Vacuum freeze-drying protective agent aiming at lactobacillus plantarum LS-41 as well as preparation method and application of vacuum freeze-drying protective agent

Abstract

The invention relates to the field of freeze-drying protective agents, and particularly discloses a vacuum freeze-drying protective agent for lactobacillus plantarum LS-41. The vacuum freeze drying protective agent for the lactobacillus plantarum LS-41 comprises the following components: 15 parts of dried skim milk, 3 parts of cane sugar, 1 part of PBS dry powder, 6-10 parts of trehalose, 0.2-0.4 part of ascorbic acid, 0.06-0.10 part of sodium glutamate, 8-12 parts of sorbitol and 100 parts of ultrapure water. Through the synergistic effect of the trehalose, the ascorbic acid, the sodium glutamate, the sorbitol, the skimmed milk powder and the cane sugar, and improvement and reasonable matching of the category and the concentration of the protective agents, the damage of the lactobacillus plantarum LS-41 in the vacuum freeze-drying process can be reduced, and the freeze-drying survival rate of the lactobacillus plantarum LS-41 is increased.

Description

Technical Field

[0001] The invention belongs to the field of freeze-drying protective agents, and particularly relates to a vacuum freeze-drying protective agent for Lactobacillus plantarum LS-41, a preparation method and application thereof. Background Art

[0002] Microbial strains can degrade their desirable traits during use or storage due to internal and external factors. This is primarily due to spontaneous mutations, which alter genetic material and cause negative mutations in relevant genes, leading to cell death and subsequent degradation during use or storage. Continuous subculture can also lead to strain degradation. This is primarily due to the increased frequency of spontaneous mutations during subculture. The probability of spontaneous mutations increases with the number of subcultures, and the probability of negative mutations in particular increases significantly. Finally, strain degradation can be caused by an unsuitable culture environment. The bacterial growth environment includes temperature, humidity, ventilation, light, pH, and excessively rich or poor culture media. Temperature has the greatest impact on biological metabolism and is the primary environmental factor contributing to strain degradation. Extremely unsuitable temperatures can alter the genetic characteristics of strains, resulting in slower growth rates, reduced yields, and altered colony morphology and color. Microbial resources are widely distributed in natural ecosystems, accounting for approximately 17% of Earth's biomass. They are abundant, fast-growing, and easily transformable, making them closely related to industries such as industry, agriculture, food, brewing, medicine, energy, and the environment. However, the consequences of strain degradation have limited their application in these areas. Therefore, the effective preservation of microbial strains is essential.

[0003] Currently, commonly used bacterial strain preservation methods include periodic transplantation, mineral oil preservation, carrier preservation, liquid nitrogen freezing, -80°C freezer freezing, and vacuum freeze-drying. While the periodic transplantation method is simple to operate, easy to observe, and requires minimal operator expertise, requiring minimal specialized equipment, resulting in low cost and wide application, its drawbacks are high labor intensity, limited ability to preserve strains for extended periods, and the need for frequent subculture and transplantation. Furthermore, the transplantation process can easily lead to contamination and degradation. Mineral oil preservation can prolong the life of microorganisms and maintain their excellent virulence, but it also requires high operator skill, is labor-intensive, and is expensive. Care must be taken to prevent contamination and fire hazards when using mineral oil, and it is inconvenient to carry. While carrier preservation is simple to operate and requires minimal equipment, it requires the strain to be adsorbed onto sterile filter paper, making it unsuitable for vegetative cells and limiting its scope of application. Liquid nitrogen freezing allows for a longer preservation period, a higher freeze-dried survival rate, and resistance to mutation and degradation. However, its disadvantages are relatively complex operation, requiring high operator skill, expensive equipment, and the need for regular replenishment of liquid nitrogen, resulting in high preservation costs. The -80°C freezer method involves suspending the strain in a protective agent and freezing it in a -80°C freezer. This method offers advantages such as ease of use and a longer shelf life, but is difficult to transport and carry, and requires high equipment requirements, including a -80°C freezer. Vacuum freeze-drying is one of the most effective methods for preserving bacterial strains. The pre-frozen bacterial liquid is placed in a vacuum and low-temperature environment to sublimate into a dry powder or crystal state, which can inhibit the growth and reproduction of the bacterial strain, put the bacterial strain in an inactive metabolic state, and try to maintain the original physiological and biochemical characteristics of the bacterial strain. This method can generally be preserved for 40 years. Compared with other methods for preserving bacterial strains, it has significant advantages in storage and transportation, with better preservation effect and high survival rate, which is conducive to the research and application of bacterial strains. However, during the freeze-drying process, due to the stress of freezing and drying, the bacterial cells are damaged or even die. Although there are some freeze-drying protectants on the market, not all of them are suitable for freeze-drying protection of strains. Moreover, due to the differences in physiological characteristics, metabolic performance, enzyme composition, etc. of different strains, the existing freeze-drying protectants for strains are not applicable to the freeze-drying of all strains. For example, the freeze-drying protectant suitable for Debaryomyces hansenii is not suitable for Lactobacillus plantarum. Therefore, it is necessary to develop suitable vacuum freeze-drying protectants specifically for specific strains. Summary of the Invention

