Lactobacillus plantarum freeze-drying protective agent, freeze-drying bacterial powder as well as preparation method and application of freeze-drying bacterial powder

Abstract

The invention discloses a lactobacillus plantarum freeze-drying protective agent, freeze-drying bacterial powder as well as a preparation method and application of the freeze-drying bacterial powder, and belongs to the technical field of microorganisms. The lactobacillus plantarum freeze-drying protective agent comprises the following raw materials: saccharides, milk powder and sodium salt. The mass ratio of the saccharides to the milk powder to the sodium salt is (4-6): (14-16): (4-6). The lactobacillus plantarum freeze-drying protective agent can relieve damage caused by ice crystals in the freeze-drying process of lactobacillus plantarum and maintain and stabilize protein and cell structures so as to effectively protect thalli, and the viable count and the cell survival rate of freeze-dried lactobacillus plantarum are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a Lactobacillus plantarum freeze-drying protective agent, freeze-dried bacterial powder and a preparation method and application thereof. BACKGROUND

[0002] Lactobacillus plantarum belongs to the genus of Lactobacillus and mainly exists in fermented products of butter, meat and vegetables. Most of the strains are isolated from plant raw materials, so it is named Lactobacillus plantarum. Lactobacillus plantarum has probiotic properties on the human body, can regulate intestinal flora, has antioxidant activity, antibacterial activity and can resist the acidic environment of the gastrointestinal tract, and is widely used in food fermentation such as dairy products, meat products and vegetable products. However, lactic acid bacteria have weak resistance to the external environment, and bacterial agents are not easy to store, resulting in high production and application costs of lactic acid bacteria bacterial agents. Freeze-drying is one of the most commonly used and effective methods for preserving lactic acid bacteria, which has the advantages of high survival rate of microorganisms, maintaining original properties, easy rehydration, not easy to oxidize, wide application range, long preservation period, avoiding contamination of other bacteria, convenient transportation and the like.

[0003] However, the freeze-drying process will cause a certain degree of damage to the cells, thereby affecting the play of lactic acid bacteria bacterial agents. In order to reduce the damage, it is usually necessary to add appropriate protective agents during freeze-drying. Because the structures of different strains are different, in order to minimize the damage, their requirements for protective agents and freeze-drying processes are also quite different. However, there is no freeze-drying protective agent suitable for Lactobacillus plantarum at present, resulting in inconvenience in storage and transportation of Lactobacillus plantarum. Therefore, it is of great significance to provide a freeze-drying protective agent suitable for Lactobacillus plantarum to reduce the damage thereof during freeze-drying. SUMMARY

[0004] In view of the above problems existing in the prior art, the present application provides a Lactobacillus plantarum freeze-drying protective agent, freeze-dried bacterial powder and a preparation method and application thereof. During freeze-drying, the Lactobacillus plantarum can maintain normal physiological functions, and the viable count and cell survival rate of the Lactobacillus plantarum after freeze-drying are improved.

[0005] The technical scheme of the present application is as follows: The present application provides a Lactobacillus plantarum freeze-drying protective agent, freeze-dried bacterial powder and a preparation method and application thereof. During freeze-drying, the Lactobacillus plantarum can maintain normal physiological functions, and the viable count and cell survival rate of the Lactobacillus plantarum after freeze-drying are improved. The mass ratio of the sugar, the milk powder and the sodium salt is (4-6):(14-16):(4-6).

[0006] Preferably, the raw materials of the Lactobacillus plantarum freeze-drying protective agent further comprise water. Preferably, the Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components by mass percentage: 4-6% of a saccharide, 14-16% of milk powder, 4-6% of a sodium salt, and the balance of water.

[0007] Preferably, the saccharide comprises at least one of xylo-oligosaccharide, trehalose, inulin, and malt dextrin; Preferably, the milk powder comprises at least one of skim milk powder and whey powder; Preferably, the sodium salt comprises at least one of sodium glutamate, sodium alginate, and sodium ascorbate.

[0008] The second aspect of the present application protects a preparation method of the Lactobacillus plantarum freeze-drying protective agent of the first aspect, and the preparation method comprises the following steps: weighing the formula amount of the saccharide, the milk powder, and the sodium salt, dissolving them in water, and uniformly mixing to obtain the Lactobacillus plantarum freeze-drying protective agent; Preferably, the water is sterile water.

[0009] The third aspect of the present application protects a preparation method of Lactobacillus plantarum freeze-dried bacteria powder, which comprises the following steps: S1, culturing Lactobacillus plantarum to obtain a culture liquid, centrifuging and washing to obtain a bacterial slurry; S2, mixing the bacterial slurry with a freeze-drying protective agent, pre-freezing, and then freeze-drying to obtain the Lactobacillus plantarum freeze-dried bacteria powder; The freeze-drying protective agent is the Lactobacillus plantarum freeze-drying protective agent of the first aspect or the Lactobacillus plantarum freeze-drying protective agent prepared by the preparation method of the second aspect.

[0010] Preferably, in S1, the Lactobacillus plantarum comprises Lactobacillus plantarum ZJ-23; Preferably, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 4-6:10.

