CN102952755A - Lactobacillus bulgaricus freeze-drying protecting agent and preparation method thereof - Google Patents
Lactobacillus bulgaricus freeze-drying protecting agent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a lactobacillus bulgaricus freeze-drying protecting agent and a preparation method thereof. The lactobacillus bulgaricus freeze-drying protecting agent comprises the following components in parts by mass: 20-30 parts of lactose, 20-30 parts of dried skim milk, 3-5 parts of sodium ascorbate, 0.2-0.6 part of polyethylene glycol 4000, 0.2-0.8 part of polyvinylpyrrolidone, 0.5-2.0 parts of bovine serum albumin and 100 parts of distilled water. The freeze-drying protecting agent is added in a powder preparation process, so that the anti-freeze capacity of the thalli can be improved, thereby improving the freeze-dry survival rate of the powder and guaranteeing long storage period and low probability for reduction of the survival rate.
Description
Technical field
The invention belongs to lactobacillus bulgaricus Techniques of preserving field, relate to a kind of lactobacillus bulgaricus lyophilized vaccine and preparation method thereof.
Background technology
Lactobacillus bulgaricus (Lactobacillus bulgaricus) is one of physiology flora very important in normal human's enteron aisle, have the human body of keeping microecological balance, promoting digestion, cancer-resisting, strengthening immunity, the anti-ageing effect of waiting for a long time, closely bound up with body health.
Vacuum Freezing ﹠ Drying Technology is mainly adopted in the at present preparation of lactobacillus bulgaricus bacterium powder, if but lactobacillus bulgaricus directly carries out lyophilize, can cause inevitably the dead or damage of part cell, and very unfavorable to producing.Report that its survival rate is only about 10%-30% if its nutrient solution is directly carried out lyophilize.
And the factor that affects the cryodesiccated survival rate of thalline is many-sided.Comprise bacterial strain, cell size and shape, growth phase and growth velocity, initial bacteria suspension concentration, substratum composition, pH value, protective material, pre-freeze condition, rate of temperature fall, lyophilisation condition, cell water content, cytolemma composition and rehydration condition etc., wherein protectant impact is the most outstanding.A kind of effective protective material requires can provide provide protection in freezing dry process; physics when it can change the sample lyophilize, chemical environment; alleviate or prevent freezing dry process to the infringement of thalline, keep as far as possible the original various physio-biochemical characteristics of thalline and biological activity.
Have much about the protectant report of lactobacillus bulgaricus, mainly concentrate on medical science and field of biology.Pu Lili etc. have studied the provide protection of kinds of protect agent to lactobacillus bulgaricus, find that the protection effect of trehalose is best, and it can significantly suppress the morphological instability that ice crystal generates.In fermented liquid, add protective material can the freezing-inhibiting drying process in the generation of eutectic phenomena, improve the freezing tolerance of thalline.Hou Minna etc. study the lactobacillus bulgaricus protective material with orthogonal experimental method.The result shows that 10% sucrose, 5% lactose, 3% glycerine and 0.4% Sodium Glutamate are the optimum formula combination.The lactobacillus bulgaricus lyophilized vaccine has also been optimized in the screening of the employing response surface analysis methods such as Zhang Zesheng, finds that skimming milk, trehalose, inulin, maltodextrin are given prominence to the bacterial classification provide protection in numerous materials.Tian Fengwei etc. have studied the impact of lyophilized vaccine composition on lactobacillus bulgaricus 18 lyophilize survival rates and vigor.Experiment shows that 3% restores the lyophilized vaccine of skimming milk, 3% lactose, 3% glycerine composition, makes the freeze-drying survival rate reach 68.9%, has faster acid producing ability.A quintessence of Northeast Agricultural University waits by single factor comparison and orthogonal experimental design, determines to be fit to the best frozen-dried protective agent prescription of lactobacillus bulgaricus: skimmed milk powder 8%, trehalose 1%, glycerine 2%, Sodium Glutamate 1.5%.
Summary of the invention
The problem that the present invention solves is to provide a kind of lactobacillus bulgaricus lyophilized vaccine and preparation method thereof; for the preparation of high reactivity bacterium powder; in the process of preparation bacterium powder, add lyophilized vaccine, can improve the freezing tolerance of thalline, thereby improve the freeze-drying survival rate of bacterium powder.
