CN112063547A - Method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 - Google Patents
Method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to a method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12, which comprises the following steps: inoculating Lactobacillus gasseri LG-G12 in an MRSA culture medium, and transferring 2% to another MRSA culture medium to obtain a Lactobacillus gasseri LG-G12 seed solution; inoculating 2% of seed liquid into a fermentation culture medium, preparing another fermentation culture medium, inoculating 2-3% of the seed liquid of lactobacillus gasseri LG-G12, stopping fermentation when the pH value is reduced to about 4.4, sampling and centrifuging to obtain bacterial sludge; according to the bacterial sludge: the protective agent is 1:2, emulsifying and carrying out vacuum freeze drying to obtain freeze-dried powder of the lactobacillus gasseri LG-G12. Compared with the prior art, the live bacteria number of the fermentation liquid of the lactobacillus gasseri LG-G12 is high, no impurity is precipitated, the yield of the freeze-dried powder is doubled compared with the prior level, the live bacteria number of the freeze-dried powder is obviously improved, the survival rate and the stability of the bacterial powder are good, and the production level is obviously improved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12.
Background
Lactobacillus gasseri (Lactobacillus gasseri) is a common species of probiotic bacteria, which is widely present in the gastrointestinal tract of humans and animals. The lactobacillus gasseri belongs to gram-positive bacillus, lactobacillus, does not have catalase, oxidase and motility, can grow in aerobic and anaerobic environments, belongs to facultative heterogeneous acidic strains, does not generate gas during glucose metabolism, and has a spherical or oval shape, no spores and no capsules. Can grow in an acid environment with the pH value of 4.0-5.0 or even lower, the optimal growth temperature is 35-38 ℃, and the fermentation product L- (+) -lactic acid is the main.
The lactobacillus gasseri is obtained by separating and purifying digestive tracts of healthy newborns, belongs to a human body protospecies, and can exist in gastric juice and secrete lactic acid, acetic acid and the like to promote the digestive ability of the stomach; in addition, the lactobacillus gasseri can adjust the balance of intestinal flora, inhibit the proliferation of undesirable microorganisms in the intestinal tract and generate antagonism on intestinal pathogenic bacteria. As probiotics, the Lactobacillus gasseri also has the functions of enhancing the immunity of the organism, promoting the absorption of nutrients, improving the hematopoietic function, discharging enterotoxins, delaying aging, resisting allergy and the like. Because probiotics have the advantages of no residue, no drug resistance and the like, the probiotics are generally applied to the production of animal feeds at present and are generally considered as promising substitutes of antibiotics. The factors influencing the quality of the lactobacillus gasseri powder comprise the number of viable bacteria of the powder, the stability of the powder and the survival rate of gastrointestinal fluid, and the number of the viable bacteria of the powder is determined by the survival rate of the lactobacillus gasseri in the fermentation and freeze-drying processes.
Therefore, it is necessary to design a method for realizing high-density fermentation of lactobacillus gasseri and improving the quality of bacterial powder.
Disclosure of Invention
The invention breaks through the difficult problems in the prior art and designs a method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12.