[0004] In view of the defects and problems of current freeze-drying protective agents, the present invention provides a vacuum freeze-drying protective agent for Lactobacillus plantarum, a preparation method and application thereof.

[0005] The invention provides a vacuum freeze-drying protective agent, which comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6-10 parts of trehalose, 0.2-0.4 parts of ascorbic acid, 0.06-0.10 parts of sodium glutamate, 8-12 parts of sorbitol and 100 parts of ultrapure water.

[0006] The vacuum freeze-drying protective agent comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6-8 parts of trehalose, 0.2-0.3 parts of ascorbic acid, 0.06-0.08 parts of sodium glutamate, 8-10 parts of sorbitol, and 100 parts of ultrapure water.

[0007] The above-mentioned vacuum freeze-drying protective agent includes the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 8 parts of trehalose, 0.3 parts of ascorbic acid, 0.08 parts of sodium glutamate, 10 parts of sorbitol, and 100 parts of ultrapure water.

[0008] The above-mentioned vacuum freeze-drying protective agent includes the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6 parts of trehalose, 0.2 parts of ascorbic acid, 0.08 parts of sodium glutamate, 10 parts of sorbitol, and 100 parts of ultrapure water.

[0009] The vacuum freeze-drying protective agent is prepared by accurately weighing skim milk powder, sucrose, PBS dry powder, trehalose, ascorbic acid, sodium glutamate, and sorbitol, dissolving the above raw materials in ultrapure water, and sterilizing to obtain a freeze-drying protective agent solution.

[0010] Application of any of the above-mentioned vacuum freeze-drying protective agents in vacuum freeze-drying of Lactobacillus plantarum LS-41.

[0011] The present invention also provides a preservation method for Lactobacillus plantarum LS-41, comprising the following steps: (1) Under a sterile environment, Lactobacillus plantarum LS-41 bacterial slurry is mixed with any of the above-mentioned vacuum freeze-drying protective agents in a ratio of 1:8 to prepare a bacterial suspension; (2) The bacterial suspension is pre-frozen and freeze-dried, and then placed in an environment at -20°C or below.

[0012] The preservation method of the above-mentioned Lactobacillus plantarum LS-41 is as follows: in step (2), the bacterial suspension is sealed and pre-frozen at -80°C for 16 hours, taken out, a plurality of holes are pierced at the mouth of the tube with a needle, and placed in a vacuum freeze dryer and freeze-dried at -80°C for 24 hours.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the vacuum freeze-drying protective agent for Lactobacillus plantarum LS-41 of the present invention can reduce the damage of Lactobacillus plantarum LS-41 during the vacuum freeze-drying process and improve the freeze-drying survival rate of the bacteria through the synergistic effect of trehalose, ascorbic acid, sodium glutamate, sorbitol, skim milk powder and sucrose, and through the improvement and reasonable combination of the type and concentration of the protective agent. DETAILED DESCRIPTION

[0014] The present invention provides a vacuum freeze-drying protective agent for Lactobacillus plantarum LS-41 and its application. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention.