The present invention is achieved through the following technical solutions:
A kind of lactobacillus bulgaricus lyophilized vaccine in mass fraction, comprises following component:
100 parts of 20～30 parts of lactose, 20～30 parts of skimmed milk powders, 3～5 parts of sodium ascorbates, 0.2～0.6 part of Macrogol 4000,0.2～0.8 part of polyvinylpyrrolidone, 0.5～2.0 part of bovine serum albumin and distilled water.
Further, in mass fraction, comprise following component:
100 parts of 20～26 parts of lactose, 24～30 parts of skimmed milk powders, 4.4～5 parts of sodium ascorbates, 0.2～0.4 part of Macrogol 4000,0.3～0.6 part of polyvinylpyrrolidone, 0.5～1.0 part of bovine serum albumin and distilled water.
A kind of preparation method of lactobacillus bulgaricus lyophilized vaccine may further comprise the steps:
1) in mass fraction, in distilled water, add 20～30 parts lactose and 20～30 parts skimmed milk powder, be 6.9～7.1 with regulating pH after the heating for dissolving, sterilization is cooled to room temperature;
2) in mass fraction, 0.2～0.8 part polyvinylpyrrolidone is dissolved in the distilled water, sterilization is cooled to room temperature;
3) in mass fraction, 3～5 parts sodium ascorbates, 0.2～0.6 part Macrogol 4000 and 0.5～2.0 part bovine serum albumin are dissolved in the stroke-physiological saline solution, then use the membrane filtration degerming;
4) with step 1), 2), 3) prepared solution mixes, and the total mass umber of adjusting water is 100 parts, obtains the lactobacillus bulgaricus lyophilized vaccine.
The preparation method of described lactobacillus bulgaricus lyophilized vaccine may further comprise the steps:
1) in mass fraction, in distilled water, add 20～26 parts lactose and 24～30 parts skimmed milk powder, be 6.9～7.1 with regulating pH after the heating for dissolving, sterilization is cooled to room temperature;
2) in mass fraction, 0.3～0.6 part polyvinylpyrrolidone is dissolved in the distilled water, sterilization is cooled to room temperature;
3) in mass fraction, 4.4～5 parts sodium ascorbates, 0.2～0.4 part Macrogol 4000 and 0.5～1.0 part bovine serum albumin are dissolved in the stroke-physiological saline solution, then use the membrane filtration degerming;
4) with step 1), 2), 3) prepared solution mixes, and the total mass umber of adjusting water is 100 parts, obtains the lactobacillus bulgaricus lyophilized vaccine.
Described step 1), step 2) sterilization be 100～115 ℃ the sterilization 10～15min.
Described lactobacillus bulgaricus lyophilized vaccine and lactobacillus bulgaricus mud equal-volume are mixed, in-40 ℃ of pre-freeze 3～12h, then-50～-56 ℃ lyophilize 18～24h obtains lactobacillus bulgaricus bacterium powder.
Compared with prior art, the present invention has following useful technique effect:
Lactobacillus bulgaricus lyophilized vaccine provided by the invention adds lyophilized vaccine in the process of preparation bacterium powder, can improve the freezing tolerance of thalline, thereby improve the freeze-drying survival rate of bacterium powder, and preservation term is longer, and survival rate is difficult for reducing.
Lactobacillus bulgaricus lyophilized vaccine provided by the invention, behind the adding lyophilized vaccine, the freeze-drying survival rate reaches 53.5～58.567% in the prepared lactobacillus bulgaricus bacterium powder in the process of preparation bacterium powder, and bacterium powder viable count is 0.916 * 10
11～1.066 * 10
11Cfu/g.
Lactobacillus bulgaricus lyophilized vaccine provided by the invention, its main raw material are lactose and skimmed milk powder, and raw material is easy to get, cost is low.The lactobacillus bulgaricus freeze dried fermenting preparation that adopts this composite protectant to make can be used as the throw type leaven of producing the fermentation using lactobacillus bulgaricus milk-product and uses.
