Direct vat set starter for lactic acid bacteria as well as preparation method and application of direct vat set starter

Abstract

The invention provides a direct vat set starter for lactic acid bacteria as well as a preparation method and application of the direct vat set starter. The method comprises the following steps: inoculating an activated lactic acid bacteria strain into a nucleotide culture medium, and carrying out enlarged culture to obtain a high-density lactic acid bacteria agent; the method comprises the following steps: centrifuging a high-density lactic acid bacterium agent, mixing thalli with a freeze-drying protective agent, and freeze-drying to obtain a lactic acid bacterium direct vat set starter; wherein the nucleotide culture medium comprises the following components: 0.3 g / L to 0.8 g / L of nucleotide, 2 g / L to 10 g / L of beef extract powder, 25 g / L to 35 g / L of soy peptone, 10 g / L to 17 g / L of cane sugar, 1 g / L to 6 g / L of anhydrous sodium acetate, 2 g / L to 8 g / L of triammonium citrate, 1.0 g / L to 1.6 g / L of monopotassium phosphate, 0.1 g / L to 0.8 g / L of magnesium sulfate monohydrate, 0.023 g / L to 0.029 g / L of manganese sulfate and 2 g / L to 6 g / L of Tween 80; the nucleotide comprises at least one of guanine, adenine, xanthine or uracil. According to the method provided by the invention, the activity and stress resistance of the lactic acid bacteria strain can be improved, and the adaptive capacity of the lactic acid bacteria strain to a severe environment is improved, so that the lactic acid bacteria direct vat set starter with relatively high density and freeze-drying survival rate is obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a lactic acid bacteria direct-injection starter and a preparation method and application thereof. Background Art

[0002] Optimizing the production of probiotic starter cultures, especially for microorganisms such as lactic acid bacteria (LAB) that have multiple health benefits, is a current research hotspot in the field of food science and biotechnology. In order to maximize the probiotic benefits of LAB, researchers continue to explore and improve preparation techniques, of which high-density fermentation and freeze-drying are the two core technologies. High-density fermentation technology ensures a sufficient number of live bacteria by precisely controlling culture conditions such as temperature, pH value, and nutrient supply, which is the basis for the effectiveness of probiotic products. Freeze-drying technology effectively extends the shelf life of probiotics through a process of rapid freezing and reduced pressure drying, facilitates transportation and storage, and reduces the risk of bacteriophage contamination, which is a key step in maintaining the activity of probiotics.

[0003] However, when faced with extreme conditions such as low temperature and dryness, the physiological structure and function of probiotics are easily damaged, such as reduced activity of key enzymes, damaged cell membrane integrity, and even bacterial death, which directly affects the quality and effect of probiotic products. Therefore, improving the survival rate of probiotics during the freeze-drying process has become an urgent problem to be solved. In response to this challenge, researchers have proposed a variety of strategies, including adjusting the culture conditions of the strains, screening and optimizing freeze-drying protective agents, and fine-tuning the freeze-drying process parameters. Among them, changing the composition of the culture medium, especially adding synergistic factors that are crucial to the growth and metabolism of probiotics, is considered to be a very promising method.

[0004] In view of the various limitations of the prior art regarding lactic acid bacteria proliferation density method technology and freeze-drying technology, in order to effectively improve the activity of lactic acid bacteria direct-injection starter, improve the survival rate of the bacteria during freeze-drying and preservation, and optimize its application quality, the present invention provides a method for improving the activity of lactic acid bacteria direct-injection starter and the freeze-drying survival rate. Summary of the invention

[0005] In order to solve all or part of the above technical problems, the present invention provides the following technical solutions:

[0006] One of the purposes of the present invention is to provide a method for preparing a lactic acid bacteria direct-injection starter, comprising:

[0007] The activated lactic acid bacteria strains are inoculated into a nucleotide culture medium for expansion culture to obtain a high-density lactic acid bacteria agent;

