Composite protective agent for spray drying of pediococcus acidilactici as well as preparation method and application of composite protective agent

Abstract

The invention discloses a composite protective agent for spray drying of pediococcus acidilactici as well as a preparation method and application of the composite protective agent. The composite protective agent consists of skimmed milk, cordyceps militaris polysaccharide and sodium glutamate. Experimental results show that the spray drying survival rate of pediococcus acidilactici without adding the protective agent is 17.60%, while the survival rate of the pediococcus acidilactici after spray drying is remarkably improved by adding the composite protective agent disclosed by the invention, and the survival rate reaches 71.72%. The survival rate of the pediococcus acidilactici after spray drying can be increased, and the digestion and storage stability of in-vitro simulated gastrointestinal fluid can be improved.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic spray drying, and particularly relates to a composite protective agent for spray drying Pediococcus acidilactici, a preparation method thereof, and an application thereof. Background Art

[0002] Pediococcus acidilactici has spherical cell morphology, generally grows in pairs, rarely grows singly, and does not arrange in chains. It is Gram-positive, non-motile, does not form spores, is catalase-negative, does not produce cytochromes, and is facultatively anaerobic. Its colonies on MRS solid medium are small and milky white. It can grow in the temperature range of 20 - 40 °C and the pH range of 4.2 - 8.0, and the optimal growth pH value is 5.5 - 6.5; it has high salt tolerance and can still grow when the NaCl concentration is 9 - 10%. Therefore, Pediococcus acidilactici has very high potential application value.

[0003] With the improvement of living standards and the popularization of the concept of healthy diet in China, probiotic powder has been favored by consumers because of its rich nutritional value and physiological functions. Commercially, vacuum freeze-drying and spray drying are the two most widely used technologies for producing probiotic powder. Compared with freeze-drying, spray drying is simple in operation, more cost-effective and less time-consuming, suitable for continuous production, and is considered a promising method for producing probiotic preparations. The powder prepared by this method has a relatively uniform shape, good sphericity, and a relatively small particle size (usually less than 100 μm). However, during the spray drying process, probiotic cells will be subjected to different pressures, which will cause damage to the integrity of the cell membrane of the strain, protein denaturation, loss of enzyme activity, and reduction in the stability of macromolecules such as ribosomes and RNA, resulting in damage or even death of the bacteria. Although various stress factors will seriously affect the viability of probiotics during and after spray drying, these factors can be overcome by selecting appropriate protection strategies. In order to improve the survival rate of probiotics during spray drying, researchers at home and abroad mainly intervene from the following means:

[0004] (1) Growth stage: Probiotics are usually harvested in the logarithmic phase or stationary phase, at which time the maximum yield can be obtained. However, the stationary phase is more frequently mentioned and used compared to the logarithmic phase because the collected cells also show higher viability during the drying process. Corcoran et al. determined the survival rate of Lactobacillus rhamnosus GG cultures after spray drying at different growth stages (lag, logarithmic, and stationary phases) in order to establish the optimal growth stage for spray drying cultures. The results showed that using stationary-phase cells obtained a survival rate of more than 50%, while the survival rate of logarithmic-phase cultures was 14%, and lag-phase cells were most vulnerable to spray drying, with a survival rate of only 2%.

[0005] (2) Harvesting technology: The separation of bacterial cells is an important intermediate step in the preparation of probiotic preparations. The centrifugation method is still the most widely used harvesting technology at present. The collection process of lactic acid bacteria is affected by centrifugation conditions. Firstly, the mechanical action during centrifugation will cause the death of some bacterial cells. Secondly, after centrifugation of the bacterial solution, some bacterial cells will inevitably remain in the supernatant, resulting in losses. Therefore, the survival rate and recovery rate of bacterial cells are closely related to the centrifugation process. It is necessary to conduct detailed research on factors such as the centrifugal force, centrifugation time, and centrifugation temperature, in order to obtain the optimal centrifugation conditions with the highest recovery rate of bacterial cells. Kang Jiao et al. used a single-factor combined with orthogonal experiments to optimize the centrifugation conditions of Lactobacillus fermentum FYa1, the strain used in the direct vat set starter for pickles, and determined that the optimal centrifugation conditions for this strain were a centrifugal speed of 3000 r / min, a centrifugation time of 15 min, and a centrifugation temperature of 15 °C. Moreover, the influence of centrifugation time on the survival rate of the strain was much greater than that of centrifugation temperature and centrifugal speed.