In order to achieve the aim, the invention designs a method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12, which comprises the following steps:
step 1: soaking shrimp powder and DB7801 in water for 2 hr, boiling at 105 deg.C for 30 min, centrifuging at 6000rpm for 8min, and collecting supernatant;
step 2: preparing an MRSA culture medium;
and step 3: putting 200mL of MRSA culture medium into a 250mL flat-bottomed bottle, inoculating a stored glycerol tube of the Lactobacillus gasseri LG-G12, placing the glycerol tube into a constant-temperature incubator at 37 ℃ for standing culture for 12 hours, transferring 2% of glycerol tube into another fresh 200mL of MRSA culture medium, and placing the glycerol tube into the constant-temperature incubator at 37 ℃ for standing culture for 6 to 8 hours to obtain a seed solution of the Lactobacillus gasseri LG-G12;
and 4, step 4: preparing a fermentation medium by using the supernatant and the shrimp meal which are finished in the step 1;
and 5: placing 200mL fermentation medium in 250mL flat-bottomed bottle, inoculating 2% of the Lactobacillus gasseri LG-G12 seed solution obtained in step 3, standing and culturing in a constant temperature incubator at 37 deg.C for 12 hr, and detecting OD600pH and viable count of the fermentation broth;
step 6: optimizing the formula of the culture medium according to the detection data in the step 5: preparing another fermentation medium, putting the fermentation medium into a fermentation tank for fermentation culture, adding 5G/L calcium carbonate on the basis of the another fermentation medium, wherein the content of the medium is 4L, the pH of the initial medium is 7.2, the sterilization and disinfection conditions are 121 ℃ and 25 minutes, cooling to 36-38 ℃, inoculating 2-3% of inoculum size into the Lactobacillus gasseri LG-G12 seed solution, keeping the temperature at 37 ℃, stirring at 80rpm, introducing nitrogen to maintain the pressure of the fermentation tank at 0.03MPa, detecting the light absorption value and the pH value of the fermentation liquor at the wavelength of 600nm during the period, stopping fermentation when the pH value does not fall or falls to about 4.4, sampling and centrifuging to obtain bacterial sludge;
and 7: preparing one or more protective agents, sterilizing the protective agents at 115 ℃ for 15 minutes, and quickly cooling to 10-20 ℃ for later use after sterilization;
and 8: according to the bacterial sludge: the protective agent is 1:2, emulsifying and carrying out vacuum freeze drying to obtain freeze-dried powder of lactobacillus gasseri LG-G12;
and step 9: the freeze-dried powder of the lactobacillus gasseri LG-G12 is respectively stored in the environment of 37 ℃, 25 ℃, 4 ℃ and-18 ℃, and the viable count in the MRSA culture medium is respectively sampled and detected in 0 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months, and the viable count change of the freeze-dried powder is detected.
The MRSA medium includes: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate and 0.05 g/L of manganese sulfate.
The fermentation medium comprises: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 2g/L of cysteine amine salt, 0-5 g/L of shrimp meal, 0-10 g/L of shrimp meal supernatant and 0-10 g/L of soybean protein powder DB7801 supernatant for fermentation.
In the step 6, the centrifugation conditions are that the temperature is 4 ℃, the rotating speed is 8000rpm, and the centrifugation time is 8 min.
The conditions of vacuum freeze drying in step 7 are as follows: firstly, pre-freezing for 6 hours at the temperature of minus 80 ℃, and then, freezing and drying for 25 to 32 hours at the temperature of a cold trap lower than minus 57 ℃ and under the vacuum degree of 0.1 Pa.
The formula of the protective agent in the step 8 comprises the following steps: 8-14% of trehalose, 4-8% of sucrose, 2-8% of dextrin, 1-4% of glucose, 1-4% of pectin and 73% of water.
Lactobacillus gasseri LG-G12 (Lactobacillus gasseri LG-G12) with the storage location: china center for type culture Collection, the collection numbers are: CCTCC NO of M2019829, and the preservation time is 10 months and 17 days in 2019.
Compared with the prior art, the viable count of the fermentation liquor of the lactobacillus gasseri LG-G12 is high, no impurity is precipitated, the yield of the freeze-dried powder is doubled compared with the prior level, the viable count of the freeze-dried powder is obviously improved and reaches more than 3000 hundred million cfu/G, the survival rate and the stability of the bacterial powder are good, and the production level is obviously improved.
Drawings
FIG. 1 shows the survival rate of the live bacteria of the freeze-dried bacterial powder of the present invention under different protective agents.
FIG. 2 shows the survival rates of freeze-dried powder of Lactobacillus gasseri LG-G12 of the present invention at different temperatures.
Detailed Description
The present invention will now be described.
The Lactobacillus gasseri LG-G12 (Lactobacillus gasseri LG-G12) adopted in the invention has the following preservation places: china center for type culture Collection, the collection numbers are: CCTCC NO of M2019829, and the preservation time is 10 months and 17 days in 2019. The address of the depository: wuhan in China.