Embodiment
The invention provides a kind of lactobacillus bulgaricus lyophilized vaccine and preparation method thereof, for the preparation of high reactivity bacterium powder, in the process of preparation bacterium powder, add lyophilized vaccine, can improve the freezing tolerance of thalline, thereby improve the freeze-drying survival rate of bacterium powder.The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
Embodiment 1
Consisting of of lactobacillus bulgaricus lyophilized vaccine: lactose 22g, skimmed milk powder 26g, sodium ascorbate 4.6g, Macrogol 4000 0.3g, polyvinylpyrrolidone PVP 0.4g, bovine serum albumin BSA 0.7g, distilled water 100ml.
Its preparation method:
1) will mix after lactose and the skimmed milk powder heating for dissolving according to quantity, and regulate pH 6.9 with NaOH solution, then through 110 ℃ of sterilization 15min, be cooled to room temperature;
2) take by weighing according to quantity polyvinylpyrrolidone PVP, be dissolved in the distilled water, 115 ℃ of sterilization 30min are cooled to room temperature again;
3) take by weighing respectively according to quantity sodium ascorbate, Macrogol 4000, bovine serum albumin BSA and be dissolved in the 0.85-0.9% stroke-physiological saline solution, with 0.22 μ m membrane filtration degerming;
4) with step 1), 2) solution of two moist heat sterilizations mixes according to quantity, and under aseptic condition, add sodium ascorbate, the filtration sterilization aqueous solution of Macrogol 4000, bovine serum albumin BSA, mixing namely gets the lactobacillus bulgaricus lyophilized vaccine.
Lactobacillus bulgaricus is inoculated in the MRS broth culture; activate three times; microscopy without miscellaneous bacteria and form without after unusual; with it by 4%(v/v) inoculum size access contain cultivate 18h in the MRS substratum after; the centrifugal 15min of 9000r/min; obtain lactobacillus bulgaricus bacterium mud; equal-volume adds the lactobacillus bulgaricus lyophilized vaccine; in cryogenic refrigerator in-40 ℃ of pre-freeze 4h; then put into Freeze Drying Equipment and carry out lyophilize 18h; namely get lactobacillus bulgaricus bacterium powder, the freeze-drying survival rate is 53.500%, and bacterium powder viable count is 0.916 * 10
11Cfu/g.
Embodiment 2
Consisting of of lactobacillus bulgaricus lyophilized vaccine: lactose 23g, skimmed milk powder 24g, sodium ascorbate 4.7g, Macrogol 4000 0.3g, polyvinylpyrrolidone PVP 0.3g, bovine serum albumin BSA 0.6g, distilled water 100ml.
Its preparation method:
1) will mix after lactose and the skimmed milk powder heating for dissolving according to quantity, and regulate pH 7.0 with NaOH solution, then through 110 ℃ of sterilization 15min, be cooled to room temperature;
2) take by weighing according to quantity polyvinylpyrrolidone PVP, be dissolved in the distilled water, 115 ℃ of sterilization 30min are cooled to room temperature again;
3) take by weighing respectively according to quantity sodium ascorbate, Macrogol 4000, bovine serum albumin BSA and be dissolved in the 0.85-0.9% stroke-physiological saline solution, with 0.22 μ m membrane filtration degerming;
4) with step 1), 2) solution of two moist heat sterilizations mixes according to quantity, and under aseptic condition, add sodium ascorbate, the filtration sterilization aqueous solution of Macrogol 4000, bovine serum albumin BSA, mixing namely gets the lactobacillus bulgaricus lyophilized vaccine.
Lactobacillus bulgaricus is inoculated in the MRS broth culture; activate three times; microscopy without miscellaneous bacteria and form without after unusual; with it by 4%(v/v) inoculum size access contain cultivate 18h in the MRS substratum after; the centrifugal 15min of 9000r/min; namely get lactobacillus bulgaricus bacterium mud; equal-volume adds above-mentioned compound lyophilized vaccine again; in cryogenic refrigerator in-40 ℃ of pre-freeze 5h; then put into Freeze Drying Equipment and carry out lyophilize 20h; namely get lactobacillus bulgaricus bacterium powder, the freeze-drying survival rate reaches 54.350%, and bacterium powder viable count is 0.946 * 10
11Cfu/g.