[0006] (3) Heat shock treatment of probiotic cells before drying: Some studies have shown that appropriate heat stress treatment can effectively improve the heat resistance of strains. Heat shock treatment, also known as heat stress, places probiotics in an environment above their optimal growth temperature, which will induce the expression of genes encoding heat shock proteins (HSPs). These proteins help to refold damaged intracellular proteins, maintain the normal living level of cells, and at the same time can change the cell physiological structure to enhance its tolerance to high-temperature environments. Paéz et al. found that when Lactobacillus casei Nad and Lactobacillus plantarum 8329 were heat-pretreated at 52 °C for 15 min, their survival rates were higher. Zhang et al. found that after heat shock treatment of Limosilactobacillus salivarius B-30514 at 50 °C for 15 min, the viable cell count after spray drying of the strain increased by 1.6 log CFU / mL compared with the control group.

[0007] (4) Adding protectants: Probiotics are not heat-resistant and will experience heat damage and dehydration damage during spray drying. Some studies have shown that the addition of protectants can improve the survival rate of bacterial cells during spray drying. The protection mechanisms of various protectants are different, so their protection effects on probiotics are also different. In addition, studies have also found that the effect of using protectants in combination is significantly better than that of using a single protectant. When Khem et al. used skim milk and gum arabic together as protectants for spray drying, they were able to improve the survival rate of Bifidobacterium animalis subsp. lactis BB12.

[0008] However, due to the specificity of strains, optimization needs to be carried out for each strain, rather than directly transferring from one strain to another. Summary of the Invention

[0009] The object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a composite protective agent for spray drying of Pediococcus acidilactici.

[0010] Another object of the present invention is to provide a preparation method of the above-mentioned composite protective agent.

[0011] Still another object of the present invention is to provide the application of the above-mentioned composite protective agent.

[0012] The object of the present invention is achieved by the following technical solutions:

[0013] A composite protective agent for spray drying of Pediococcus acidilactici, which is composed of the following components in parts by mass: 5-10 parts of skim milk, 4-6 parts of Cordyceps militaris polysaccharide, and 2-4 parts of sodium glutamate.

[0014] Further, the composite protective agent is composed of the following components in parts by mass: 7-8 parts of skim milk, 4.5-5.5 parts of Cordyceps militaris polysaccharide, and 2.5-3.5 parts of sodium glutamate.

[0015] Further, the composite protective agent is composed of the following components in parts by mass: 7.5 parts of skim milk, 5 parts of Cordyceps militaris polysaccharide, and 3 parts of sodium glutamate.

[0016] The preparation method of the above-mentioned composite protective agent is to weigh skim milk, Cordyceps militaris polysaccharide and sodium glutamate according to the said parts by mass, dissolve them in water and sterilize.

[0017] Further, the ratio of skim milk, Cordyceps militaris polysaccharide, sodium glutamate and water is 5-10 g: 4-6 g: 2-4 g: 100 mL.

[0018] Further, the sterilization conditions are: temperature 85±5°C, time 30±5 min.

[0019] The application of the above-mentioned composite protective agent in improving the spray drying survival rate and / or stability of Pediococcus acidilactici.

[0020] Further, the stability includes stability in simulated gastrointestinal fluid and storage stability.

[0021] Further, the application is as follows: preparing a bacterial suspension of Pediococcus acidilactici, performing heat shock treatment on the bacterial suspension, centrifuging to obtain bacterial sludge, mixing the bacterial sludge with an aqueous solution of the above-mentioned composite protective agent at a mass ratio of 1:3-7, preferably 1:5, and then performing spray drying. The parameters of spray drying are: inlet air temperature 90±2°C, outlet air temperature controlled at 60°C-70°C, peristaltic pump speed 12±3 r / min, atomization pressure 0.2±0.05 MPa, and fan power 50±5 Hz.

