Preparation method and application of freeze-dried powder of lactobacillus salivarius

Abstract

The invention discloses a preparation method of freeze-dried powder of lactobacillus salivarius. The preparation method specifically comprises the following steps: step 1, determining an optimal growth period by drawing a growth curve of lactobacillus salivarius; 2, screening an excellent freeze-drying protective agent through a single factor; 3, freeze-drying conditions of the bacterial powder are determined through an orthogonal test; and step 4, adding an antioxidant to store the freeze-dried bacterial powder. According to the preparation method of the freeze-dried lactobacillus salivarius powder, a single-factor test is adopted to select an excellent freeze-drying protective agent, then a three-factor four-level orthogonal test is adopted to determine freeze-drying conditions, finally, an antioxidant is added before freeze-drying, the protective effects of different antioxidants on long-term storage of the powder are evaluated, and a long-acting protection mechanism is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic powder preparation, relates to a method for preparing freeze-dried Lactobacillus salivarius powder and also relates to application of the freeze-dried Lactobacillus salivarius powder. Background Art

[0002] Lactobacillus salivarius is a valuable probiotic that is widely distributed in the human digestive system, such as the oral cavity and intestines. It can significantly improve the host's intestinal microbial environment and promote health. In recent years, as people's understanding of probiotics deepens, Lactobacillus salivarius has been used more and more widely in the fields of food, health products, and medicine. However, how to maintain the activity and stability of Lactobacillus salivarius has become one of the main challenges hindering its widespread application.

[0003] At present, freeze-drying technology is developing rapidly and is used for the storage and transportation of probiotics. However, during the freeze-drying process, the main factors for the loss of cell viability are the formation of intracellular ice crystals, high osmotic pressure and membrane damage caused by high concentrations of intracellular solutes, denaturation of macromolecules, and evaporation of water. These will affect the properties of many hydrophilic macromolecules in the cells, resulting in a sharp drop in survival rate. Therefore, it is very important to choose a suitable freeze-drying protective agent to improve the freeze-dried survival rate of Lactobacillus salivarius. Existing studies have shown that sucrose, trehalose, skim milk and other substances can be used as freeze-drying protective agents for probiotics, but their protective effects vary depending on external conditions and added ratios. There is even less research on the development of specific protective agents for Lactobacillus salivarius.

[0004] Lactobacillus salivarius has multiple functions, such as improving bad breath, protecting dental health, preventing the growth of harmful bacteria, and maintaining the normal functioning of the digestive system. In the food industry, Lactobacillus salivarius has been widely used in the production of fermented dairy products, beverages, and baked goods, giving these products unique health attributes and flavors. In the field of health care products, Lactobacillus salivarius, as a key probiotic ingredient, is used in the research and development of multiple health functions such as regulating intestinal flora and enhancing immunity. In addition, in the medical field, Lactobacillus salivarius shows potential application prospects, such as in the preparation of anti-allergic drugs.

[0005] In summary, Lactobacillus salivarius, as an important type of probiotic, has broad application prospects in many fields. However, the improvement of its freeze-dried survival rate is still an urgent problem to be solved. To this end, this study conducted an in-depth study on the preparation parameters of Lactobacillus salivarius and the composition of the freeze-drying protective agent, in order to find an effective method for preparing freeze-dried powder of Lactobacillus salivarius, and to provide a theoretical basis and practical guidance for its application in various fields. Summary of the invention

[0006] The invention aims to provide a method for preparing freeze-dried powder of Lactobacillus salivarius, which solves the problem of low freeze-drying survival rate of Lactobacillus salivarius in the process of preparing the bacteria.

[0007] The present invention also aims to provide application of freeze-dried Lactobacillus salivarius powder.

[0008] The first technical solution adopted by the present invention is a method for preparing freeze-dried Lactobacillus salivarius powder, and the specific operating steps are as follows: Step 1: Determine the optimal growth period by drawing a growth curve of Lactobacillus salivarius; Step 2: Screening excellent lyophilization protectants by single factor; Step 3: Determine the freeze-drying conditions of bacterial powder through orthogonal test; Step 4: Add antioxidants to store the freeze-dried bacterial powder.