[0015] The present invention uses Lactobacillus plantarum LS-41 as a test strain. Lactobacillus plantarum LS-41 is a beneficial bacterium widely distributed in nature. It has probiotic properties such as anti-oxidation, regulating dyslipidemia, improving intestinal inflammation and barrier function, regulating intestinal homeostasis, immunomodulation, and antibacterial properties, and has good health-promoting effects. Lactobacillus plantarum LS-41 is vacuum-freeze-dried and prepared into a freeze-dried powder, which makes it more convenient in terms of use, protection, transportation, circulation, etc., thereby allowing Lactobacillus plantarum LS-41 to be more widely used.

[0016] The freeze-drying survival rate of Lactobacillus plantarum LS-41 was measured to be 22.95% when no freeze-drying protectant was added (negative control).

[0017] The test materials used in this invention are all common commercial products and can be purchased on the market. The test materials used in this invention are as follows: 1. Strains: Lactobacillus plantarum LS-41: Microbiology Laboratory, College of Food Science and Engineering, Xinyang Agriculture and Forestry University; Debaryomyces hansenii: Microbiology Laboratory, College of Food Science and Engineering, Xinyang Agriculture and Forestry University; Aspergillus oryzae C0-26: Microbiology Laboratory, College of Food Science and Engineering, Xinyang Agriculture and Forestry University.

[0018] 2. Culture medium (1) MRS broth medium: peptone 10.00 g, beef extract 8.00 g, yeast extract 4.00 g, glucose 20.00 g, sodium acetate 5.00 g, diammonium hydrogen citrate 2.00 g, dipotassium hydrogen phosphate 2.00 g, magnesium sulfate 0.20 g, manganese sulfate 0.04 g, Tween-80 1.00 mL, distilled water 1000 mL; (2) MRS agar medium: add 18.00 g of agar to MRS broth medium; (3) YPD medium: 1% yeast extract powder, 2% glucose, 2% peptone, 2% agar powder, and 1000 ml water; (4) YPD liquid medium: 1% yeast extract powder, 2% glucose, 2% peptone, and 1000 ml water; (5) Slant culture medium: 1.5% glucose, 0.2% yeast extract, 2% agar; (6) Shake flask fermentation medium: 10% glucose, 0.05% MgSO4·7H2O, 2.5% yeast extract, 0.05% KCl, 0.1% KH2PO4, 0.01% FeSO4, 6% anhydrous ethanol.

[0019] The present invention evaluates the effect of each lyoprotectant by freeze-drying survival rate, and the freeze-drying survival rate determination method is: (1) Activation of bacterial strains: Take out the frozen Lactobacillus plantarum LS-41 from the -80℃ ultra-low temperature freezer, thaw it at room temperature, shake it evenly, use a pipette to draw 100 μL of bacterial solution and spread it on MRS solid culture medium, culture it at 37℃ for 48 h, pick up a single colony with an inoculation loop, streak it on MRS solid culture medium, and culture it under the same conditions as above. Make sure that the bacteria are not contaminated during the storage process, inoculate the single colony into a slant test tube, culture it under the same conditions as above, and store it at 4℃ for later use.

[0020] (2) Expansion culture of bacterial strains Pick 1 loop of the strain preserved on the slant and inoculate it into 10 mL of MRS liquid medium. Culture it at 37°C for 12 h to prepare the seed solution. Inoculate the seed solution into 100 mL of MRS liquid medium at 1% and culture it at 37°C for 12 h until the logarithmic growth phase. Dilute the solution with sterile water to obtain the OD value. 600 The value is 0.600±0.105 for use.

[0021] (3) Counting viable bacteria before freeze-drying: Shake the bacterial solution in (2) and dilute it by 10 -1 ~10 -7 (Select plates with colony counts between 30 and 300 for viable bacteria counting), spread 100 μL onto MRS solid medium, and culture inverted at 37°C for 48 h.