[0011] Preferably, in S2, the pre-freezing temperature is -80 to -20℃; Preferably, the pre-freezing time is 8-12h.

[0012] Preferably, in S2, the freeze-drying time is 22-26h; Preferably, the freeze-drying temperature is -80 to -20℃.

[0013] The fourth aspect of the present application protects a Lactobacillus plantarum freeze-dried bacteria powder, which is prepared by the preparation method of the third aspect.

[0014] The fifth aspect of the present application protects the application of the Lactobacillus plantarum freeze-dried bacteria powder of the fourth aspect or the Lactobacillus plantarum freeze-dried bacteria powder prepared by the preparation method of the third aspect in food fermentation.

[0015] The application has the beneficial technical effects that: The application provides a freeze-drying protective agent for Lactobacillus plantarum, which can relieve damage caused by ice crystals in the freeze-drying process of Lactobacillus plantarum, maintain and stabilize protein and cell structure, thereby effectively protecting the bacterial body, and improve the viable count and cell survival rate of Lactobacillus plantarum after freeze-drying.

[0016] The application further provides a preparation method of freeze-dried Lactobacillus plantarum powder, wherein the freeze-drying protective agent is added during preparation, and the Lactobacillus plantarum is pre-frozen, and the addition amount of the freeze-drying protective agent and the process conditions of pre-freezing are optimized, so that the bacterial body can maintain normal physiological functions, and the viable count and cell survival rate of Lactobacillus plantarum after freeze-drying are improved. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a morphology diagram of the experimental group bacterial powder in the application under a scanning electron microscope at 10 k magnification.

[0018] Figure 2 It is a morphology diagram of the experimental group bacterial powder in the application under a scanning electron microscope at 20 k magnification.

[0019] Figure 3 It is a morphology diagram of the experimental group bacterial powder in the application under a scanning electron microscope at 50 k magnification.

[0020] Figure 4 It is a morphology diagram of the control group bacterial powder in the application under a scanning electron microscope at 10 k magnification.

[0021] Figure 5 It is a morphology diagram of the control group bacterial powder in the application under a scanning electron microscope at 20 k magnification.

[0022] Figure 6 It is a morphology diagram of the control group bacterial powder in the application under a scanning electron microscope at 50 k magnification.

[0023] Figure 7 It is a cell membrane integrity observation diagram of the bacterial body in the experimental group and the control group under a laser confocal microscope in the application.

[0024] In the figure: (A) is the bacterial body in the experimental group, and (B) is the bacterial body in the control group.

[0025] Figure 8 It is a β-galactosidase activity diagram of the bacterial body in the experimental group, the control group and the blank group in the application.

[0026] Figure 9 It is a lactate dehydrogenase activity diagram of the bacterial body in the experimental group, the control group and the blank group in the application.

[0027] Figure 10Na+ in the bacterial bodies of the experimental group, the control group and the blank group in the application + -K + ATPase activity graph.

[0028] Figure 11 Ca2+ in the bacterial bodies of the experimental group, the control group and the blank group in the application 2+ -Mg 2+ ATPase activity graph.

[0029] Figure 12 Total ATPase activity graph in the bacterial bodies of the experimental group, the control group and the blank group in the application.

[0030] Figure 13 Difference gene distribution scatter plot (A) and significant difference volcano plot (B) of the experimental group and the control group in the application.

[0031] Figure 14 GO function annotation diagram of the difference genes of the experimental group and the control group in the application. DETAILED DESCRIPTION

[0032] The application will be described in detail below with reference to the accompanying drawings.

[0033] The first aspect of the application provides a freeze-drying protective agent for Lactobacillus plantarum, comprising the following raw materials: a sugar, milk powder, and a sodium salt: The mass ratio of the sugar, the milk powder, and the sodium salt is (4-6):(14-16):(4-6), including but not limited to 4:14:4, 4:14:6, 4:16:4, 4:16:6, 6:14:4, 6:14:6, 6:16:4, and 6:16:6.

[0034] In some embodiments, the freeze-drying protective agent for Lactobacillus plantarum further comprises water.

[0035] In some embodiments, the freeze-drying protective agent for Lactobacillus plantarum comprises the following raw material components by mass percentage: 4-6% of the sugar, 14-16% of the milk powder, 4-6% of the sodium salt, and the balance of water. The mass percentage of the sugar includes but is not limited to 4%, 5%, and 6%; the mass percentage of the milk powder includes but is not limited to 14%, 15%, and 16%; and the mass percentage of the sodium salt includes but is not limited to 4%, 5%, and 6%.

[0036] During the freeze-drying process, water in the intracellular and extracellular environment will form ice crystals, which will pierce the cell membrane of Lactobacillus plantarum, causing the osmotic pressure imbalance between the intracellular and extracellular environments during the dehydration process and leading to the collapse of the membrane structure. The freeze-drying protective agent can alleviate the damage.

[0037] It can be understood that the saccharide, the milk powder and the sodium salt can maintain and stabilize the protein and the cell structure during the freeze-drying of the lactic acid bacteria, thereby effectively protecting the bacteria and improving the cell survival rate.