[0022] Furthermore, the heat shock treatment conditions are as follows: placing the bacterial suspension in a water bath at 45-60°C for heat shock for 5-20 min; preferably, heat shock at 55°C for 10 min.

[0023] Furthermore, the centrifugation conditions are centrifuging at 3000-6000 rpm for 8-20 min; preferably, centrifuging at 5000 rpm for 10 min.

[0024] Furthermore, the method for preparing the bacterial suspension is as follows: activating the strain to obtain single colonies; inoculating and culturing the single colonies to obtain a seed solution; transferring the seed solution to an MRS liquid medium for subculture to obtain a subcultured solution; centrifuging the subcultured solution to collect bacterial sludge, and resuspending it in sterilized 0.85% physiological saline to obtain the bacterial suspension.

[0025] The conditions for the subculture are culturing at 37-45°C for 10-14 h, preferably, culturing at 37°C for 12 h.

[0026] The centrifugation conditions are centrifuging at 3000-6000 rpm for 8-20 min; preferably, centrifuging at 5000 rpm for 15 min.

[0027] A Pediococcus acidilactici powder is prepared by the above spray drying. The Pediococcus acidilactici powder has stability in simulated gastrointestinal fluids and storage stability.

[0028] The present invention has the following advantages and effects compared with the prior art:

[0029] The present invention provides a composite protective agent for spray drying of Pediococcus acidilactici. Experimental results show that the spray drying survival rate of Pediococcus acidilactici without adding a protective agent is 17.60%, while adding the composite protective agent significantly improves the survival rate of Pediococcus acidilactici after spray drying, reaching 71.72%. Scanning electron microscopy shows that after adding the composite protective agent, the overall contour is clear, the surface is relatively flat, the structure is compact, and no visible cracks or fissures are shown on the particle surface, thus ensuring greater protection for probiotics. Inverted fluorescence microscopy shows that compared with without adding a protective agent, the red fluorescence significantly decreases after adding the composite protective agent, thus improving the cell membrane integrity of Pediococcus acidilactici. During the digestion process in simulated gastrointestinal fluids, adding the composite protective agent can, to a certain extent, resist the damage of low pH of gastric acid, and release the bacteria during the intestinal digestion stage, thus playing a probiotic role. When stored at -20°C, 4°C, and 25°C for one month, it is found that the survival rate decreases most significantly at 25°C. The survival rate of the bacterial powder without adding a protective agent decreases by nearly 50%, while the decrease of the composite protective agent is only less than 10%. Description of the Drawings

[0030] Figure 1It is the SEM image (10000×) of Pediococcus acidilactici after spray drying in physiological saline without adding protective agent;

[0031] Figure 2 It is the SEM image (10000×) of Pediococcus acidilactici after spray drying with the optimal composite protective agent added;

[0032] Figure 3 It is the inverted fluorescence microscope image (40×) of Pediococcus acidilactici after spray drying in physiological saline without adding protective agent;

[0033] Figure 4 It is the inverted fluorescence microscope image (40×) of Pediococcus acidilactici after spray drying without adding the optimal composite protective agent;

[0034] Figure 5 It is the graph of the determination results of the survival rate of Pediococcus acidilactici after spray drying with a single protective agent; among them, a. the effect of skim milk concentration on the survival rate of the bacterial powder; b. the effect of Cordyceps militaris polysaccharide concentration on the survival rate of the bacterial powder; c. the effect of sodium glutamate concentration on the survival rate of the bacterial powder, and different lowercase letters indicate significant differences between the data (P<0.05). Detailed implementation manners

[0035] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0036] The culture media and bacterial powders involved in the following embodiments:

[0037] (1) Preparation of culture media and main reagents

[0038] MRS culture medium: 10 g of beef extract, 10 g of peptone, 5 g of yeast extract powder, 20 g of glucose, 1 g of Tween-80, 5 g of anhydrous sodium acetate, 2 g of dipotassium hydrogen phosphate, 2 g of ammonium citrate hydrogen, 0.2 g of magnesium sulfate heptahydrate, 0.054 g of manganese sulfate monohydrate, made up to 1 L with sterile water, and the pH was adjusted to 6.5±0.1. Among them, 15-20 g of agar powder needs to be added to the solid culture medium, and it was sterilized at 121°C for 15-20 min.