[0022] (4) The bacterial strain protective agent solution and bacterial sludge were mixed evenly at a ratio of bacterial sludge to freeze-drying protective agent (m:v) of 1:8 to obtain bacterial suspension, which was then divided into 10 mL centrifuge tubes and frozen at -80°C for 20 h. The tube mouth was covered with plastic wrap and 10 small holes were pierced. The working conditions of the vacuum freeze dryer were set to -80°C and 20 MPa, and vacuum drying was carried out under these conditions for 18 h.

[0023] (5) Counting viable bacteria after freeze-drying: Add the same volume of sterile saline to the freeze-dried bacterial slurry as the bacterial solution before centrifugal freeze-drying, resuscitate for 3 minutes, vortex and shake for 3 minutes to mix, and count the bacteria in the same way as above.

[0024] (6) Freeze-dried survival rate: The calculation formula is:

[0025] The present invention will be further described below with reference to specific examples. All experiments were performed in triplicate.

[0026] Examples 1-10: This example provides 10 sets of lyoprotectant components and ratios. Each example uses 100 ml of ultrapure water. The preparation method is as follows: accurately weigh skim milk powder, sucrose, PBS dry powder, trehalose, ascorbic acid, sodium glutamate, and sorbitol, dissolve the above ingredients in ultrapure water, and sterilize to obtain a lyoprotectant solution. The freeze-drying survival rate of each group of freeze-drying protectants was determined using Lactobacillus plantarum LS-41, as follows.

[0027]

[0028] To further verify the reliability of the optimal ratio results, three parallel tests were carried out using the ratio of Example 4. The measured results were 89.59%, 87.17%, and 90.80%, respectively. The average value (89.19%) was close to 90.36%, indicating that the freeze-drying protection effect of this ratio on Lactobacillus plantarum LS-41 was stable and reliable.

[0029] Comparative Example 1: The lyoprotectant of this comparative example includes: 15 g skim milk powder, 3 g sucrose, 1 g PBS dry powder, 10 g trehalose, and 100 ml ultrapure water.

[0030] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 45.40%.

[0031] Comparative Example 2: The lyoprotectant of this comparative example includes: 15 g skim milk powder, 3 g sucrose, 1 g PBS dry powder, 8 g trehalose, and 100 ml ultrapure water.

[0032] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 74.33%.

[0033] Comparative Example 3: The freeze-drying protective agent of this comparative example includes: 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 10g oligofructose, and 100ml ultrapure water.

[0034] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 58.09%.

[0035] Comparative Example 4: The freeze-drying protective agent of this comparative example includes: 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 15g oligofructose, and 100ml ultrapure water.

[0036] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 71.64%.

[0037] Comparative Example 5: The freeze-drying protective agent of this comparative example includes: 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 15g glucose, and 100ml ultrapure water.

[0038] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 56.28%.

[0039] Comparative Example 6: The freeze-drying protective agent of this comparative example includes: 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 1g glucose, and 100ml ultrapure water.

[0040] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 71.95%.

[0041] Comparative Example 7: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 1 g of sodium thiosulfate, and 100 ml of ultrapure water.

[0042] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 48.88%.

[0043] Comparative Example 8: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 4 g of sodium thiosulfate, and 100 ml of ultrapure water.

[0044] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 57.97%.

[0045] Comparative Example 9: The freeze-drying protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 1g ascorbic acid, and 100ml ultrapure water.

[0046] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 57.05%.

[0047] Comparative Example 10: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 0.3 g of ascorbic acid, and 100 ml of ultrapure water.

[0048] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 71.88%.

[0049] Comparative Example 11: The freeze-drying protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 1g carotene, and 100ml ultrapure water.

[0050] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 41.38%.

[0051] Comparative Example 12: The freeze-drying protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 0.13g β-carotene, and 100ml ultrapure water.

[0052] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 59.00%.

[0053] Comparative Example 13: The freeze-drying protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 0.8g glutamic acid, and 100ml ultrapure water.