The invention designs a method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12, which comprises the following steps:
step 1: soaking shrimp powder and DB7801 in water for 2 hr, boiling at 105 deg.C for 30 min, centrifuging at 6000rpm for 8min, and collecting supernatant;
step 2: preparing an MRSA culture medium; the MRSA medium includes: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate and 0.05 g/L of manganese sulfate;
and step 3: putting 200mLMRSA culture medium into a 250mL flat-bottomed bottle, inoculating a stored glycerol tube of the Lactobacillus gasseri LG-G12, placing the glycerol tube into a constant-temperature incubator at 37 ℃ for standing culture for 12 hours, transferring 2 percent of glycerol tube into another fresh 200mL MRSA culture medium, placing the glycerol tube into the constant-temperature incubator at 37 ℃ for standing culture for 6 to 8 hours to obtain a seed solution of the Lactobacillus gasseri LG-G12;
and 4, step 4: preparing a fermentation medium by using the supernatant and the shrimp meal which are finished in the step 1; the fermentation medium comprises: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 2g/L of cysteine amine salt, 0-5 g/L of shrimp meal, 0-10 g/L of shrimp meal supernatant and 0-10 g/L of soybean protein powder DB7801 supernatant for fermentation;
and 5: placing 200mL fermentation medium in 250mL flat-bottomed bottle, inoculating 2% of the Lactobacillus gasseri LG-G12 seed solution obtained in step 3, standing and culturing in a constant temperature incubator at 37 deg.C for 12 hr, and detecting OD600pH and viable count of the fermentation broth;
step 6: optimizing the formula of the culture medium according to the detection data in the step 5: preparing another fermentation medium, putting the fermentation medium into a fermentation tank for fermentation culture, adding 5G/L calcium carbonate on the basis of the fermentation medium, wherein the content of the culture medium is 4L, the pH of the initial culture medium is 7.2, the sterilization and disinfection conditions are that the temperature is 121 ℃ and the time is 25 minutes, cooling to 36-38 ℃, inoculating 2-3% of inoculum size into the Lactobacillus gasseri LG-G12 seed solution, keeping the temperature at 37 ℃, stirring at the rotating speed of 80rpm, introducing nitrogen to maintain the pressure of the fermentation tank at 0.03MPa, and detecting the light absorption value (OD) of the fermentation liquid at the wavelength of 600nm during the period600) And the pH value is reduced to about 4.4 or not, the fermentation is stopped for about 12-14 h, and the bacteria mud is obtained after the sampling and centrifugation are carried out under the conditions that the temperature is 4 ℃, the rotating speed is 8000rpm and the centrifugation time is 8 min;
and 7: preparing one or more protective agents, sterilizing the protective agents at 115 ℃ for 15 minutes, and quickly cooling to 10-20 ℃ for later use after sterilization, wherein the formula of the protective agents comprises: 8-14% of trehalose, 4-8% of sucrose, 2-8% of dextrin, 1-4% of glucose, 1-4% of pectin and 73% of water, and the specific formula is shown in the following table:
| protectant numbering | A | B | C | D | E | F |
| Seaweed (Sargassum)Candy | 8% | 10% | 11% | 12% | 13% | 14% |
| Sucrose | 8% | 6% | 5% | 4% | 3% | 2% |
| Dextrin | 7% | 8% | 6% | 5% | 4% | 6% |
| Glucose | 2% | 1% | 3% | 3% | 4% | 4% |
| Pectin | 4% | 2% | 2% | 3% | 3% | 1% |
| Water (W) | 73% | 73% | 73% | 73% | 73% | 73% |
And 8: according to the bacterial sludge: the protective agent is 1:2, emulsifying and vacuum freeze-drying, wherein the conditions of vacuum freeze-drying are as follows: firstly, pre-freezing for 4 hours at the temperature of minus 80 ℃, and then freeze-drying for 22-30 hours at the temperature of a cold trap lower than minus 55 ℃ and under the vacuum degree of 0.1Pa to obtain freeze-dried powder of Lactobacillus gasseri LG-G12;
and step 9: respectively storing in the environment of 37 deg.C, 25 deg.C, 4 deg.C and-18 deg.C, respectively sampling and detecting viable count in MRSA culture medium at 0 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months, and detecting viable count change of lyophilized powder of Lactobacillus gasseri LG-G12.