[0039] Beef extract, peptone, yeast extract powder, glucose, anhydrous sodium acetate, dipotassium hydrogen phosphate, ammonium citrate hydrogen, magnesium sulfate heptahydrate, manganese sulfate monohydrate, Tween: analytical pure, Sinopharm Chemical Reagent Co., Ltd.

[0040] Preparation of simulated gastric fluid (SGF): It consists of 0.32% (w / w) pepsin and 0.2% (w / w) sodium chloride, and the pH was adjusted to 2.0 with 1M hydrochloric acid.

[0041] Preparation of simulated intestinal fluid (SIF): It is composed of 0.1% (w / w) pancreatin and 0.08% (w / w) bile salt, and is prepared in PBS buffer (0.2 M, pH 7.0).

[0042] (2) Pediococcus acidilactici

[0043] Pediococcus acidilactici strain HI5 has been publicly available in NCBI GenBank with the accession number CP184530.1.

[0044] (3) Preparation of Pediococcus acidilactici powder

[0045] The activated Pediococcus acidilactici was inoculated into MRS liquid medium at an inoculation amount of 5%, and cultured statically at 37 °C for 12 h until the stationary phase. The bacterial sludge was collected by centrifugation at 4 °C and 5000 rpm for 15 min, washed 1-2 times with 0.85% normal saline, and resuspended in sterilized 0.85% normal saline to obtain a bacterial suspension, which was subjected to heat shock treatment in a 55 °C water bath for 10 min. After the heat shock treatment, centrifugation was performed again to obtain the final bacterial sludge. An aqueous solution of the protective agent that had been sterilized (sterilized at 85 °C for 30 min) was added according to the mass ratio of bacterial sludge to protective agent of 1:5, and the mixture was thoroughly mixed to obtain a sample solution. Spray drying was carried out under the conditions of an inlet air temperature of 90 °C, an outlet air temperature of 65 ± 3 °C, a peristaltic pump speed of 12 r / min, an atomization pressure of 0.2 Mpa, and a fan power of 50 HZ.

[0046] (4) Determination of viable cell count and survival rate: The viable cell count was calculated using the national standard "GB4789.35-2016 National Food Safety Standard Microbiological Examination of Foods Detection of Lactic Acid Bacteria", and the formula for calculating the survival rate is as follows:

[0047]

[0048] In the formula, A1 is the viable cell count per gram of bacterial powder after spray drying; M is the gram number of bacterial powder after spray drying; A2 is the viable cell count per milliliter of sample solution before spray drying; V is the volume of sample solution before spray drying.

[0049] Example 1: Spray-dried composite protective agent for Pediococcus acidilactici

[0050] (1) An orthogonal experiment with four factors and three levels was carried out using skim milk, Cordyceps militaris polysaccharide, and sodium glutamate. Taking the survival rate of Pediococcus acidilactici as the index, the spray-dried composite protective agent for Pediococcus acidilactici was optimized. The influence factor levels of the orthogonal experiment are shown in Table 1. The range analysis and variance analysis results of the orthogonal experiment are shown in Tables 2 and 3.

[0051] Table 1 Influence factor level table of orthogonal experiment

[0052]

[0053] Table 2 Results of Orthogonal Experiment Design of Spray-Drying Protectants

[0054]

[0055]

[0056] Note: K1, K2, and K3 represent the spray-drying survival rates of each factor at each level; k1, k2, and k3 represent the average spray-drying survival rates of each factor at each level; R is the range, indicating the magnitude of the influence of this factor on the survival rate of Pediococcus acidilactici powder