[0009] The present invention is also characterized in that: Step 1 is as follows: The activated Lactobacillus salivarius seed liquid was inoculated into the MRS medium at a 2% inoculation rate and cultured at 37°C for 24 hours; the bacterial liquid was collected every 2 hours, the bacterial population concentration was determined by the dilution plate method, and the absorbance was determined at OD600 to draw the growth curve of Lactobacillus salivarius to determine the time when the bacterial population concentration reached the logarithmic growth phase when the inoculation rate of Lactobacillus salivarius was 2%; The MRS culture medium includes 10 g / L peptone, 10 g / L beef powder, 5 g / L yeast powder, 20 g / L glucose, 2.6 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L triammonium citrate trihydrate, 3 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 ml / L Tween 80.

[0010] Step 2 is as follows: Step 2.1: Inoculate the activated Lactobacillus salivarius into MRS medium at a rate of 2%, and culture at a constant temperature of 37°C until the logarithmic growth phase according to the growth curve of Lactobacillus salivarius; Step 2.2: collect the bacterial liquid, centrifuge at 4000×g and 4°C for 5 min, discard the supernatant, and wash twice with sterile saline to obtain Lactobacillus salivarius bacterial slurry; Step 2.3: resuspend the Lactobacillus salivarius slurry in sterile saline to prepare a bacterial suspension; and determine the bacterial concentration using the dilution plate method; Step 2.4: Add different types of freeze-dried protective agents to the bacterial suspension, mix each freeze-dried protective agent with the bacterial suspension at a volume ratio of 5:1, transfer to a culture dish, and freeze at -80°C for 12 hours, then put into a vacuum freeze dryer for drying for 24 hours, and obtain freeze-dried bacterial powder after freeze drying, and weigh the mass of the obtained bacterial powder; Step 2.5: Accurately weigh the unit mass of freeze-dried bacterial powder to count the bacteria and calculate the survival rate.

[0011] Step 3 is as follows: According to the freeze-drying and survival rate of each lyoprotectant on Lactobacillus salivarius obtained in step 2.5, three lyoprotectants with better protection effects are selected for compounding, and these three protectants are used as basic cryoprotectants for compounding, and the protection effect of the basic protectants is calculated.

[0012] Step 3 is as follows: the freeze-drying conditions of the bacterial powder were explored through orthogonal experiments. The orthogonal experiment was designed based on the factors composed of the type of amino acids added in the basic protective agent, the pre-freezing temperature and the pre-freezing time. A three-factor, three-level orthogonal experiment was established, with a total of 9 groups of experiments. The protective agent and the bacterial suspension were evenly mixed at a volume ratio of 5:1. Three replicates were set for each group. The bacterial mud was freeze-dried according to different freeze-drying times. The initial number of viable bacteria before frozen storage and the number of viable bacteria after frozen storage were detected, and the survival rate was calculated. On the premise of meeting the powdering requirements and the freeze-drying survival rate, the freeze-drying protective agent with the largest survival rate was selected as the optimal condition for preparing the bacterial powder.

[0013] The excellent lyoprotectant determined in step 3 includes, by mass percentage, 10% skim milk powder, 10% whey protein, 8% sucrose, 1% aspartic acid, 1% vitamin E, and the rest is water.

[0014] The freeze-dried survival rate of step 3 is not less than 5%.

[0015] Step 4 is as follows: freeze-drying the Lactobacillus salivarius slurry to prepare bacterial powder and storing it at room temperature, adding an antioxidant to the bacterial solution before freeze-drying, the antioxidant added in an amount of 1% of the mass of the bacterial solution; the antioxidant includes any one of vitamin C, vitamin E and sodium thiosulfate.

[0016] The second technical solution commonly used in the present invention is to use freeze-dried powder of Lactobacillus salivarius as intestinal probiotics and conduct application evaluation on feeding laying hens.

[0017] The beneficial effects of the present invention are: The method for preparing freeze-dried Lactobacillus salivarius powder of the present invention provides a theoretical basis and practical guidance for its application in various fields by determining various parameters in the preparation process of freeze-dried Lactobacillus salivarius powder; specifically: 1. Selection of lyophilized protective agents: Use single-factor experiments to determine the protective effect of a single protective agent, select three protective agents with better effects for compounding, determine the basic protective agent, and screen the optimal lyophilized protective agent by measuring the lyophilization protection rate.

[0018] 2. Selection of freeze-drying conditions: The factors that affect the freeze-drying effect of Lactobacillus salivarius mainly include pre-freezing time, pre-freezing temperature and the type of amino acid added. The optimal freeze-drying conditions of the bacterial powder were determined through a three-factor four-level orthogonal experiment.