[0008] After centrifuging the high-density lactic acid bacteria agent, the bacterial cells are mixed with a composite freeze-drying protective agent and freeze-dried to obtain a lactic acid bacteria direct-injection fermentation agent;

[0009] The components of the nucleotide culture medium include: 0.3-0.8 g / L nucleotides, 2-10 g / L beef extract powder, 25-35 g / L soy peptone, 10-17 g / L sucrose, 1-6 g / L anhydrous sodium acetate, 2-8 g / L triammonium citrate, 1.0-1.6 g / L potassium dihydrogen phosphate, 0.1-0.8 g / L magnesium sulfate monohydrate, 0.023-0.029 g / L manganese sulfate and 2-6 g / L Tween 80;

[0010] The nucleotides include one or a combination of multiple of guanine, adenine, xanthine or uracil.

[0011] The method provided by the present invention can improve the activity and stress resistance of the strain, improve the ability of lactic acid bacteria to adapt to harsh environments, and obtain a lactic acid bacteria direct-injection starter with a high density and freeze-dried survival rate. The lactic acid bacteria density reaches 10 10 CFU / mL, the freeze-dried survival rate increased to over 85.6%.

[0012] In some preferred embodiments, the nucleotide medium includes uracil.

[0013] In some preferred embodiments, the nucleotide culture medium includes uracil and further includes at least one of guanine, adenine, and xanthine.

[0014] In some preferred embodiments, the nucleotide culture medium includes uracil and guanine, and the concentration ratio of the two is (1-3): (1-3). Alternatively, the nucleotide culture medium includes uracil and xanthine, and the concentration ratio of the two is (1-4): (1-4). Alternatively, the nucleotide culture medium includes uracil: xanthine, and the concentration ratio is (1-3): (1-3). Alternatively, the nucleotide culture medium includes uracil: guanine: adenine: xanthine, and the concentration ratio is (1-3): (1-3): (1-3): (1-3).

[0015] In some embodiments, the method specifically includes: inoculating the activated lactic acid bacteria into the nucleotide culture medium at a volume concentration of 1 to 5%, culturing at 25 to 37° C. for 12 to 18 hours, centrifuging at 5000 to 9000 rpm for 10 to 15 minutes, washing the high-density lactobacillus agent obtained by centrifugation with sterile saline, and then freeze-drying.

[0016] In some embodiments, the method specifically includes: inoculating lactic acid bacteria into an activation medium at a volume concentration of 1 to 5%, culturing at 25 to 37° C. for 12 to 18 hours to obtain activated lactic acid bacteria strains; and then inoculating the activated lactic acid bacteria strains into the nucleotide medium. The activation medium can be an MRS medium.

[0017] In some embodiments, the ingredients of the composite lyophilization protectant include: 5-20wt% sugar, 10-30wt% skim milk powder, 8-25wt% proline, and the rest includes distilled water.

[0018] In some embodiments, the sugar includes one or a combination of glucose, trehalose, sorbitol or sucrose.

[0019] In some embodiments, the preparation method of the composite lyoprotectant includes: mixing and grinding the sugar, proline and distilled water for 5-15 minutes, stirring at 40-100°C and 50-300rpm for 8-12 hours to form a uniform, viscous, transparent liquid eutectic solvent, and then mixing with a skimmed milk powder solution to obtain the composite lyoprotectant.

[0020] In some embodiments, the freeze-drying specifically includes: centrifuging the high-density lactic acid bacteria agent to obtain the bacteria, and adding a freeze-drying protective agent with an equal volume to that before centrifugation to form a mixture, pre-freezing the mixture at -50°C for 2 to 10 hours, and then gradually heating the mixture to -40°C for 1 to 5 hours, -30°C for 1 to 5 hours, -20°C for 1 to 4 hours, -10°C for 1 to 3 hours, 0°C for 0 to 3 hours, and 10°C for 5 to 20 hours, thereby obtaining the lactic acid bacteria direct-injection fermentation agent.

[0021] In some embodiments, the lactic acid bacteria are Lactobacillus plantarum YR07, Lactobacillus sakei L48 or Lactobacillus curvatum D2.