In the specific implementation, the existing viable count of the lactobacillus gasseri fermentation liquor, viable count of the bacterial powder and yield of the bacterial powder are used as controls and compared with experimental groups; and (3) inspecting the stability of the obtained high-quality Lactobacillus gasseri LG-G12 powder, and the tolerance and the survival rate of the powder in artificial simulated gastrointestinal fluid.
The preserved Lactobacillus gasseri LG-G12 Glycine 1 group was used as the primordial seed for the control group and the experimental group.
Soaking shrimp powder and fermented soybean protein powder DB7801 in water for 2 hr, boiling at 105 deg.C for 30 min, centrifuging at 6000rpm for 8min, and collecting supernatant.
Adopting an MRSA culture medium formula: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate and 0.05 g/L of manganese sulfate, and the fermentation medium is prepared into L1.
5g/L of shrimp meal to be used, 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate and 2g/L of cysteine amine salt are combined to prepare a fermentation culture medium L2.
Taking 10g/L of shrimp powder supernatant to be used, 10g/L of soybean protein powder DB7801 supernatant for fermentation, 20 g/L of combined glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium hydrogen phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate and 2g/L of cysteine amino acid salt, and preparing a fermentation culture medium L3.
And respectively putting 200mL of the three groups of culture media into three 250mL flat-bottomed bottles, inoculating 2% of Lactobacillus gasseri LG-G12 seed solution, placing the seed solution in a constant-temperature incubator at 37 ℃ for standing culture for 12 hours, and detecting the light absorption value, the pH value and the viable count of the fermentation liquor at the wavelength of 600 nm. The following table is obtained:
| fermentation medium | L1 | L2 | L3 |
| pH | 4.56 | 4.39 | 4.40 |
| OD600 | 5.13 | A small amount of precipitate | 6.16 |
| Fermentation liquor viable bacteria cfu/mL | 10.2 hundred million | 20.5 hundred million | 19.8 hundred million |
From the data in the table, it is clear that the viable cell counts of L2 and L3 are significantly higher than those of L1 compared to the control L1. Performing fermentation tank culture according to the formula of three groups of fermentation culture media, adding 5G/L calcium carbonate on the basis of the three groups of fermentation culture media, wherein the loading amount of the culture media is 4L, the pH of an initial culture medium is 7.2, the sterilization temperature is 121 ℃, the sterilization temperature is 25min, and after cooling, inoculating 2-3% of inoculation amount of the lactobacillus gasseri LG-G12 seed solution; keeping the temperature at 37 ℃, stirring at the rotating speed of 80rpm, introducing nitrogen to maintain the pressure of the fermentation tank at 0.03MPa, detecting the light absorption value and pH value of the fermentation liquor at the wavelength of 600nm during the period, stopping fermentation when the pH value does not drop or drops to about 4.4, sampling, centrifuging and emulsifying, wherein the centrifugation conditions are as follows: centrifuging at 4 deg.C and 8000rpm for 8min to obtain bacterial sludge. According to the bacterial sludge: the protective agent is 1:2, emulsifying and vacuum freeze-drying, wherein the freeze-drying conditions are as follows: pre-freezing for 4h at-80 deg.C, freeze-drying for 22-30 hr at cold trap temperature lower than-55 deg.C and vacuum degree of 0.1Pa to obtain lyophilized powder, and comparing viable count and yield of three groups of lyophilized powder:
| control group | Experimental group | Experimental group | |
| Fermentation medium | L1 | L2 | L3 |
| Fermentation period/h | 13.5 | 14 | 14.5 |
| Yield g/L of wet bacterial sludge | 6 | 13 | 9 |
| Live bacteria of fermentation liquor are hundreds of millions/mL | 13.9 | 39.3 | 38.6 |
| Freeze-dried powder g/L | 4.2 | 8.5 | 7.8 |
| Bacteria powder with hundreds of millions of cfu/g of live bacteria | 1600 | 2800 | 3000 |
According to data analysis in the table, compared with a control group L1, the number of live bacteria in the fermentation liquid of the L2 experimental group is the highest, impurities exist in the fermentation liquid, and the yield of freeze-dried powder is the highest; the freeze-dried powder of the L3 experimental group has the highest viable count, and the fermentation liquid has no impurities. The viable count of L3 groups of lyophilized bacteria powder reaches 3000 hundred million cfu/g, which is significantly improved compared with the existing production level, and the L3 group of fermentation medium is optimal under comprehensive consideration.