Embodiment 3
Consisting of of lactobacillus bulgaricus lyophilized vaccine: lactose 26g, skimmed milk powder 24g, sodium ascorbate 4.8g, Macrogol 4000 0.2g, polyvinylpyrrolidone PVP 0.5g, bovine serum albumin BSA 0.8g, distilled water 100ml.
Its preparation method:
1) will mix after lactose and the skimmed milk powder heating for dissolving according to quantity, and regulate pH 7.1 with NaOH solution, then through 110 ℃ of sterilization 15min, be cooled to room temperature;
2) take by weighing according to quantity polyvinylpyrrolidone PVP, be dissolved in the distilled water, 115 ℃ of sterilization 30min are cooled to room temperature again;
3) take by weighing respectively according to quantity sodium ascorbate, Macrogol 4000, bovine serum albumin BSA and be dissolved in the 0.85-0.9% stroke-physiological saline solution, with 0.22 μ m membrane filtration degerming;
4) with step 1), 2) solution of two moist heat sterilizations mixes according to quantity, and under aseptic condition, add sodium ascorbate, the filtration sterilization aqueous solution of Macrogol 4000, bovine serum albumin BSA, mixing namely gets the lactobacillus bulgaricus lyophilized vaccine.
Lactobacillus bulgaricus is inoculated in the MRS broth culture; activate three times; microscopy without miscellaneous bacteria and form without after unusual; with it by 4%(v/v) inoculum size access contain cultivate 18h in the MRS substratum after; the centrifugal 15min of 9000r/min; namely get lactobacillus bulgaricus bacterium mud; equal-volume adds above-mentioned compound lyophilized vaccine again; in cryogenic refrigerator in-40 ℃ of pre-freeze 5h; then put into Freeze Drying Equipment and carry out lyophilize 20h; namely get lactobacillus bulgaricus bacterium powder, the freeze-drying survival rate reaches 57.475%, and bacterium powder viable count is 0.984 * 10
11Cfu/g.
Embodiment 4
Consisting of of lactobacillus bulgaricus lyophilized vaccine: lactose 24g, skimmed milk powder 28g, sodium ascorbate 4.8g, Macrogol 4000 0.4g, polyvinylpyrrolidone PVP 0.5g, bovine serum albumin BSA 0.5g, distilled water 100ml.
Its preparation method:
1) will mix after lactose and the skimmed milk powder heating for dissolving according to quantity, and regulate pH 6.9 with NaOH solution, then through 110 ℃ of sterilization 15min, be cooled to room temperature;
2) take by weighing according to quantity polyvinylpyrrolidone PVP, be dissolved in the distilled water, 115 ℃ of sterilization 30min are cooled to room temperature again;
3) take by weighing respectively according to quantity sodium ascorbate, Macrogol 4000, bovine serum albumin BSA and be dissolved in the 0.85-0.9% stroke-physiological saline solution, with 0.22 μ m membrane filtration degerming;
4) with step 1), 2) solution of two moist heat sterilizations mixes according to quantity, and under aseptic condition, add sodium ascorbate, the filtration sterilization aqueous solution of Macrogol 4000, bovine serum albumin BSA, mixing namely gets the lactobacillus bulgaricus lyophilized vaccine.
Lactobacillus bulgaricus is inoculated in the MRS broth culture; activate three times; microscopy without miscellaneous bacteria and form without after unusual; with it by 4%(v/v) inoculum size access contain cultivate 18h in the MRS substratum after; the centrifugal 15min of 9000r/min; namely get lactobacillus bulgaricus bacterium mud; equal-volume adds above-mentioned compound lyophilized vaccine again; in cryogenic refrigerator in-40 ℃ of pre-freeze 6h; then put into Freeze Drying Equipment and carry out lyophilize 20h; namely get lactobacillus bulgaricus bacterium powder, the freeze-drying survival rate reaches 58.567%, and bacterium powder viable count is 1.066 * 10
11Cfu/g.
Claims (6)
1. a lactobacillus bulgaricus lyophilized vaccine is characterized in that, in mass fraction, comprises following component:
100 parts of 20～30 parts of lactose, 20～30 parts of skimmed milk powders, 3～5 parts of sodium ascorbates, 0.2～0.6 part of Macrogol 4000,0.2～0.8 part of polyvinylpyrrolidone, 0.5～2.0 part of bovine serum albumin and distilled water.