[0019] 3. Selection of antioxidants before freeze-drying: In addition to the storage environment, the factors that affect the storage effect of freeze-dried bacterial powder are the addition of an appropriate amount of antioxidants in the early stage, which also has a significant effect on extending the storage period. Through single-factor comparative experiments, the activity of bacterial powder is regularly measured, and the protective effect of different antioxidants on the long-term storage of bacterial powder is evaluated to achieve a long-term protection mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the growth curve of Lactobacillus salivarius of the present invention. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1 The method for preparing freeze-dried Lactobacillus salivarius powder of the present invention comprises the following specific steps: Step 1: Plot a growth curve of Lactobacillus salivarius (e.g. Figure 1 As shown in the figure, the optimal growth period was determined to be 2% inoculation and cultured at 37°C for 10 h; Step 2: Screening excellent lyophilization protectants by single factor; Step 3: Determine the freeze-drying conditions of bacterial powder through orthogonal test; Step 4: Add antioxidants to store the freeze-dried bacterial powder to improve the storage effect.

[0023] Example 2 Based on Example 1, Step 1 is as follows: The activated Lactobacillus salivarius seed liquid was inoculated into the MRS medium at a 2% inoculation rate and cultured at 37°C for 24 hours; the bacterial liquid was collected every 2 hours, the bacterial population concentration was determined by the dilution plate method, and the absorbance was determined at OD600 to draw the growth curve of Lactobacillus salivarius to determine the time when the bacterial population concentration reached the logarithmic growth phase when the inoculation rate of Lactobacillus salivarius was 2%; MRS culture medium includes 10 g / L peptone, 10 g / L beef powder, 5 g / L yeast powder, 20 g / L glucose, 2.6 g / L potassium dihydrogen phosphate trihydrate, 2 g / L triammonium citrate trihydrate, 3 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 ml / L Tween 80.

[0024] Example 3 Based on Example 2, Step 2 is as follows: Step 2.1: Inoculate the activated Lactobacillus salivarius into MRS medium at a rate of 2%, and culture at a constant temperature of 37°C until the logarithmic growth phase (10 hours) according to the growth curve of Lactobacillus salivarius; Step 2.2: collect the bacterial liquid, centrifuge at 4000×g and 4°C for 5 min, discard the supernatant, and wash twice with sterile saline to obtain Lactobacillus salivarius bacterial slurry; Step 2.3: resuspend the Lactobacillus salivarius slurry in sterile saline to prepare a bacterial suspension; and determine the bacterial concentration using the dilution plate method; Step 2.4: Add different types of freeze-dried protective agents to the bacterial suspension, mix each freeze-dried protective agent with the bacterial suspension in proportion, transfer to a culture dish, and freeze at -80°C for 12 hours, then put into a vacuum freeze dryer for 24 hours, and obtain freeze-dried bacterial powder after freeze drying, and weigh the mass of the obtained bacterial powder; Step 2.5: Accurately weigh a unit mass of freeze-dried bacterial powder for bacterial counting. After 2 weeks of storage at room temperature, accurately weigh a unit mass of freeze-dried bacterial powder again for bacterial counting. Finally, the freeze-drying and long-term storage survival rate of Lactobacillus salivarius for each lyoprotectant was determined; the volume ratio of lyoprotectant to bacterial suspension was 5:1.

[0025] Example 4 Based on Example 3, Step 3 is as follows: According to the freeze-drying protection rate of each freeze-drying protectant for Lactobacillus salivarius obtained in step 2.5, three freeze-drying protectants with better protection effects are selected for compounding as basic protectants, and the freeze-drying protection effects of the basic protectants are calculated.

[0026] An orthogonal experiment was designed based on the factors composed of the types of amino acids added to the basic protective agent, pre-freezing temperature and pre-freezing time. A three-factor, three-level orthogonal experiment was established, with a total of 9 groups of experiments. The protective agent and bacterial suspension were evenly mixed in a volume ratio of 5:1. Three replicates were set up in each group. The bacterial sludge was freeze-dried according to different freeze-drying times. The initial number of viable bacteria before frozen storage and the number of viable bacteria after frozen storage were tested, and the survival rate was calculated. Under the premise of meeting the powdering requirements and freeze-drying survival rate (freeze-drying survival rate not less than 5%), the freeze-drying protective agent with the largest survival rate was selected as the optimal ratio.

[0027] The optimal ratio of the lyoprotectant finally determined in step 3 is, in terms of mass percentage, 10% skim milk powder, 10% whey protein, 8% sucrose, 1% aspartic acid, 1% vitamin E, and the rest is water.

