 2014-05-30 | Agency For Science, Technology And Research | Highly Efficient Production Of Lactic Acid From Hemicellulose Sugars By Newly Isolated Thermophilic Bacillus Coagulans Strains |
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