[0057] As can be seen from Table 2, different combinations of protectant formulations affect the survival rate of Pediococcus acidilactici powder. The order of the influence of single protectants on the survival rate of Pediococcus acidilactici powder from large to small is: A > B > C, that is, skim milk > Cordyceps militaris polysaccharide > sodium glutamate. The optimal composite protectant formulation is A2B2C2, that is, 7.5% skim milk, 5% Cordyceps militaris polysaccharide, and 3% sodium glutamate

[0058] Through the measurement results of the survival rate, the spray-drying survival rate of Pediococcus acidilactici without adding protectant is 17.60%, while adding the optimal composite protectant significantly improves the survival rate of Pediococcus acidilactici after spray drying, reaching 71.72%

[0059] Table 3 Analysis of Variance of Spray-Drying Protectants

[0060]

[0061] Note: * indicates a significant difference at the P < 0.05 level, and ** indicates a highly significant difference at the P < 0.01 level

[0062] As can be seen from Table 3, the P value of skim milk is less than 0.01, and the P values of Cordyceps militaris polysaccharide and sodium glutamate are less than 0.05, indicating that skim milk has a highly significant influence on the survival rate of Pediococcus acidilactici spray-dried powder, while Cordyceps militaris polysaccharide and sodium glutamate have a significant influence on the survival rate of Pediococcus acidilactici spray-dried powder

[0063] (2) Scanning Electron Microscope (SEM)

[0064] Stick the carbon adhesive label onto the aluminum sheet. Dip a toothpick into a small amount of Pediococcus acidilactici spray-dried powder and sprinkle it on the sample stage with conductive adhesive material. Spread it evenly with a spatula, and carefully remove the excess powder with an ear syringe. Then, gold-plate the plate in a small ion diffractometer for 2.5 min. Finally, place the prepared sample under the scanning electron microscope at a voltage of 10 KV, and select an appropriate magnification by adjusting the distance between the sample and the probe to obtain the scanning electron microscope image of the sample

[0065] Using the spray-dried powder of Pediococcus acidilactici without added protectant as a control, the microscopic morphology of the powder under the optimal composite protectant was observed by scanning electron microscopy. The results are shown in Figure 1 and Figure 2 . As can be seen from the figure, the cells of the powder without added protectant are piled up, which may be caused by cell deformation or rupture and leakage of intracellular substances. Compared with the control group, the powder added with the optimal composite protectant has a clear overall outline, a relatively flat surface, a compact structure, and no visible cracks or fissures on the particle surface, thus ensuring greater protection for probiotics. Combining with the determination results of survival rate, it can be proved that the composite protectant can more effectively encapsulate probiotics and protect cells from damage caused by heat stress and oxidative stress.

[0066] (5) Cell membrane integrity

[0067] Dissolve the spray-dried powder of Pediococcus acidilactici in sterile PBS, adjust the bacterial concentration to 10 7 cfu / mL. Take 1 mL of the bacterial solution and mix it with 30 μL of propidium iodide (PI) solution, and incubate it in the dark at 37 °C for 30 min. Centrifuge (5000 rpm, 10 min) to collect the bacteria, wash them three times with sterile PBS to remove the unbound dye, and resuspend them in 1 mL of sterile PBS for standby. Dip a drop and observe it under an inverted fluorescence microscope.

[0068] Using the spray-dried powder of Pediococcus acidilactici without added protectant as a control, the damage of the powder under the optimal composite protectant was observed by inverted fluorescence microscope. The results are shown in Figure 3 and Figure 4 . As can be seen from the figure, the red fluorescence indicates that the cell membrane of Pediococcus acidilactici is damaged or even dead after spray drying. Compared with the control group, the red fluorescence significantly decreases after adding the optimal composite protectant, which indicates that the optimal composite protectant can improve the integrity of the cell membrane of Pediococcus acidilactici. By combining with the survival rate results, the addition of the optimal composite protectant increased the drying survival rate of Pediococcus acidilactici by 54.12%.