Preparing one or more protective agents, and using the bacterial sludge obtained by the experimental group L3 according to the ratio of bacterial sludge: emulsifying and vacuum freeze-drying the protective agent in a ratio of 1:2, wherein the freeze-drying conditions are as follows: firstly, pre-freezing for 4h at the temperature of minus 80 ℃, then, freezing and drying for 22 to 30 hours at the temperature of a cold trap lower than minus 55 ℃ and under the vacuum degree of 0.1Pa to obtain various freeze-dried bacterial powders, and inspecting the living bacterial survival rate of the various freeze-dried bacterial powders, as shown in figure 1. Through experiments, the protective agent with the highest survival rate in the freeze-drying process of the emulsified lactobacillus gasseri LG-G12 bacterial mud is obtained to comprise: 11% of trehalose, 5% of sucrose, 6% of dextrin, 3% of glucose, 2% of pectin and 73% of water; the viable count of the freeze-dried powder of the lactobacillus gasseri LG-G12 can reach 3500 hundred million cfu/G at most.
200G of freeze-dried powder of the prepared Lactobacillus gasseri LG-G12 are randomly extracted, 50G of the freeze-dried powder is correspondingly stored in the environment with the temperature of 37 ℃, 25 ℃, 4 ℃ and-18 ℃ respectively, and the viable count in the MRSA culture medium is detected in 0 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months. Through the detection of the obtained data, as can be seen from the figure 2, the freeze-dried powder of lactobacillus gasseri LG-G12 has a good storage effect in the environment of 25 ℃, 4 ℃ and-18 ℃, and basically has a survival rate of 50% after being placed for 2 months and tends to be stable; after being stored in an environment at 37 ℃ and being placed for 3 months, the survival rate of the product is 10 percent and is obviously better than that of the similar products under the condition.
Inoculating the preserved Lactobacillus gasseri LG-G12 glycerol tube seed into an MRSA culture medium, culturing at 37 ℃ for 12h, carrying out amplification culture on the culture medium for 2 times, taking Lactobacillus gasseri LG-G12 fermentation liquor, centrifuging at 8000rpm for 8 minutes, collecting thallus, and suspending in artificial simulated gastric juice with the pH of 2.5, wherein the artificial simulated gastric juice is as follows: MRSA culture medium containing 1.5% pepsin and pH2.5, culturing at 37 deg.C, sampling at 0h, 1h, 2h and 3h, respectively, pouring and culturing with MRSA agar culture medium, counting plates, measuring viable bacteria count and calculating survival rate. Taking the fermentation liquor of Lactobacillus gasseri LG-G12, centrifuging at 8000rpm for 8 minutes, collecting the thallus, and suspending in artificial simulated intestinal fluid which is: culturing in MRSA culture medium containing 0.5% ox bile salt, 1.5% trypsin, and pH8.0 at 37 deg.C for 0h, 1h, 2h, 3h, 4h, and 5h, respectively, pouring and culturing in MRSA agar culture medium, counting plates, determining viable bacteria count, and calculating survival rate. The survival rate is the ratio of the viable count at the time of sampling in the medium to the viable count at 0h, expressed in%, and the specific data are shown in the following table. As can be seen from the data in the two tables, the Lactobacillus gasseri LG-G12 has better tolerance and survival rate in artificial simulated gastrointestinal fluids.