[0028] Step 4 is as follows: freeze-drying the Lactobacillus salivarius bacterial slurry to prepare bacterial powder and storing it at room temperature, adding an antioxidant to the bacterial solution before freeze-drying, the antioxidant added in an amount of 1% of the mass of the bacterial solution; the antioxidant includes any one of vitamin C, vitamin E and sodium thiosulfate.

[0029] Example 5 The method for preparing freeze-dried Lactobacillus salivarius powder provided by the present invention comprises the following specific steps: 1. Preparation of Lactobacillus salivarius suspension Prepare MRS medium, peptone 10g / L, beef powder 10g / L, yeast powder 5g / L, glucose 20g / L, potassium phosphate trihydrate 2.6 g / L, ammonium citrate trihydrate 2 g / L, sodium acetate 3 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1ml / L. Inoculate one thousandth of Lactobacillus salivarius seed liquid and culture at 37℃ for 10h to reach the logarithmic growth phase. Take 50ml of culture liquid in a centrifuge tube and centrifuge at 20℃ for 4min at 4000g / min to obtain bacterial mud, wash twice with sterile saline, and add 1ml PBS to obtain bacterial suspension.

[0030] 2. Add lyophilization protective agent Prepare freeze-dried protective agent, 10% skim milk powder, 10% whey protein, 8% sucrose, 1% aspartic acid and 1% vitamin E (tocopherol). Add 5 ml of protective agent to the bacterial suspension and mix well, and freeze quickly in -196 liquid nitrogen for 12 hours.

[0031] 3. Freeze-drying of Lactobacillus salivarius slurry After the bacterial suspension was quick-frozen for 12 hours, it was placed in a vacuum freeze dryer for freeze drying, with a well temperature of -53°C, a sample temperature of -30°C, and a vacuum degree of 453 Pa. After freeze drying for 24 hours, the water was completely lost. The sample was ground using a grinder to obtain Lactobacillus salivarius powder, which was diluted with maltodextrin and placed at room temperature for long-term storage.

[0032] 4. Determination of bacterial powder activity Take 0.1g sample and dissolve it in 900μl PBS, perform gradient dilution, and determine the bacterial powder activity value by the coating plate method. Example 6 By drawing the growth curve of Lactobacillus salivarius, the time required for it to reach the logarithmic growth phase during the culture process is determined. Studies have shown that the logarithmic phase strains divide quickly, increase in number, have the most vigorous growth metabolism, have the most typical bacterial morphology and physiology, have a higher ability to resist the external environment, and have higher vitality. By selecting the logarithmic phase strains for the preparation of bacterial powder, better freezing results will be achieved.

[0033] Freeze-drying is an important part of the bacterial powder preparation process. The type and proportion of freeze-drying protective agents will also have an important impact on the freeze-drying effect. Studies have shown that glycerol, low-molecular sugars and alcohols have more than 5 hydroxyl groups, which can be linked to free radicals on the surface of bacteria to prevent the bacteria from being exposed to the medium. They can also form hydrogen bonds with proteins to replace water, ensuring the stability of proteins. High-molecular protective agents such as skimmed milk powder and soluble starch can form a protective layer on the surface of bacteria, reduce the leakage of intracellular substances caused by cell wall damage, and thus play a protective role. Protective agents can also act as buffers to repair damage to bacteria and maintain the nutritional status of bacteria; at the same time, they can prevent the generation of free radicals and reduce the oxidation reaction of bacteria during the drying process. Therefore, by determining the specific type of protective agent and its addition ratio, the freeze-drying process of the strain can be effectively protected.

[0034] The time required for freeze-drying varies for different protective agents. On the one hand, it is necessary to ensure complete drying and powdering effect. At the same time, the freeze-drying time should be properly controlled to prevent a major impact on the vitality of the strain. Therefore, determining the appropriate freeze-drying time can effectively improve the freeze-drying effect of the bacterial powder.

[0035] Single factor test Table 1 Single lyoprotectant concentration gradient

[0036] Table 2 Protection effect of single lyophilization protective agent

[0037] Table 3 Protection effect of composite (basic) freeze-dried protective agent

[0038] 3. Determine the freeze-drying conditions of bacterial powder through orthogonal test According to the results of single-factor experimental screening, three cryoprotectants with better protective effects were selected from the cryoprotectants for compounding, and finally 10% skim milk powder + 10% whey protein + 8% sucrose were determined as the basic protective agent. An orthogonal experimental design was carried out with the type of amino acid addition, pre-freezing temperature and pre-freezing time as the difference factors. An orthogonal table with three factors and three levels (see Table 4 and Table 5) was established to conduct an orthogonal experiment. Three replicates were set up in each group to detect the initial number of viable bacteria before cryopreservation and the number of viable bacteria after cryopreservation, and the survival rate was calculated. The results showed that adding 1% aspartic acid to the basic protective agent and pre-freezing at -196℃ for 12 hours had the best protective effect.