[0069] (6) In vitro simulated gastrointestinal fluid digestion

[0070] Preheat the simulated gastrointestinal fluid in a 37°C water bath and sterilize it through a 0.22 μm filter membrane for standby. First, add 0.1 g of spray-dried bacterial powder to 9.9 mL of simulated gastric juice, shake it evenly with a vortex oscillator, and place it in a 37°C constant temperature shaking incubator for digestion. Take 1 mL of the mixed digestive fluid at 1 h and 2 h respectively, fully lyse it, and then dilute it for plate counting. Immediately after 2 h of digestion in simulated gastric juice, adjust its pH to 7.0 with 1 M sodium hydroxide, then add 10 mL of simulated intestinal fluid, shake it evenly with a vortex oscillator, and place it in a 37°C constant temperature shaking incubator for digestion. Take 1 mL of the mixed solution at 1, 2, 3, and 4 h respectively, dilute it, and then perform plate counting.

[0071] Table 4 Survival rate of Pediococcus acidilactici powder during in vitro simulated digestion

[0072]

[0073] As can be seen from Table 4, when digested in simulated gastric juice for 2 h, the survival rate of the bacterial powder without added protectant is 38.15%, while the survival rate of the bacterial powder with the optimal composite protectant added is 81.16%. During the digestion in simulated intestinal fluid, the survival rate of the bacterial powder with the optimal composite protectant added slightly increases. Among them, when digested in the intestine for 4 h, the survival rate of the bacterial powder without added protectant is 22.88%, and the survival rate of the bacterial powder with the optimal composite protectant added is 101.51%. This shows that adding the optimal composite protectant can, to a certain extent, resist the damage of low pH of gastric acid and release the bacteria during the intestinal digestion stage, thereby exerting a probiotic effect.

[0074] (7) Storage stability

[0075] After separately packaging the bacterial powder without added protectant and the bacterial powder with the optimal composite protectant added in sterile aluminum foil bags, place them in a frozen environment (-20°C), a refrigerated environment (4°C), and a room temperature environment (25°C) for storage for one month. Using the 0th day as a control, calculate the survival rate of the bacterial powder at different storage temperatures.

[0076] Table 5 Survival rate of Pediococcus acidilactici powder during storage

[0077]

[0078] As can be seen from Table 5, during the 30-day storage period, the survival rates of the bacterial powder without adding protective agents and with the optimal composite protective agent both decreased to varying degrees. Specifically, the survival rate was the highest at -20°C, followed by 4°C, and the survival rate decreased most significantly at 25°C. The survival rate of the bacterial powder without adding protective agents decreased by nearly 50%, while that of the optimal composite protective agent was less than 10%. The higher the storage temperature, the more a series of life activities will occur in the bacterial cells, resulting in a decrease in vitality. Therefore, room temperature is not suitable for the long-term storage of bacterial powder, and the best effect can be obtained by storing the bacterial powder at -20°C. The optimal composite protective agent not only improves the survival rate of the spray-dried bacteria but also has the best stability during storage.

[0079] Comparative Example 1: Single protective agent for spray drying of Pediococcus acidilactici

[0080] Prepare 2.5%, 5%, 7.5%, 10%, 12.5% skim milk aqueous solutions, 3%, 4%, 5%, 6%, 7% Cordyceps militaris polysaccharide aqueous solutions, and 1%, 2%, 3%, 4%, 5% sodium glutamate aqueous solutions respectively as single protective agents for spray drying of Pediococcus acidilactici.

[0081] The activated Pediococcus acidilactici was inoculated into MRS liquid medium at an inoculation amount of 5%, and statically cultured at 37°C for 12 h until the stationary phase. The bacterial sludge was collected by centrifugation at 4°C and 5000 rpm for 15 min, washed 1-2 times with 0.85% physiological saline, and resuspended in sterilized 0.85% physiological saline to obtain a bacterial suspension, which was subjected to a heat shock treatment in a hot water bath at 55°C for 10 min. After the heat shock treatment, the bacterial sludge was centrifuged again to obtain the final bacterial sludge. The sterilized (sterilized at 85°C for 30 min) single protective agent aqueous solution was added according to the mass ratio of bacterial sludge to protective agent of 1:5, and thoroughly mixed to obtain a sample solution, which was spray-dried under the conditions of an inlet air temperature of 90°C, an outlet air temperature of 65±3°C, a peristaltic pump speed of 12 r / min, an atomization pressure of 0.2 Mpa, and a fan power of 50 HZ. The viable count was calculated using the national standard "GB4789.35-2016 National Food Safety Standard Microbiological Examination of Foods Detection of Lactic Acid Bacteria".