[0038] The trehalose has low hygroscopicity and is not easy to crystallize, and the hydroxyl groups thereof can be combined with the polar groups of cell membrane phospholipids and proteins, for example, the trehalose can interact with the cell phospholipids through hydrogen bonds, and replace water molecules to maintain the three-dimensional structure of biological macromolecules in a dehydrated state; meanwhile, the trehalose has a high glass transition temperature and can form a stable glass state to inhibit the growth of ice crystals and cell shrinkage; in addition, the trehalose can also reduce oxidative stress damage by scavenging free radicals.

[0039] The lactose and the protein in the skim milk powder can form an amorphous glassy structure during the freeze-drying process, and wrap the bacterial cells to reduce the mechanical damage of ice crystals to the cell membrane; the whey protein in the skim milk powder can stabilize the cell membrane phospholipid bilayer structure through hydrogen bonds and hydrophobic interaction, and prevent the membrane from rupturing; meanwhile, the natural buffering capacity of the skim milk powder can reduce the stress of the bacteria caused by the sharp change of pH. The combination of the skim milk powder and the trehalose can effectively prevent the cell from dehydrating and rupturing, protect the cell membrane and further improve the freeze-drying survival rate of the bacteria.

[0040] As a compatible solute, the sodium glutamate can balance the osmotic pressure inside and outside the cell, prevent the cell from dehydrating and collapsing, and also neutralize the membrane surface charge to reduce the membrane lipid phase transition. Therefore, the combination of the trehalose, the skim milk powder and the sodium glutamate is beneficial to protecting the cells from damage caused by freeze-drying in multiple aspects, and effectively improves the freeze-drying survival rate.

[0041] The second aspect of the present application provides a preparation method of the Lactobacillus plantarum freeze-drying protective agent, and the preparation method comprises the following steps:

[0042] The third aspect of the present application provides a preparation method of Lactobacillus plantarum freeze-dried bacteria powder, which comprises the following steps: S1, culturing the Lactobacillus plantarum to obtain a culture bacterial liquid, centrifuging and washing the culture bacterial liquid to obtain a bacterial slurry; S2, mixing the bacterial slurry with the freeze-drying protective agent, pre-freezing and then freeze-drying to obtain the Lactobacillus plantarum freeze-dried bacteria powder; The freeze-drying protective agent is the Lactobacillus plantarum freeze-drying protective agent according to the first aspect, or the Lactobacillus plantarum freeze-drying protective agent prepared by the preparation method according to the second aspect.

[0043] In some embodiments, in S1, the Lactobacillus plantarum comprises Lactobacillus plantarum ZJ-23.

[0044] In some embodiments, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 4-6:10.

[0045] It can be understood that too high or too low addition amount of the freeze-drying protective agent can cause changes in the environmental osmotic pressure, resulting in reduced cell survival rate.

[0046] In some embodiments, in S2, the pre-freezing temperature is -80~-20℃, including but not limited to -80℃, -40℃, -20℃.

[0047] In some embodiments, in S2, the pre-freezing time is 8-12h, including but not limited to 8h, 10h, 12h.

[0048] It can be understood that suitable pre-freezing temperature and pre-freezing time of the strain in the freeze-drying process can quickly dehydrate the cells, maintain the cell membrane integrity and cell activity. Too high pre-freezing temperature can cause incomplete freezing of the water in the bacterial slurry, and the expansion and foaming phenomenon occurs during freeze-drying, and too low pre-freezing temperature can also cause mechanical damage to the bacterial cells, affecting the survival rate. Appropriate pre-freezing time allows the bacterial cells to adapt to the extreme environment, which is conducive to further improving the survival rate, and too long pre-freezing time can form too many and large ice crystals, damaging the cells and causing resource waste.

[0049] In some embodiments, in S2, the freeze-drying time is 22-26h, including but not limited to 22h, 23h, 24h, 25h, 26h.

[0050] In some embodiments, in S2, the freeze-drying temperature is -80~-20℃, including but not limited to -80℃, -40℃, -20℃.

[0051] The fourth aspect of the present application provides a freeze-dried Lactobacillus plantarum powder, which is prepared by the preparation method of the third aspect.

[0052] The fifth aspect of the present application provides the use of the freeze-dried Lactobacillus plantarum powder of the fourth aspect or the freeze-dried Lactobacillus plantarum powder prepared by the preparation method of the third aspect in food fermentation.

[0053] In the present application, the classification and naming of Lactobacillus plantarum ZJ-23 is Lactiplantibacillus plantarum , which was preserved in the China General Microbiological Culture Collection Center on April 21, 2025, with the preservation number of CGMCC No. 34276, and the address of the China Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China.

[0054] In the embodiment of the present application, the MRS liquid culture medium comprises: 10.0 g / L of proteose peptone, 8.0 g / L of beef powder, 4.0 g / L of yeast powder, 20.0 g / L of glucose, 2.0 g / L of potassium phosphate dibasic, 2.0 g / L of diammonium hydrogen citrate, 5.0 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.04 g / L of manganese sulfate, 1.0 g / L of Tween 80, and pH 5.7.

[0055] The present application is further described below in conjunction with examples.