[0039] Table 4: Table of levels of factors in orthogonal experiment

[0040] Table 5: Orthogonal experimental design table

[0041] K1 represents the sum of the values ​​of the test indicators corresponding to experiment number 1; K2 represents the sum of the values ​​of the test indicators corresponding to the level of experiment number 2; K3 represents the sum of the values ​​of the test indicators corresponding to the level of experiment number 3; R stands for range, which is used to indicate the magnitude of the effect of a factor on the result. R = max(K i ) - min(K i ).

[0042] It can be seen from the above table that the influence of the dominant effect is A>C>B>D, and the optimal ratio is A3B3C2.

[0043] 4. Improve the storage effect of bacterial powder by adding antioxidants According to the results of the orthogonal test, the Lactobacillus salivarius bacterial slurry was freeze-dried to prepare bacterial powder for storage at room temperature. To improve the storage effect of the bacterial powder, antioxidants 1% vitamin C, vitamin E and sodium thiosulfate were added to the bacterial solution before freeze-drying. The activity of the bacterial powder was measured on day 0, day 15 and day 30 according to Table 6.

[0044] Table 6: Storage survival rate

[0045] As can be seen from the above table, the survival rate of live bacteria in the control group without added antioxidants was poor after 30 days, only 22.62%. The survival rate of live bacteria added with vitamin C, sodium thiosulfate or vitamin E was above 28.57%, among which the survival rate with added vitamin E was the highest, reaching 47.59%.

[0046] 5. Application of freeze-dried bacterial powder According to the above orthogonal test results and storage effects, 10% skim milk powder + 10% whey protein + 8% sucrose + 1% aspartic acid + 1% vitamin E (tocopherol) + 70% distilled water was used to prepare a freeze-drying protective agent, which was quick-frozen at -196 degrees Celsius (liquid nitrogen) for 8 hours, and then vacuum-freeze-dried to prepare Lactobacillus salivarius freeze-dried powder. We evaluated the application of Lactobacillus salivarius on laying hens as an intestinal probiotic. 480 healthy, 50-week-old Hy-Line gray laying hens with basically the same physical condition were selected and randomly divided into 4 groups, with 8 replicates in each group and 12 chickens in each replicate. The trial period was 14 weeks, including a 2-week pre-trial period and a 12-week formal period; the specific groups were divided as follows: Control group CON: basic diet group; Low-dose group LD: (basal diet + 0.3g / kg quercetin + 7.5×10 7 CFU / kg Lactobacillus salivarius); Medium dose group MD: (basal diet + 0.4g / kg quercetin + 1×10 8 CFU / kg Lactobacillus salivarius group); High-dose group HD: (basal diet + 0.5g / kg quercetin + 1.25×10 8 CFU / kg Lactobacillus salivarius group); During the feeding process, the production performance of laying hens was calculated, the egg quality was measured, and the effect of Lactobacillus salivarius application was evaluated. The results are shown in the following table.

[0047] Table 7 Effects of Lactobacillus salivarius on production performance of laying hens

[0048] Table 8 Effect of Lactobacillus salivarius on egg quality

[0049] It can be found from Table 7 that, from the perspective of full-term production performance, compared with the CON group, the LD group, MD group and HD group can significantly increase the egg production rate and reduce the feed-egg ratio from 50 to 61 weeks; the HD group can significantly reduce the average daily feed intake from 50 to 61 weeks. Compared with the CON group, the LD group, MD group and HD group can significantly reduce the eggshell thickness from 54 to 57 weeks, while the BM group did not show significant changes; Table 8 shows that compared with the CON group, the HD group can significantly reduce the albumen height, Haugh unit and eggshell thickness from 58 to 61 weeks. This shows that adding an appropriate amount of freeze-dried Lactobacillus salivarius powder to the diet can have a good effect on improving the production performance and egg quality of laying hens.