[0054] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 45.10%.

[0055] Comparative Example 14: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 1 g of glutamic acid, and 100 ml of ultrapure water.

[0056] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 44.52%.

[0057] Comparative Example 15: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 0.8 g of glycine, and 100 ml of ultrapure water.

[0058] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 45.94%.

[0059] Comparative Example 16: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 0.6 g of glycine, and 100 ml of ultrapure water.

[0060] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 44.43%.

[0061] Comparative Example 17: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 0.8 g of sodium glutamate, and 100 ml of ultrapure water.

[0062] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 56.44%.

[0063] Comparative Example 18: The freeze-drying protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 0.08g sodium glutamate, and 100ml ultrapure water.

[0064] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 77.13%.

[0065] Comparative Example 19: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 10 g of mannitol, and 100 ml of ultrapure water.

[0066] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 52.49%.

[0067] Comparative Example 20: The lyoprotectant of this comparative example includes 15 g of skim milk powder, 3 g of sucrose, 1 g of PBS dry powder, 10 g of sorbitol, and 100 ml of ultrapure water.

[0068] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 73.64%.

[0069] Comparative Example 21: The freeze-dried protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 10g glycerol, and 100ml ultrapure water.

[0070] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 58.01%.

[0071] Comparative Example 22: The freeze-dried protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, 4g glycerol, and 100ml ultrapure water.

[0072] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 41.53%.

[0073] Comparative Example 23: The freeze-dried protective agent of this comparative example includes 15g skim milk powder, 3g sucrose, 1g PBS dry powder, and 100ml ultrapure water.

[0074] The freeze-dried survival rate of Lactobacillus plantarum LS-41 was measured to be 67.13%.

[0075] From the perspective of comprehensive embodiments of the present invention 1-10 and comparative example 1-23, adopt the composite lyophilization protective agent of the embodiment of the present invention, the freeze-drying survival rate of plant lactobacillus LS-41 is generally higher, illustrate that the composite trehalose, ascorbic acid, sodium glutamate, the sorbitol of adding appropriate proportion in skim milk powder, sucrose, PBS dry powder can improve the freeze-drying survival rate of plant lactobacillus LS-41.Wherein the composite lyophilization protective agent of embodiment 4 and embodiment 9 is significantly higher than other embodiments and comparative examples to the freeze-drying survival rate of plant lactobacillus LS-41, illustrate that when ultrapure water is 100ml, add 8g trehalose, 0.3g ascorbic acid, 0.08g sodium glutamate, 10g sorbitol in 15g skim milk powder, 3g sucrose, 1gPBS dry powder, or add 6g trehalose, 0.2g ascorbic acid, 0.08g sodium glutamate, 10g sorbitol can play synergistic effect, improve the freeze-drying survival rate of plant lactobacillus LS-41.

[0076] Combining Comparative Examples 1-6 with Comparative Example 23, the freeze-dried survival rates of the cells added with 8g of trehalose (Comparative Example 2), 15g of oligofructose (Comparative Example 4), and 1g of glucose (Comparative Example 6) to the positive control of Comparative Example 23 were all higher than the 67.13% of Comparative Example 23. However, the effect of adding 8g of trehalose to Comparative Example 2 was significantly better, indicating that the combination of 8g of trehalose with 15g of skim milk powder and 3g of sucrose can improve the freeze-dried survival rate of Lactobacillus plantarum LS-41. The reason why the addition of 10g of trehalose to the positive control compared to 8g of trehalose was less protective may be that the trehalose concentration was too high, resulting in an excessively strong vitrified structure, which affected the structure of the strain cells themselves and led to a decrease in survival rate. However, the addition of 8g of trehalose to the positive control in the present invention exerted a certain synergistic effect with sucrose, thereby reducing the concentration of trehalose that exerted the optimal protective effect. The freeze-drying survival rates of the positive control when 10 g of trehalose, 10 g of oligofructose, and 10 g of glucose were added were lower than those of the positive control, indicating that not all sugars that can be used as freeze-drying protective agents are suitable for Lactobacillus plantarum LS-41, and the concentration of the same sugar is also very critical.