[0022] The second object of the present invention is to provide a lactic acid bacteria direct-injection starter, which is prepared by any of the methods described above.

[0023] The third object of the present invention is to provide the application of the lactic acid bacteria direct-injection starter in fermenting sausages.

[0024] In some embodiments, the application includes: uniformly mixing the lactic acid bacteria direct-injection starter with minced pork at an addition concentration of 1 to 5 wt%, fermenting for 12 to 48 hours in an environment of 10 to 40° C. and 30 to 90% humidity, and then air-drying to obtain sausages.

[0025] In some embodiments, the minced pork further contains auxiliary materials, which include salt, glucose, nitrite, garlic powder, fennel powder, black pepper powder and sausage flavor.

[0026] The direct-injection starter prepared by the special composite nucleotide culture medium and the composite freeze-drying protective agent of the present invention shows good fermentation characteristics in the production of fermented sausages. The growth state in the fermented sausages is extremely good, the number of live bacteria far exceeds that of other groups, and the activity is strong, the acid production is rapid, and the flavor substances can be produced faster and in larger quantities. It shows significant advantages in the production of fermented sausages. The bacteria in the starter can continue to thrive and metabolize efficiently in a high-salt environment, giving the sausages a unique aroma and flavor.

[0027] Compared with traditional fermentation agents, the direct-throw fermentation agent of the present invention can significantly shorten the fermentation time of meat products, and the fermentation process can be completed in as fast as 9 days, and the fermentation efficiency is increased by 25%. Therefore, the sausages fermented with the direct-throw fermentation agent of the present invention not only have a unique flavor and high quality, but also score the highest in sensory evaluation, which fully verifies the excellent performance and practical value of the present invention.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects: the method provided by the present invention can improve the stress resistance of lactic acid bacteria, reduce damage to its cell wall and cell membrane, enhance the activity of lactic acid bacteria LDH enzyme and ATP enzyme, improve the sugar metabolism ability of bacteria, and reduce the damage to the secondary structure of protein after freeze-drying. It can also increase the relative content of long-chain unsaturated fatty acids in the cell membrane of lactic acid bacteria, increase the compactness of the cell membrane of the strain, and make the cell membrane structure of the strain more stable, so that it can better cope with environmental stress and stress damage during the freeze-drying stage. It is thus possible to obtain a lactic acid bacteria direct-injection fermentation agent with a higher density and freeze-drying survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 Pareto Chart for different cultivation optimization factors;

[0031] Figure 2 , Figure 3 The response surface diagram and contour diagram of the effects of beef powder + soy peptone and ammonium citrate tribasic on bacterial growth are shown respectively;

[0032] Figure 4 , Figure 5 The response surface diagram and contour diagram of the effects of beef powder + soy peptone and sodium acetate on bacterial growth are shown respectively;

[0033] Figure 6 , Figure 7 These are the response surface diagram and contour diagram of the effects of sodium acetate and triammonium citrate on bacterial growth;

[0034] Figure 8 The effect of different synergistic factors on the viable count of Lactobacillus plantarum YR07;

[0035] Fig. 9 The effects of different culture media on the viable count and freeze-dried survival rate of Lactobacillus plantarum YR07;

[0036] Fig.10 , Fig.11 The effects of MRS medium, optimized medium and uracil medium on the lysozyme sensitivity and sodium chloride sensitivity of Lactobacillus plantarum YR07 were shown respectively;

[0037] Fig.12 The effects of MRS medium, optimized medium and uracil medium on the ATPase activity (Na + K + -ATPase);

[0038] Fig.13 The effects of MRS medium, optimized medium and uracil medium on the ATPase activity (Ca 2+ Mg 2- ATPase);

[0039] Fig.14 The effects of MRS medium, optimized medium and uracil medium on the protein structure of Lactobacillus plantarum YR07;