The tolerance and survival rate of Lactobacillus gasseri LG-G12 in simulated gastric fluid are shown in the following table:
the tolerance and survival rate of Lactobacillus gasseri LG-G12 in artificially simulated intestinal fluid are shown in the following table:
Claims (7)
1. a method for high-density fermentation and bacterial powder quality improvement of Lactobacillus gasseri LG-G12 is characterized by comprising the following steps: the method comprises the following steps:
step 1: soaking shrimp powder and DB7801 in water for 2 hr, boiling at 105 deg.C for 30 min, centrifuging at 6000rpm for 8min, and collecting supernatant;
step 2: preparing an MRSA culture medium;
and step 3: putting 200mL of MRSA culture medium into a 250mL flat-bottomed bottle, inoculating a stored glycerol tube of the Lactobacillus gasseri LG-G12, placing the glycerol tube into a constant-temperature incubator at 37 ℃ for standing culture for 12 hours, transferring 2% of glycerol tube into another fresh 200mL of MRSA culture medium, and placing the glycerol tube into the constant-temperature incubator at 37 ℃ for standing culture for 6 to 8 hours to obtain a seed solution of the Lactobacillus gasseri LG-G12;
and 4, step 4: preparing a fermentation medium by using the supernatant and the shrimp meal which are finished in the step 1;
and 5: placing 200mL fermentation medium in 250mL flat-bottomed bottle, inoculating 2% of the Lactobacillus gasseri LG-G12 seed solution obtained in step 3, standing and culturing in a constant temperature incubator at 37 deg.C for 12 hr, and detecting OD600pH and viable count of the fermentation broth;
step 6: optimizing the formula of the culture medium according to the detection data in the step 5: preparing another fermentation medium, putting the fermentation medium into a fermentation tank for fermentation culture, adding 5G/L calcium carbonate on the basis of the another fermentation medium, wherein the content of the medium is 4L, the pH of the initial medium is 7.2, the sterilization and disinfection conditions are 121 ℃ and 25 minutes, cooling to 36-38 ℃, inoculating 2-3% of inoculum size into the Lactobacillus gasseri LG-G12 seed solution, keeping the temperature at 37 ℃, stirring at 80rpm, introducing nitrogen to maintain the pressure of the fermentation tank at 0.03MPa, detecting the light absorption value and the pH value of the fermentation liquor at the wavelength of 600nm during the period, stopping fermentation when the pH value does not fall or falls to about 4.4, sampling and centrifuging to obtain bacterial sludge;
and 7: preparing one or more protective agents, sterilizing the protective agents at 115 ℃ for 15 minutes, and quickly cooling to 10-20 ℃ for later use after sterilization;
and 8: according to the bacterial sludge: emulsifying and vacuum freeze-drying the protective agent at a ratio of 1:2 to obtain freeze-dried powder of Lactobacillus gasseri LG-G12;
and step 9: the freeze-dried powder of the lactobacillus gasseri LG-G12 is respectively stored in the environment of 37 ℃, 25 ℃, 4 ℃ and-18 ℃, and the viable count in the MRSA culture medium is respectively sampled and detected in 0 day, 1 week, 2 weeks, 3 weeks, 1 month, 2 months and 3 months, and the viable count change of the freeze-dried powder is detected.
2. The method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 according to claim 1, wherein the method comprises the following steps: the MRSA culture medium comprises: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate and 0.05 g/L of manganese sulfate.
3. The method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 according to claim 1, wherein the method comprises the following steps: the fermentation medium comprises: 20 g/L of glucose, 10g/L of beef extract powder, 10g/L of peptone, 5g/L of yeast extract, 5g/L of anhydrous sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 801 g/L of tween-801, 0.25 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 2g/L of cysteine amine salt, 0-5 g/L of shrimp meal, 0-10 g/L of shrimp meal supernatant and 0-10 g/L of soybean protein powder DB7801 supernatant for fermentation.
4. The method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 according to claim 1, wherein the method comprises the following steps: the centrifugation conditions in the step 6 are that the temperature is 4 ℃, the rotating speed is 8000rpm, and the centrifugation time is 8 min.