2. lactobacillus bulgaricus lyophilized vaccine as claimed in claim 1 is characterized in that, in mass fraction, comprises following component:
100 parts of 20～26 parts of lactose, 24～30 parts of skimmed milk powders, 4.4～5 parts of sodium ascorbates, 0.2～0.4 part of Macrogol 4000,0.3～0.6 part of polyvinylpyrrolidone, 0.5～1.0 part of bovine serum albumin and distilled water.
3. the preparation method of a lactobacillus bulgaricus lyophilized vaccine is characterized in that, may further comprise the steps:
1) in mass fraction, in distilled water, add 20～30 parts lactose and 20～30 parts skimmed milk powder, be 6.9～7.1 with regulating pH after the heating for dissolving, sterilization is cooled to room temperature;
2) in mass fraction, 0.2～0.8 part polyvinylpyrrolidone is dissolved in the distilled water, sterilization is cooled to room temperature;
3) in mass fraction, 3～5 parts sodium ascorbates, 0.2～0.6 part Macrogol 4000 and 0.5～2.0 part bovine serum albumin are dissolved in the stroke-physiological saline solution, then use the membrane filtration degerming;
4) with step 1), 2), 3) prepared solution mixes, and the total mass umber of adjusting water is 100 parts, obtains the lactobacillus bulgaricus lyophilized vaccine.
4. the preparation method of lactobacillus bulgaricus lyophilized vaccine as claimed in claim 3 is characterized in that, may further comprise the steps:
1) in mass fraction, in distilled water, add 20～26 parts lactose and 24～30 parts skimmed milk powder, be 6.9～7.1 with regulating pH after the heating for dissolving, sterilization is cooled to room temperature;
2) in mass fraction, 0.3～0.6 part polyvinylpyrrolidone is dissolved in the distilled water, sterilization is cooled to room temperature;
3) in mass fraction, 4.4～5 parts sodium ascorbates, 0.2～0.4 part Macrogol 4000 and 0.5～1.0 part bovine serum albumin are dissolved in the stroke-physiological saline solution, then use the membrane filtration degerming;
4) with step 1), 2), 3) prepared solution mixes, and the total mass umber of adjusting water is 100 parts, obtains the lactobacillus bulgaricus lyophilized vaccine.
5. the preparation method of lactobacillus bulgaricus lyophilized vaccine as claimed in claim 3 is characterized in that, described step 1), step 2) sterilization be 100～115 ℃ the sterilization 10～15min.
6. the preparation method of lactobacillus bulgaricus lyophilized vaccine as claimed in claim 3; it is characterized in that; lactobacillus bulgaricus lyophilized vaccine and lactobacillus bulgaricus mud equal-volume are mixed; in-40 ℃ of pre-freeze 3～12h; then-50～-56 ℃ lyophilize 18～24h obtains lactobacillus bulgaricus bacterium powder.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387939A (en) * | 2013-07-23 | 2013-11-13 | 甘肃农业大学 | Method for preparing dry powder of lactobacillus delbrueckii subsp. Bulgaricus |
| CN103923835A (en) * | 2014-04-25 | 2014-07-16 | 陕西科技大学 | Method for preparing lactobacillus bulgaricus freeze-dried powder |
| CN105709235A (en) * | 2016-03-31 | 2016-06-29 | 深圳市国赛生物技术有限公司 | Cryoprotectant and lyophilization method for latex coupled antibody |
| CN106754525A (en) * | 2016-12-28 | 2017-05-31 | 石家庄君乐宝乳业有限公司 | Freeze drying protectant, lactobacillus paracasei N1115 freeze-dried powder leavenings and preparation method thereof |
| CN107475117A (en) * | 2017-07-03 | 2017-12-15 | 桐乡恒基生物股份有限公司 | A kind of freeze drying protectant for lactobacillus reuteri and lyophilized store method |
| CN107815416A (en) * | 2017-11-06 | 2018-03-20 | 合肥民祯健康科技有限公司 | A kind of cryoprotectants for lactic acid bacteria |