[0077] Combining Comparative Examples 7-12 with Comparative Example 23, the freeze-dried survival rate of 71.88% when 0.3g ascorbic acid was added to the positive control of Comparative Example 23 (Comparative Example 10) was higher than 67.13% of Comparative Example 23, indicating that the freeze-dried survival rate of Lactobacillus plantarum LS-41 can be improved by adding 1g sodium thiosulfate, 4g sodium thiosulfate, 1g sodium ascorbate, 1g carotene and 0.13g β -Carotene has a negative effect. The possible reason is that 0.3g of sodium ascorbate may have a stronger free radical scavenging ability, thus more effectively protecting the microbial cell membrane and protein structure. It may also more effectively inhibit ice crystal growth by regulating the osmotic pressure of the solution, thereby reducing damage to the bacteria during freeze-drying. Sodium thiosulfate reduces oxidation reactions by consuming oxygen in the environment through its own oxidation. Its protective effect depends on the oxygen consumption rate and the ambient oxygen content. β Carotene is a fat-soluble antioxidant and may not be able to effectively penetrate the cell membrane or regulate extracellular osmotic pressure, leading to excessive intracellular solute concentrations and osmotic damage. The reason why the addition of 1g of ascorbic acid to the positive control was less protective than 0.3g of ascorbic acid may be because high sodium ascorbate concentrations can alter bacterial cell structure.

[0078] Combining Comparative Examples 13-18 with Comparative Example 23, the freeze-drying survival rate of adding 0.08 g of sodium glutamate (Comparative Example 18) to the positive control of Comparative Example 23 was higher than 67.13% of the positive control, while the others were all lower than 67.13%. This shows that compared with glycine and glutamic acid, although both are conventional choices of amino acid freeze-drying protectants, adding 0.08 g of sodium glutamate to the positive control helps to improve the freeze-drying survival rate of Lactobacillus plantarum LS-41. The possible reason is that sodium glutamate has a small molecular weight (169 g / mol) and is reducing, and can easily pass through the cell membrane into the cell, directly protecting the protein and cell structure. After sodium glutamate enters the cell membrane, it changes the supercooled state of the cell, making the intracellular pressure close to the extracellular pressure, reducing the degree and speed of cell dehydration shrinkage, and reducing cell damage during freeze-drying; however, when the content of sodium glutamate entering the cell is too high, the intracellular osmotic pressure will be too high, thereby inactivating the intracellular substances, thereby reducing the survival rate of the bacteria and reducing the freeze-drying survival rate. Therefore, the concentration of sodium glutamate is very critical. Glycine (75 g / mol) has a small molecular weight, but may lack the ability to interact with cellular components, making it unable to effectively protect cells; glutamate may lack sodium ion-mediated reducing effect, and its protective effect is weaker.

[0079] Combining Comparative Examples 19-22 with Comparative Example 23, the freeze-drying survival rate of adding 10g of sorbitol to the positive control of Comparative Example 23 (Comparative Example 20) was higher than 67.13%, while the others were all lower than 67.13%, indicating that compared with mannitol and glycerol, although both are conventional choices of alcohol freeze-drying protectants, adding 10g of sorbitol to the positive control helps to improve the freeze-drying survival rate of Lactobacillus plantarum LS-41. The possible reason is that sorbitol contains multiple hydroxyl groups, which can interact with the phosphate groups of cell membrane phospholipids, lower the phase transition temperature (Tm) of the cell membrane, and thus reduce the mechanical damage of ice crystals to the cell membrane during freeze-drying; however, excessive sorbitol will combine with water molecules, eventually causing cell membrane rupture, thereby reducing the freeze-drying survival rate. Glycerol, as an osmotic protective agent, has a relatively good freeze-drying protection effect when added to the reagent. However, when added to bacteria, it can only reduce the damage caused by ice crystals inside the cell membrane, and has little effect on protecting the bacterial cell membrane from damage. In addition, glycerol is a viscous liquid. If too high a concentration of glycerol protective agent is added when freeze-drying bacterial powder, some bacteria will not be completely dried, and the activity of the bacterial powder will be seriously lost. Although mannitol is an alcohol protective agent, it mainly protects cells by forming a support structure through crystallization. However, this protection may have a negative impact on cell metabolic activities, thereby inhibiting bacterial growth and reducing the freeze-drying survival rate.