[0050] The present invention includes but is not limited to the above implementation cases. The above examples and descriptions are for better understanding and use of the invention. At the same time, technicians and researchers familiar with the field can modify the above implementation cases according to their own understanding to improve the effect and reduce the cost. However, modifications and improvements made without departing from the scope of the present invention are within the protection scope of the present invention.

Claims

1. A method for preparing freeze-dried Lactobacillus salivarius powder, characterized in that: The specific steps are as follows: Step 1: Determine the optimal growth period by drawing a growth curve of Lactobacillus salivarius; Step 2: Screening excellent lyophilization protectants by single factor; Step 3: Determine the freeze-drying conditions of bacterial powder through orthogonal test; Step 4: Add antioxidants to store the freeze-dried bacterial powder.

2. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 1, wherein: Step 1 is as follows: The activated Lactobacillus salivarius seed liquid was inoculated into the MRS medium at a 2% inoculation rate and cultured at 37°C for 24 hours; the bacterial liquid was collected every 2 hours, the bacterial population concentration was determined by the dilution plate method, and the absorbance was determined at OD600 to draw the growth curve of Lactobacillus salivarius to determine the time when the bacterial population concentration reached the logarithmic growth phase when the inoculation rate of Lactobacillus salivarius was 2%; The MRS culture medium includes 10 g / L peptone, 10 g / L beef powder, 5 g / L yeast powder, 20 g / L glucose, 2.6 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L triammonium citrate trihydrate, 3 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 ml / L Tween 80.

3. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 2, wherein: Step 2 is as follows: Step 2.1: Inoculate the activated Lactobacillus salivarius into MRS medium at a rate of 2%, and culture at a constant temperature of 37°C until the logarithmic growth phase according to the growth curve of Lactobacillus salivarius; Step 2.2: Collect the bacterial liquid, centrifuge at 4000×g and 4°C for 5 min, discard the supernatant, and wash twice with sterile saline to obtain Lactobacillus salivarius bacterial slurry; Step 2.3: resuspend the Lactobacillus salivarius slurry in sterile saline to prepare a bacterial suspension; and determine the bacterial concentration using the dilution plate method; Step 2.4: Add different types of freeze-dried protective agents to the bacterial suspension, mix each freeze-dried protective agent with the bacterial suspension in proportion, transfer to a culture dish, and freeze at -80°C for 12 hours, then put into a vacuum freeze dryer for 24 hours, and obtain freeze-dried bacterial powder after freeze drying, and weigh the mass of the obtained bacterial powder; Step 2.5: Accurately weigh the unit mass of freeze-dried bacterial powder to count the bacteria and calculate the survival rate.

4. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 2, wherein: The volume ratio of the lyophilization protectant and bacterial suspension in step 2.4 is 5:

1.

5. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 2, wherein: Step 3 is as follows: According to the freeze-drying and survival rate of Lactobacillus salivarius obtained by each freeze-drying protectant in step 2.5, three freeze-drying protectants with better protection effects are selected for compounding, and these three protectants are used as the components of the basic cryoprotectant to design an orthogonal experimental design, and a three-factor, three-level orthogonal experiment is established, with a total of 9 groups of experiments. The protectant and the bacterial suspension are evenly mixed in a volume ratio of 1:

1. Three replicates are set for each group. The bacterial mud is freeze-dried according to different freeze-drying times, and the initial number of viable bacteria before and after frozen storage are detected. The number of viable bacteria after frozen storage is calculated, and the survival rate is calculated. On the premise of meeting the powdering requirements and the freeze-drying survival rate, the freeze-drying protectant with the largest survival rate is selected as the optimal ratio.

6. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 5, characterized in that: The excellent lyoprotectant determined in step 3 includes, by mass percentage, 10% skim milk powder, 10% whey protein, 8% sucrose, 1% aspartic acid, 1% vitamin E, and the rest is water.

7. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 6, characterized in that: The freeze-dried survival rate of step 2 is not less than 5%.

8. The method for preparing freeze-dried Lactobacillus salivarius powder according to claim 1, characterized in that: Step 4 is as follows: freeze-drying the Lactobacillus salivarius mud to prepare bacterial powder for storage at room temperature, adding an antioxidant to the bacterial solution before freeze-drying, the antioxidant added in an amount of 1% of the mass of the bacterial solution; the antioxidant includes any one of vitamin C, vitamin E and sodium thiosulfate.

9. The application of freeze-dried Lactobacillus salivarius powder is characterized in that: The freeze-dried powder of Lactobacillus salivarius was evaluated as an intestinal probiotic in feeding laying hens.

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