[0056] Example 1 A freeze-drying protective agent for Lactobacillus plantarum comprises, by mass percentage, the following raw material components: 4% of trehalose, 14% of skimmed milk powder, 4% of sodium glutamate, and the balance being water.

[0057] The preparation method of the freeze-drying protective agent for Lactobacillus plantarum comprises the following steps: weighing the formula amount of trehalose, skimmed milk powder, and sodium glutamate, dissolving them in sterile water, and mixing them uniformly with a vortex instrument to obtain the freeze-drying protective agent for Lactobacillus plantarum.

[0058] A preparation method of freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 comprises the following steps: S1, after the glycerol-preserved Lactobacillus plantarum ZJ-23 is streaked on an MRS plate culture medium and incubated at 37°C overnight, the incubation is repeated 2-3 times and the Lactobacillus plantarum ZJ-23 is stored in a 4°C refrigerator; before use, a single colony is picked and recovered in an MRS liquid culture medium, 100 μL of the culture solution is removed after 24 h and cultured in 15 mL of the MRS liquid culture medium for another 24 h, the process is repeated 2-3 times before use, and the last time of the bacterial solution is inoculated into the MRS liquid culture medium at a 2% (v / v) inoculation amount, cultured for 14 h to reach the logarithmic phase, 40 mL of the culture bacterial solution is obtained, the culture bacterial solution is centrifuged at 8000 x g at 4°C for 15 min, and the bacterial slurry is obtained after being washed twice with 0.85% sterile normal saline.

[0059] S2, according to the mass / volume ratio g / mL of 6:10 of the bacterial slurry and the freeze-drying protective agent, the freeze-drying protective agent is added to the bacterial slurry and mixed uniformly with a vortex instrument, the bacterial body after pre-freezing at -80°C for 8 h is transferred to a freeze dryer and freeze-dried at -55°C for 24 h to obtain the freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23.

[0060] Example 2 The same as example 1, except that the formula of the freeze-drying protective agent for Lactobacillus plantarum and the preparation method of the freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 are different.

[0061] A freeze-drying protective agent for Lactobacillus plantarum comprises, by mass percentage, the following raw material components: 4% of trehalose, 16% of skimmed milk powder, 6% of sodium glutamate, and the balance being water.

[0062] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0063] Example 3 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0064] The Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 6%, skimmed milk powder 16%, and sodium glutamate 5%, and the rest is water.

[0065] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0066] Comparative Example 1 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0067] The Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 4%, skimmed milk powder 12%, and sodium glutamate 4%, and the rest is water.

[0068] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0069] Comparative Example 2 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent in the present example is used, and meanwhile, in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing temperature is-20℃, and the rest of the operations are the same as those in Example 1.

[0070] The Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 4%, skimmed milk powder 12%, and sodium glutamate 5%, and the rest is water.

[0071] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing time is 12 h, and the rest of the operations are the same as in Example 1.

[0072] Comparative Example 3 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing time is 12 h, and the rest of the operations are the same as in Example 1.

[0073] A Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 4%, skim milk powder 16%, and sodium glutamate 5%, and the rest is water.

[0074] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the pre-freezing temperature is -40℃, and the pre-freezing time is 4 h, and the rest of the operations are the same as in Example 1.

[0075] Comparative Example 4 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing time is 12 h, and the rest of the operations are the same as in Example 1.

[0076] A Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 6%, skim milk powder 12%, and sodium glutamate 4%, and the rest is water.

[0077] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the pre-freezing temperature is -40℃, and the pre-freezing time is 12 h, and the rest of the operations are the same as in Example 1.

[0078] Comparative Example 5 The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 5:10; the pre-freezing time is 12 h, and the rest of the operations are the same as in Example 1.

[0079] A Lactobacillus plantarum freeze-drying protective agent comprises the following raw material components in percentage by mass: trehalose 6%, skim milk powder 12%, and sodium glutamate 6%, and the rest is water.

[0080] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacterial slurry to the freeze-drying protective agent is 4:10; the rest of the operations are the same as in Example 1.

[0081] Comparative Example 6 The same as Example 1, except that the formula of the Lactobacillus plantarum freeze-drying protective agent and the preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder.

[0082] A Lactobacillus plantarum freeze-drying protective agent, comprising the following raw material components by mass percentage: trehalose 6%, skim milk powder 14%, sodium glutamate 6%, and the balance being water.

[0083] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder is the same as Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the pre-freezing temperature is -20℃, the pre-freezing time is 4h, and the rest of the operations are the same as Example 1.

[0084] Comparative Example 7 The same as Example 1, except that the formula of the Lactobacillus plantarum freeze-drying protective agent and the preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder.

[0085] A Lactobacillus plantarum freeze-drying protective agent, comprising the following raw material components by mass percentage: trehalose 6%, skim milk powder 16%, sodium glutamate 4%, and the balance being water.

[0086] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder is the same as Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacteria slurry to the freeze-drying protective agent is 5:10; the pre-freezing time is 4h, and the rest of the operations are the same as Example 1.

[0087] Comparative Example 8 The same as Example 1, except that the formula of the Lactobacillus plantarum freeze-drying protective agent and the preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder.