| CN108018211A (en) * | 2018-02-06 | 2018-05-11 | 内蒙古双奇药业股份有限公司 | A kind of culture medium and its application process for improving lactobacillus bulgaricus freexing tolerance |
| CN110106114A (en) * | 2019-05-10 | 2019-08-09 | 上海市计量测试技术研究院(中国上海测试中心、华东国家计量测试中心、上海市计量器具强制检定中心) | A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance |
| CN110184191A (en) * | 2019-06-04 | 2019-08-30 | 内蒙古双奇药业股份有限公司 | A kind of raising lactobacillus bulgaricus is stored at room temperature and the freeze drying protectant of anti-pressure ability and its application |
| CN110982699A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Vibrio vulnificus stabilizer and application thereof |
| CN111286469A (en) * | 2018-12-10 | 2020-06-16 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN112342172A (en) * | 2020-11-27 | 2021-02-09 | 沈阳化工研究院有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387939A (en) * | 2013-07-23 | 2013-11-13 | 甘肃农业大学 | Method for preparing dry powder of lactobacillus delbrueckii subsp. Bulgaricus |
| CN103923835A (en) * | 2014-04-25 | 2014-07-16 | 陕西科技大学 | Method for preparing lactobacillus bulgaricus freeze-dried powder |
| CN103923835B (en) * | 2014-04-25 | 2016-02-10 | 陕西科技大学 | A kind of lactobacillus bulgaricus freeze-dried vaccine powder, preparation method thereof |
| CN105709235B (en) * | 2016-03-31 | 2019-02-26 | 深圳市国赛生物技术有限公司 | A kind of freeze drying process of freeze drying protectant and latex coupled antibody |
| CN105709235A (en) * | 2016-03-31 | 2016-06-29 | 深圳市国赛生物技术有限公司 | Cryoprotectant and lyophilization method for latex coupled antibody |
| CN106754525A (en) * | 2016-12-28 | 2017-05-31 | 石家庄君乐宝乳业有限公司 | Freeze drying protectant, lactobacillus paracasei N1115 freeze-dried powder leavenings and preparation method thereof |
| CN106754525B (en) * | 2016-12-28 | 2020-11-03 | 石家庄君乐宝乳业有限公司 | Lactobacillus paracasei N1115 freeze-dried powder leavening agent and preparation method thereof |
| CN107475117A (en) * | 2017-07-03 | 2017-12-15 | 桐乡恒基生物股份有限公司 | A kind of freeze drying protectant for lactobacillus reuteri and lyophilized store method |
| CN107815416A (en) * | 2017-11-06 | 2018-03-20 | 合肥民祯健康科技有限公司 | A kind of cryoprotectants for lactic acid bacteria |
| CN108018211A (en) * | 2018-02-06 | 2018-05-11 | 内蒙古双奇药业股份有限公司 | A kind of culture medium and its application process for improving lactobacillus bulgaricus freexing tolerance |
| CN111286469A (en) * | 2018-12-10 | 2020-06-16 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN111286469B (en) * | 2018-12-10 | 2022-11-01 | 东北农业大学 | Preparation method of lactobacillus paracasei freeze-dried powder |
| CN110106114A (en) * | 2019-05-10 | 2019-08-09 | 上海市计量测试技术研究院(中国上海测试中心、华东国家计量测试中心、上海市计量器具强制检定中心) | A kind of freeze drying protectant and freeze-drying store method and application for Listeria monocytogenes standard substance |
| CN110184191A (en) * | 2019-06-04 | 2019-08-30 | 内蒙古双奇药业股份有限公司 | A kind of raising lactobacillus bulgaricus is stored at room temperature and the freeze drying protectant of anti-pressure ability and its application |
| CN110184191B (en) * | 2019-06-04 | 2023-02-10 | 内蒙古双奇药业股份有限公司 | Freeze-drying protective agent for improving normal-temperature storage and pressure resistance of lactobacillus bulgaricus and application thereof |
| CN110982699A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Vibrio vulnificus stabilizer and application thereof |
| CN110982699B (en) * | 2020-01-04 | 2023-04-11 | 广东环凯生物科技有限公司 | Vibrio vulnificus stabilizer and application thereof |
| CN112342172A (en) * | 2020-11-27 | 2021-02-09 | 沈阳化工研究院有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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