5. The method for high-density fermentation and bacterial powder quality improvement of Lactobacillus gasseri LG-G12 according to claim 1, wherein the protective agent formulation in step 7 comprises: 8-14% of trehalose, 4-8% of sucrose, 2-8% of dextrin, 1-4% of glucose, 1-4% of pectin and 73% of water.
6. The method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 according to claim 1, wherein the method comprises the following steps: the vacuum freeze-drying conditions in the step 8 are as follows: firstly, pre-freezing for 6 hours at the temperature of minus 80 ℃, and then, freezing and drying for 25 to 32 hours at the temperature of a cold trap lower than minus 57 ℃ and under the vacuum degree of 0.1 Pa.
7. The method for high-density fermentation and bacterial powder quality improvement of lactobacillus gasseri LG-G12 according to claim 1, wherein the method comprises the following steps: the Lactobacillus gasseri LG-G12 (Lactobacillus gasseri LG-G12) has the following storage places: china center for type culture Collection, the collection numbers are: CCTCC NO of M2019829, and the preservation time is 10 months and 17 days in 2019.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114145460A (en) * | 2020-09-08 | 2022-03-08 | 鲁南制药集团股份有限公司 | Lactic acid bacteria, composition containing same and application |
| CN116574629A (en) * | 2022-11-16 | 2023-08-11 | 润盈生物工程（上海）有限公司 | Probiotics beneficial to reproductive health of obese men and application thereof |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008052468A1 (en) * | 2006-11-01 | 2008-05-08 | Sine Pharmaceutical Laboratories | New lactobacillus rhamnosus strain, its pharmaceutical composition and the uses thereof, and the method for preparation |
| CN101880644A (en) * | 2010-05-28 | 2010-11-10 | 广州立达尔生物科技有限公司 | Liquid submerged fermentation method for lactobacillus gasseri |
| CN106754482A (en) * | 2016-11-28 | 2017-05-31 | 江苏梁丰食品集团有限公司 | A kind of preparation method of the probiotics leaven for producing functional sugar |
| CN107841463A (en) * | 2017-12-18 | 2018-03-27 | 江南大学 | A kind of high activity lactobacillus bacterium powder vacuum freezedrying production technology and application |
| CN107868768A (en) * | 2017-12-18 | 2018-04-03 | 江南大学 | A kind of high activity lactobacillus bacterium powder vacuum drying production technology and application |
| CN108018235A (en) * | 2017-12-18 | 2018-05-11 | 江南大学 | A kind of high activity lactobacillus bacterium powder spray drying production technology and application |
| CN108559717A (en) * | 2017-11-30 | 2018-09-21 | 天津生机集团股份有限公司 | A kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method |
| CN109355231A (en) * | 2018-11-28 | 2019-02-19 | 江苏微康生物科技有限公司 | The preparation method of organic probiotics bacterial powder |
| KR101951893B1 (en) * | 2018-11-27 | 2019-02-25 | 주식회사 셀로닉스 | Method for producing high concentration of probiotic active Lactobacillus paracasei SRCM102343 strain derived from traditional fermented food |
| CN109666599A (en) * | 2018-11-22 | 2019-04-23 | 杭州娃哈哈科技有限公司 | A kind of lactobacillus reuteri high density fermentation culture medium and fermentation process, application |
| CN109749957A (en) * | 2019-01-11 | 2019-05-14 | 江苏省苏微微生物研究有限公司 | A kind of preparation and application of the lactobacillus gasseri preparation with aquatic pathogenic bacterium antagonistic properties |
| CN109749964A (en) * | 2019-02-26 | 2019-05-14 | 润盈生物工程（上海）有限公司 | Improve the medium treatment method of lactobacillus acidophilus fermentation and freeze drying viable microorganism number |