[0088] A Lactobacillus plantarum freeze-drying protective agent, comprising the following raw material components by mass percentage: trehalose 8%, skim milk powder 12%, sodium glutamate 5%, and the balance being water.

[0089] The preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder is the same as Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the pre-freezing temperature is -20℃, and the rest of the operations are the same as Example 1.

[0090] Comparative Example 9 The same as Example 1, except that the formula of the Lactobacillus plantarum freeze-drying protective agent and the preparation method of the Lactobacillus plantarum ZJ-23 freeze-dried bacteria powder.

[0091] A freeze-drying protective agent of Lactobacillus plantarum comprises the following raw material components in percentage by mass: trehalose 8%, skimmed milk powder 12%, sodium glutamate 6%, and the balance being water.

[0092] A method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the temperature of pre-freezing is -40 DEG C, the pre-freezing time is 4h, and the rest of the operations are the same as those in Example 1.

[0093] Comparative Example 10 The method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the formula of the freeze-drying protective agent of Lactobacillus plantarum and the method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 are different.

[0094] A freeze-drying protective agent of Lactobacillus plantarum comprises the following raw material components in percentage by mass: trehalose 8%, skimmed milk powder 14%, sodium glutamate 4%, and the balance being water.

[0095] A method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacteria slurry to the freeze-drying protective agent is 5:10; the temperature of pre-freezing is -20 DEG C, the pre-freezing time is 12h, and the rest of the operations are the same as those in Example 1.

[0096] Comparative Example 11 The method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the formula of the freeze-drying protective agent of Lactobacillus plantarum and the method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 are different.

[0097] A freeze-drying protective agent of Lactobacillus plantarum comprises the following raw material components in percentage by mass: trehalose 8%, skimmed milk powder 14%, sodium glutamate 5%, and the balance being water.

[0098] A method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the freeze-drying protective agent of the present comparative example is used, and in step S2, the mass-volume ratio g / mL of the bacteria slurry to the freeze-drying protective agent is 4:10; the pre-freezing time is 4h, and the rest of the operations are the same as those in Example 1.

[0099] Comparative Example 12 The method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 is basically the same as that in Example 1, except that the formula of the freeze-drying protective agent of Lactobacillus plantarum and the method for preparing freeze-dried bacteria powder of Lactobacillus plantarum ZJ-23 are different.

[0100] A freeze-drying protective agent of Lactobacillus plantarum comprises the following raw material components in percentage by mass: trehalose 8%, skimmed milk powder 16%, sodium glutamate 4%, and the balance being water.

[0101] The preparation method of Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that: the freeze-drying protectant of this comparative example is used, and in step S2, the mass-volume ratio of bacterial sludge to freeze-drying protectant is 4:10 (g / mL); the pre-freezing temperature is -40℃, and the remaining operations are the same as in Example 1.

[0102] Comparative Example 13 It is basically the same as Example 1, except for the formulation of the Lactobacillus plantarum freeze-drying protectant and the preparation method of Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder.

[0103] A freeze-drying protectant for Lactobacillus plantarum, comprising the following raw material components by mass percentage: trehalose 8%, skim milk powder 16%, monosodium glutamate 6%, and the balance being water.

[0104] The preparation method of Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder is basically the same as that in Example 1, except that the freeze-drying protectant of this comparative example is used, and the pre-freezing time in step S2 is 12 hours. The rest of the operation is the same as in Example 1.

[0105] The viable bacteria before and after freeze-drying in the above examples and comparative examples were counted, and the freeze-drying survival rate was calculated. The results are shown in the table below. The counting method is as follows: 1 mL of culture solution was transferred and mixed with 9 mL of sterile physiological saline for serial dilution. 100 μL of a suitable gradient solution was spread on solid MRS medium and incubated at 37 ℃ for 48 h. The colony count was then recorded as the viable bacteria count before freeze-drying. The freeze-dried bacterial powder was dissolved in sterile physiological saline to the original volume of the culture solution, and the above operation was repeated to record the viable bacteria count after freeze-drying. The freeze-drying survival rate was calculated according to the following formula: Freeze-dried survival rate (%) = (Number of viable bacteria after freeze-drying / Number of viable bacteria before freeze-drying) × 100% The viable count before freeze-drying refers to the number of viable bacteria in the culture medium.

[0106] Table 1: Viable bacterial counts and freeze-dried survival rates in examples and comparative examples

[0107] As shown in Table 1, when preparing Lactobacillus plantarum ZJ-23 freeze-dried bacterial powder, adding the Lactobacillus plantarum freeze-drying protectant of the present invention in proportion and following the pre-freezing process of the present invention can significantly improve the freeze-drying survival rate of Lactobacillus plantarum ZJ-23, which can reach 50.75%.

[0108] The freeze-dried bacteria powder obtained in Example 1 is taken as an experimental group; only the freeze-drying protective agent in Example 1 is replaced with an equal amount of sterile water to prepare a freeze-dried bacteria powder, which is taken as a control group; the culture bacteria liquid obtained in Example 1 through step S1 is taken as a blank group; and the freeze-drying protection mechanism of the freeze-drying protective agent of the Lactobacillus plantarum on the Lactobacillus plantarum ZJ-23 is explored.