[0082] The results are as Figure 5 shown, and the protective effect of the single protective agent for spray drying of Pediococcus acidilactici is far inferior to that of the composite protective agent.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A composite protective agent for spray drying of Pediococcus acidilactici, characterized in that: It is composed of the following components by mass parts: 5-10 parts of skim milk, 4-6 parts of Cordyceps militaris polysaccharide, and 2-4 parts of sodium glutamate.

2. The composite protective agent for spray drying of Pediococcus acidilactici according to claim 1, characterized in that: The composite protective agent is composed of the following components by mass parts: 7-8 parts of skim milk, 4.5-5.5 parts of Cordyceps militaris polysaccharide, and 2.5-3.5 parts of sodium glutamate.

3. The composite protective agent for spray drying of Pediococcus acidilactici according to claim 1, characterized in that: The composite protective agent is composed of the following components by mass parts: 7.5 parts of skim milk, 5 parts of Cordyceps militaris polysaccharide, and 3 parts of sodium glutamate.

4. The preparation method of the composite protective agent according to any one of claims 1-3, characterized in that: Weigh skim milk, Cordyceps militaris polysaccharide and sodium glutamate according to the said mass parts, dissolve them in water and sterilize.

5. The preparation method of the composite protective agent according to claim 4, characterized in that: The ratio of the skim milk, Cordyceps militaris polysaccharide, sodium glutamate and water is 5-10 g: 4-6 g: 2-4 g: 100 mL; The sterilization conditions are: temperature 85±5°C, time 30±5 min.

6. The application of the composite protective agent according to any one of claims 1-3 in improving the spray drying survival rate and / or stability of Pediococcus acidilactici.

7. The application according to claim 6, characterized in that: The said application is: preparing a bacterial suspension of Pediococcus acidilactici, performing heat shock treatment on the bacterial suspension, centrifuging to obtain bacterial sludge, mixing the bacterial sludge with the aqueous solution of the composite protective agent at a mass ratio of 1:3-7, preferably 1:5, and then performing spray drying. The parameters of spray drying are: inlet air temperature 90±2°C, outlet air temperature controlled at 60°C-70°C, peristaltic pump speed 12±3 r / min, atomization pressure 0.2±0.05 MPa, and fan power 50±5 Hz.

8. The application according to claim 6, characterized in that: The heat shock treatment conditions are: placing the bacterial suspension in a water bath at 45-60°C for heat shock for 5-20 min; The preparation method of the bacterial suspension is: activating the strain to obtain single colonies; Inoculating and culturing the single colonies to obtain a seed solution; Transferring the seed solution to an MRS liquid medium for subculture to obtain a subcultured solution; centrifuging the subcultured solution to collect bacterial sludge, and resuspending it in sterilized 0.85% physiological saline to obtain a bacterial suspension; the subculture conditions are culturing at 37-45°C for 10-14 h; The centrifugation conditions are centrifuging at 3000-6000 rpm for 8-20 min.

9. The application according to claim 8, characterized in that: The heat shock treatment conditions are: placing the bacterial suspension in a water bath at 55°C for heat shock for 10 min; The preparation method of the bacterial suspension is: activating the strain to obtain single colonies; Inoculating and culturing the single colonies to obtain a seed solution; Transfer the seed liquid to MRS liquid medium for subculture to obtain subcultured liquid; centrifuge the subcultured liquid to collect the bacterial sludge, and resuspend it in sterilized 0.85% physiological saline to obtain a bacterial suspension; the conditions for the subculture are culturing at 37 °C for 12 h; The centrifugation conditions are centrifuging at 5000 rpm for 15 min.

10. A pediococcus acidilactici powder, characterized in that: It is prepared by spray drying according to any one of claims 6-9.

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