[0080] To sum up, there are obvious differences in the freeze-drying protection effects of different types of lyoprotectants on Lactobacillus plantarum. Even the same type of lyoprotectant is not suitable for Lactobacillus plantarum LS-41. Different components of the same type have different effects. Moreover, even the same type of lyoprotectant has obvious differences in the effects of different concentrations. This shows that for Lactobacillus plantarum LS-41, the type and concentration are very critical when choosing a lyoprotectant, and it is not a random choice.

[0081] Test Example: In order to further verify whether there is strain difference in the composite lyoprotectant for Lactobacillus plantarum LS-41 of the present invention, this test example uses Debaryomyces hansenii and Aspergillus oryzae C0-26 to verify the lyoprotective effect of the lyoprotectants of Examples 4 and 9, respectively. The results are shown in Table 2 below.

[0082]

[0083] As can be seen from Table 2, the preferred lyoprotectant prepared in this example has relatively poor lyoprotective effects on Debaryomyces hansenii and Aspergillus oryzae C0-26. The main reason is that there is specificity between different bacterial species, and the required substances and substance concentrations are different.

[0084] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A vacuum freeze-drying protective agent, characterized in that: The invention comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6-10 parts of trehalose, 0.2-0.4 parts of ascorbic acid, 0.06-0.10 parts of sodium glutamate, 8-12 parts of sorbitol and 100 parts of ultrapure water.

2. The vacuum freeze-drying protective agent according to claim 1, characterized in that: The invention comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6-8 parts of trehalose, 0.2-0.3 parts of ascorbic acid, 0.06-0.08 parts of sodium glutamate, 8-10 parts of sorbitol and 100 parts of ultrapure water.

3. The vacuum freeze-drying protective agent according to claim 2, characterized in that: The invention comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 8 parts of trehalose, 0.3 parts of ascorbic acid, 0.08 parts of sodium glutamate, 10 parts of sorbitol and 100 parts of ultrapure water.

4. The vacuum freeze-drying protective agent according to claim 2, characterized in that: The invention comprises the following components: 15 parts of skim milk powder, 3 parts of sucrose, 1 part of PBS dry powder, 6 parts of trehalose, 0.2 parts of ascorbic acid, 0.08 parts of sodium glutamate, 10 parts of sorbitol and 100 parts of ultrapure water.

5. The vacuum freeze-drying protective agent according to claim 1, characterized in that: the preparation method comprises: accurately weighing skim milk powder, sucrose, PBS dry powder, trehalose, ascorbic acid, sodium glutamate, and sorbitol, dissolving the above raw materials in ultrapure water, and sterilizing to obtain a freeze-drying protective agent solution.

6. Use of the vacuum freeze-drying protective agent described in any one of 1 to 5 above in vacuum freeze-drying of Lactobacillus plantarum LS-41.

7. A preservation method for Lactobacillus plantarum LS-41, characterized in that: The following steps are involved: (1) In a sterile environment, Lactobacillus plantarum LS-41 bacterial slurry is mixed with the vacuum freeze-drying protective agent according to any one of claims 1 to 5 in a ratio of 1:8 to prepare a bacterial suspension; (2) The bacterial suspension is pre-frozen and then freeze-dried, and then placed in an environment at -20°C or below.

8. The preservation method of Lactobacillus plantarum LS-41 according to claim 7, wherein: In step (2), the bacterial suspension was sealed and pre-frozen at -80°C for 16 h, then taken out, several holes were pierced at the mouth of the tube with a needle, and placed in a vacuum freeze dryer and freeze-dried at -80°C for 24 h.

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