[0109] (1) Observation of bacterial morphology About 1-3 mm 3 After drying, the bacteria powder is fixed on a sample table by using conductive carbon glue, sprayed with gold by using an ionic sputtering instrument, and then placed under a scanning microscope at different magnifications for observation, and the results are shown in Figures 1-6 From the figure, it can be seen that the bacterial cells in the experimental group are wrapped in the protective agent and are relatively tightly arranged and distributed; the cell surface is relatively smooth and the cells are relatively complete, without rupture and fragmentation; the bacterial cells in the control group are stacked layer by layer, are mutually adhered, the cells are not complete, the bacterial cells are shrunk and ruptured, the surface is rough and presents an irregular shape, and there are leaked contents. This shows that the addition of the freeze-drying protective agent of the application during the freeze-drying process of the Lactobacillus plantarum ZJ-23 can effectively maintain the integrity of the cell morphology and structure, thereby preventing the leakage of the contents.

[0110] (2) Cell membrane integrity determination The bacteria powder in the experimental group and the control group is respectively re-dissolved to the original volume, 2 mL of bacteria liquid is taken at 4°C, 8000xg centrifuged for 3 min to obtain bacterial cells, and then washed with 1 mL of 0.9% sterile physiological saline for 2 times, dissolved in 0.85 mL of PBS buffer, and then 0.1 mL of FDA and 50 μL of PI are added and mixed thoroughly, the whole staining process is carried out in the dark (37°C, 30 min), and then washed with PBS buffer for 3 times, and the final volume is adjusted to 800 μL with PBS. 50 μL of bacterial suspension is dropped on a glass slide and observed under a laser confocal microscope to determine the integrity of the cell membrane, and the results are shown in Figure 7 . Figure 7 The results obtained by observing the bacterial cells under a laser confocal microscope at 40x magnification and 5x magnification are shown in Figure 7 It can be seen that the number of bacterial cells emitting green fluorescence in the experimental group is more than that in the control group, which shows that the addition of the freeze-drying protective agent of the application reduces the damage degree of the cell membrane of the Lactobacillus plantarum ZJ-23, and the integrity of the cell membrane is better.

[0111] (3) Intracellular key enzyme activity determination Intracellular catalytic reactions, metabolic regulation, energy conversion, and environmental utilization are closely related to intracellular enzyme activity. The extreme environment of freeze-drying can cause certain damage to enzymes in Lactobacillus plantarum cells, affecting the activity of various enzymes such as β-galactosidase, ATPase, and lactate dehydrogenase, as well as cell growth and metabolism, resulting in decreased cell activity or even death.

[0112] Extraction of key enzymes: The bacterial powder from the experimental group was reconstituted to the culture volume (40 mL). 2 mL of the bacterial suspension was centrifuged at 8000 × g for 15 min. The bacterial sludge was washed twice with 0.9% sterile physiological saline and resuspended in 1 mL of PBS buffer. The sample was placed in an ice-water bath and disrupted using a cell disruptor. The parameters were set as follows: power 200 W, sonication for 5 s with a 15 s interval, for a total disruption time of 2 min. After disruption, one portion was used for ATPase and protein quantification; the other portion was centrifuged at 8000 × g for 10 min at 4 ℃, and the supernatant was used for lactate dehydrogenase (LDH) assay.

[0113] The bacterial powder from the experimental group was reconstituted to the culture volume (40 mL). 2 mL of the bacterial suspension was centrifuged at 8000 × g for 15 min. The bacterial sludge was washed twice with 0.9% sterile physiological saline and resuspended in 1 mL of cell extract. The sample was then placed in an ice-water bath for cell disruption. The relevant parameters were set as follows: power 200 W, sonication for 5 s with a 15 s interval, and a total disruption time of 2 min. The sample was then centrifuged at 8000 × g for 10 min at 4 ℃, and the supernatant was used for the determination of β-galactosidase (β-DAL).

[0114] The extraction methods for key enzymes in the control group and blank group were the same as those in the experimental group.

[0115] Determination of key enzyme activities: The activities of the corresponding enzymes in the experimental group, control group, and blank group were determined using the β-galactosidase kit, lactate dehydrogenase assay kit, and ATPase assay kit.

[0116] (3-1) Determination of β-galactosidase β-galactosidase is involved in the physiological process of lactose degradation into glucose and galactose in cellular metabolism. As an endogenous macromolecular protein, it can leak when dehydration and drying processes cause changes in cell membrane permeability.

[0117] The intracellular β-galactosidase activity of bacteria in the blank group, freeze-dried experimental group, and control group was determined using the above method, and the results are as follows: Figure 8 As shown, according to Figure 8As can be seen, the β-galactosidase activity in the blank group is the highest, and the enzyme activity decreases significantly after freeze-drying treatment, but the enzyme activity in the experimental group is higher than that in the control group, which shows that the freeze-drying protective agent of the application can protect the integrity of the cell membrane of Lactobacillus plantarum ZJ-23 and prevent the leakage of intracellular key enzymes.