| CN110305811A (en) * | 2019-06-28 | 2019-10-08 | 天津生机集团股份有限公司 | A kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application |
| CN110577912A (en) * | 2019-09-24 | 2019-12-17 | 华南理工大学 | lactobacillus gasseri and application thereof in preparing fermented milk |
-
2020
- 2020-08-14 CN CN202010816658.5A patent/CN112063547A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008052468A1 (en) * | 2006-11-01 | 2008-05-08 | Sine Pharmaceutical Laboratories | New lactobacillus rhamnosus strain, its pharmaceutical composition and the uses thereof, and the method for preparation |
| CN101880644A (en) * | 2010-05-28 | 2010-11-10 | 广州立达尔生物科技有限公司 | Liquid submerged fermentation method for lactobacillus gasseri |
| CN106754482A (en) * | 2016-11-28 | 2017-05-31 | 江苏梁丰食品集团有限公司 | A kind of preparation method of the probiotics leaven for producing functional sugar |
| CN108559717A (en) * | 2017-11-30 | 2018-09-21 | 天津生机集团股份有限公司 | A kind of lactobacillus acidophilus high density fermentation culture medium and its bacterium powder preparation method |
| CN107841463A (en) * | 2017-12-18 | 2018-03-27 | 江南大学 | A kind of high activity lactobacillus bacterium powder vacuum freezedrying production technology and application |
| CN108018235A (en) * | 2017-12-18 | 2018-05-11 | 江南大学 | A kind of high activity lactobacillus bacterium powder spray drying production technology and application |
| CN107868768A (en) * | 2017-12-18 | 2018-04-03 | 江南大学 | A kind of high activity lactobacillus bacterium powder vacuum drying production technology and application |
| CN109666599A (en) * | 2018-11-22 | 2019-04-23 | 杭州娃哈哈科技有限公司 | A kind of lactobacillus reuteri high density fermentation culture medium and fermentation process, application |
| KR101951893B1 (en) * | 2018-11-27 | 2019-02-25 | 주식회사 셀로닉스 | Method for producing high concentration of probiotic active Lactobacillus paracasei SRCM102343 strain derived from traditional fermented food |
| CN109355231A (en) * | 2018-11-28 | 2019-02-19 | 江苏微康生物科技有限公司 | The preparation method of organic probiotics bacterial powder |
| CN109749957A (en) * | 2019-01-11 | 2019-05-14 | 江苏省苏微微生物研究有限公司 | A kind of preparation and application of the lactobacillus gasseri preparation with aquatic pathogenic bacterium antagonistic properties |
| CN109749964A (en) * | 2019-02-26 | 2019-05-14 | 润盈生物工程（上海）有限公司 | Improve the medium treatment method of lactobacillus acidophilus fermentation and freeze drying viable microorganism number |
| CN110305811A (en) * | 2019-06-28 | 2019-10-08 | 天津生机集团股份有限公司 | A kind of lactobacillus plantarum microbial inoculum and its high density fermentation preparation method and application |
| CN110577912A (en) * | 2019-09-24 | 2019-12-17 | 华南理工大学 | lactobacillus gasseri and application thereof in preparing fermented milk |
Non-Patent Citations (4)
| Title |
|---|
| 付艳茹;靳烨;: "保加利亚乳杆菌菌粉生产工艺研究", 食品科技, vol. 34, no. 07, pages 14 - 17 * |
| 张竞竞 等: "南极磷虾粉培养枯草芽孢杆菌工艺探究", 南方农业, vol. 13, no. 23, pages 1 * |
| 陈晓华;刘小鸣;王刚;田丰伟;张灏;陈卫;: "一株具有拮抗幽门螺杆菌作用的格氏乳杆菌生物学性质及其在发酵乳中应用", 食品工业科技, vol. 36, no. 21, pages 148 - 151 * |
| 黄沧海, 陈东晓: "仔猪胃源格氏乳酸杆菌生物学特性的研究", 饲料工业, vol. 26, no. 02, pages 33 - 36 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114145460A (en) * | 2020-09-08 | 2022-03-08 | 鲁南制药集团股份有限公司 | Lactic acid bacteria, composition containing same and application |
| CN116574629A (en) * | 2022-11-16 | 2023-08-11 | 润盈生物工程（上海）有限公司 | Probiotics beneficial to reproductive health of obese men and application thereof |
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