[0118] (3-2) Determination of lactate dehydrogenase (LDH) Lactate dehydrogenase (LDH) is a key enzyme in cell basic metabolism, which can mediate the conversion reaction between lactic acid and pyruvic acid, and provide energy for cell life activities in the process of glycolysis. LDH is easily affected by dehydration and low temperature in the freeze-drying process and loses activity, so it can be used as a model protein to judge the effect of freeze-drying process.

[0119] The intracellular lactate dehydrogenase activity of the blank group, the experimental group after freeze-drying and the control group was determined by the above method, and the results are shown in Figure 9 , according to Figure 9 As can be seen, the LDH activity in the blank group is the highest, and the enzyme activity decreases after freeze-drying treatment, but the enzyme activity in the experimental group is significantly higher than that in the control group, which shows that the freeze-drying protective agent of the application can more effectively maintain the LDH enzyme activity during the freeze-drying process of Lactobacillus plantarum ZJ-23.

[0120] (3-3) Determination of ATPase ATPase is a key enzyme that provides energy for cells and maintains the balance of intracellular ion osmotic pressure, and is also an enzyme that is easily damaged in the freeze-drying process. Therefore, exploring the activity of ATPase under different pre-freezing temperatures and pre-freezing times can reflect the protection effect of the protective agent on the bacteria.

[0121] The intracellular Na + -K + ATPase activity, Ca 2+ -Mg 2+ ATPase activity and total ATPase activity of the blank group, the experimental group after freeze-drying and the control group were determined by the above method, and the results are shown in Figure 10 , Figure 11 and Figure 12 , according to the three figures, it can be seen that the activities of the three enzymes in the blank group are the highest, and the activities of the three enzymes decrease after freeze-drying treatment, but the enzyme activity in the experimental group is higher than that in the control group, which shows that the freeze-drying protective agent of the application can effectively protect the activity of ATPase in Lactobacillus plantarum ZJ-23, maintain normal life metabolism of the cells and reduce the damage of freeze-drying to the bacteria.

[0122] (4) Expression of differential genes in transcriptomics The RNA of the experimental group and the control group was extracted and hybridized with microbial RNA using biotin probes, and then rRNA was removed by streptavidin magnetic adsorption. The DNA was fragmented to the range of 100-300 bp, and cDNA was obtained after reverse transcription. Then, a buffer system and double-stranded sequencing were added for linker connection, and finally high-fidelity DNA polymerase was used for library amplification. Illumina NovaSeq 6000 high-throughput sequencing platform was used for sequencing with a 2x150 bp double-end sequencing strategy. The transcriptome sequencing and analysis were completed by Shanghai Haowetai Biological Technology Co., Ltd.

[0123] The raw data obtained was subjected to sequencing data quality evaluation by Fast QC software and data analysis software R. The adapter sequences and low-quality bases were filtered out by Trim-galore software, Hisat 2 software was used for reference genome alignment, and finally RSeQC software was used for sequence sampling and expression quantification. mRNA analysis mainly includes String Tie gene expression quantification; Deseq 2 software identifies the differential genes of the experimental group and the control group, and the genes meeting P<0.05 and log2 FC (fold change)>1 are marked as differential genes, and finally GO (Gene Ontology) functional annotation is performed.

[0124] (4-1) Identification of differential genes To identify the expression and significance of differential genes in the experimental group and the control group, differential expression genes were screened according to P<0.05 and |log2 (fold change)|>1, and log2 (fold change)>1 was marked as up-regulated genes (Up); log2 (fold change)<-1 was marked as down-regulated genes (Down), and the rest were marked as non-significant differential expression genes (NotDEG). The results are as follows: a total of 131 differential genes were identified in the experimental group and the control group, including 81 up-regulated genes, accounting for 61.83% of the total differential expression genes, and 50 down-regulated genes, accounting for 38.17% of the total differential expression genes. According to the differential genes, a differential gene expression scatter plot and a differential gene expression volcano plot were drawn, as shown in Figure 13 each point in the figure represents the average expression value of a gene, red points represent genes with up-regulated expression in the experimental group relative to the control group, blue points represent genes with no significant difference, and green points represent genes with down-regulated expression in the experimental group.

[0125] The differential gene expression scatter plot reflects the logarithmic values ​​of the average expression levels of different genes in the experimental and control groups. The further a point deviates from the diagonal, the higher the relative expression level of that gene in the corresponding sample; more points deviating from the diagonal also indicate a greater difference in gene expression levels. The results showed that, compared to the control group, genes related to biological processes such as ribonucleotide synthesis, hydrolase activity, peptidase activity, pyrophosphatase activity, and hydrolase activity were significantly upregulated in the experimental group, while genes related to molecular functions such as purine ribonucleoside monophosphate metabolism, organic nitrogen synthesis, pentose phosphate decomposition, gene expression, and transcription were significantly downregulated. This indicates that when *Lactobacillus plantarum* ZJ-23 cells are freeze-dried, the cell membrane structure, intracellular biomolecules, and enzyme activity are damaged, affecting normal cellular life activities. However, after adding the freeze-drying protectant of this invention and then freeze-drying, the low temperature and dehydration environment allow some genes in the cells to actively participate in cell metabolism, thereby improving the survival rate.

[0126] (4-2) GO annotation of differentially expressed genes Based on potential functional candidate genes, the significant enrichment of representative GO functional sets in differentially expressed genes was calculated and compared with the GO database for enrichment analysis. The results are as follows: Figure 14 As shown. From Figure 14 It can be seen that the identified differentially expressed genes mainly fall into three categories and 30 functional subclasses. Among them, biological processes (BP) have 10 functional subclasses, mainly focusing on DNA replication (GO: 0006260), purine nucleoside monophosphate metabolism (GO: 0009126), and IMP biosynthesis (GO: 0006188); cellular components (CC) have 10 functional subclasses, mainly annotated as intracellular anatomical structures (GO: 0005622), cytoplasm (GO: 0005737), and intracellular organelles (GO: 0043229); and molecular functions (MF) have 10 functional subclasses, mainly annotated as nucleoside phosphate binding (GO: 1901265), carbohydrate derivative binding (GO: 0097367), and purine ribonucleotide binding (GO: 0032555).

[0127] Among the significantly up-regulated differential genes, ClpL is an ATP-dependent protease that can participate in protein homeostasis under stress conditions, and can regulate compound transport and metabolic processes. The significant up-regulation of the gene in the experimental group indicates that the cell can improve the resistance by promoting the synthesis of cell membrane in the extreme environment. TypA is an enzyme related to ribosome synthesis. The significant up-regulation of the gene in the experimental group indicates that the gene plays a key role in adapting to the low-temperature environment. XerC is related to the completion of chromosome division and maintenance of DNA fragment structure. The significant up-regulation of the gene in the experimental group may be related to the regulation of genetic material synthesis by the bacteria to improve the survival rate in the freeze-drying environment. This indicates that the freeze-drying protection mechanism of Lactobacillus plantarum ZJ-23 is a complex process in which a variety of life processes are regulated by a variety of genes.

[0128] The above only describes the preferred embodiments of the present application, and the present application is not limited to the above examples. It can be understood that other improvements and changes directly derived or thought of by those skilled in the art without departing from the spirit and concept of the present application should be considered to be included in the protection scope of the present application.

Claims

1. A freeze-drying protectant for Lactobacillus plantarum, characterized in that, Including the following ingredients: sugars, milk powder, and sodium salts; The mass ratio of the sugar, the milk powder and the sodium salt is (4-6):(14-16):(4-6).

2. The *Lactobacillus plantarum* freeze-drying protectant according to claim 1, characterized in that, The raw materials for the Lactobacillus plantarum freeze-drying protectant also include water; Preferably, the Lactobacillus plantarum freeze-drying protectant comprises, by mass percentage, the following raw material components: 4-6% sugars, 14-16% milk powder, 4-6% sodium salt, and the balance being water.

3. The *Lactobacillus plantarum* freeze-drying protectant according to any one of claims 1-2, characterized in that, The sugars include at least one of xylooligosaccharides, trehalose, inulin, and maltodextrin; And / or, the milk powder includes at least one of skim milk powder and whey powder; And / or, the sodium salt includes at least one of monosodium glutamate, sodium alginate, and sodium ascorbate.

4. A method for preparing the *Lactobacillus plantarum* freeze-drying protectant according to any one of claims 1-3, characterized in that, The preparation method includes: weighing the prescribed amounts of the sugars, the milk powder, and the sodium salt, dissolving them in water, and mixing them thoroughly to obtain the final product; Preferably, the water is sterile water.

5. A method for preparing freeze-dried Lactobacillus plantarum powder, characterized in that, Includes the following steps: S1. Expand the culture of Lactobacillus plantarum to obtain a culture solution, centrifuge and wash to obtain bacterial sludge; S2. Mix the bacterial mud with a freeze-drying protectant, pre-freeze and then freeze-dry to obtain freeze-dried Lactobacillus plantarum powder. The freeze-drying protectant is the *Lactobacillus plantarum* freeze-drying protectant according to any one of claims 1-3; or the *Lactobacillus plantarum* freeze-drying protectant prepared by the preparation method of claim 4.

6. The preparation method according to claim 5, characterized in that, In S1, the Lactobacillus plantarum includes Lactobacillus plantarum ZJ-23; And / or, the mass-to-volume ratio of the bacterial sludge to the freeze-drying protectant (g / mL) is (4~6):

10.

7. The preparation method according to claim 5, characterized in that, In S2, the pre-freezing temperature is -80~-20℃; And / or, the pre-freezing time is 8-12 hours.

8. The preparation method according to claim 5, characterized in that, In S2, the freeze-drying time is 22-26 hours; And / or, the freeze-drying temperature is -80~-20℃.

9. A freeze-dried Lactobacillus plantarum powder, characterized in that, The freeze-dried Lactobacillus plantarum powder is prepared by the preparation method according to any one of claims 5-8.

10. The application of the freeze-dried Lactobacillus plantarum powder according to claim 9, or the freeze-dried Lactobacillus plantarum powder prepared by any one of claims 5-8, in food fermentation.

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