[0040] Fig.15 The effects of MRS medium, optimized medium and uracil medium on the freeze-dried cell morphology of Lactobacillus plantarum YR07;

[0041] Fig.16 The effects of MRS medium and compound nucleotide medium on the viable counts of different lactic acid bacteria;

[0042] Fig.17 This is the effect of MRS medium and composite nucleotide medium on the freeze-drying survival rate of Lactobacillus plantarum YR07, Lactobacillus sakei L48, and Lactobacillus curvatum D2. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is described in detail below in conjunction with specific embodiments so that those skilled in the art can better understand and implement the technical solution of the present invention. The specific functional details disclosed herein should not be interpreted as limiting, but only as the basis for the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in different ways in any appropriate detailed embodiment.

[0044] In addition, unless otherwise specified, the various raw materials used in the following examples can be purchased from the market, and the various production and testing equipment used are also equipment known in the art. The testing methods are also conventional methods in the art.

[0045] Biological preservation description: Lactobacillus plantarum YR07 (Lactiplantibacillusplantarum YR07), Latilactobacillus sakei L.48 (Latilactobacillus sakei L.48), and Lactobacillus curvatus D2 (Lactobacillus curvatus D2) used in the embodiments of the present invention are deposited in the China Center for Type Culture Collection, the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation institution is abbreviated as: CCTCC, the preservation date of Lactobacillus plantarum YR07 is August 18, 2022, and the biological preservation number is CCTCC NO: M20221303; the preservation date of Lactobacillus sakei L.48 is August 18, 2022, and the biological preservation number is CCTCC NO: M20221306; the preservation date of Lactobacillus curvatus D2 is February 21, 2023, and the biological preservation number is CCTCC NO: M2023162.

[0046] The inoculation concentration percentages "%" involved in the specific embodiments of the present invention are all volume concentrations.

[0047] The guanine, adenine, xanthine and uracil used in the specific embodiment of the present invention are all purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of ≥98%.

[0048] The sensory evaluation criteria for sausages are:

[0049] Sensory evaluation form

[0050]

[0051] 1. Culture medium optimization

[0052] The invention optimizes the MRS culture medium to obtain an optimized culture medium more suitable for lactobacillus culture, specifically, a single factor combined with response surface optimization is performed on the basis of the MRS culture medium to obtain a formula of the best culture medium.

[0053] Adjust the OD of the activated bacterial solution 600 =1.0, inoculated into MRS culture medium with different initial pH at different concentrations, set the culture temperature, and cultured at 150 rpm / min for 18 h to study the effects of different initial pH (4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8); different inoculum sizes (1%, 2%, 3%, 4%, 5%); and different fermentation temperatures (20°C, 25°C, 30°C, 35°C, 37°C, 40°C) on the viable count of strain YR07.

[0054] According to the results of the single factor test, the inoculation size, temperature and initial pH were selected as influencing factors, and the concentration of viable bacteria was used as the evaluation index to design an orthogonal experiment, as shown in Table 1.

[0055] Table 1. Fermentation conditions orthogonal test factor level table

[0056]

[0057] On the basis of optimized culture conditions, single factor optimization of ingredients was carried out, including: optimization of carbon source type and content, optimization of nitrogen source type and content, optimization of potassium dihydrogen phosphate concentration, optimization of ammonium citrate concentration, optimization of sodium acetate concentration, optimization of manganese sulfate concentration, optimization of magnesium sulfate concentration, and optimization of Tween 80 concentration. On the basis of component optimization, PB test, steep slope test, and response surface experiment were carried out. The experimental design table is as follows:

[0058] Table 2 Levels of factors used in the Plackett-Burman design

[0059]

[0060] Table 3 Optimal steep slope climbing experimental design

[0061]

[0062] According to the results of the optimal steep slope test and the experimental design principle of RSM response surface, three factors, beef powder + soy peptone, sodium acetate and triammonium citrate, were selected as the research objects, and the number of viable bacteria was the response value. The experiment was designed, and the experimental data were fitted with quadratic regression. The main effect and interaction effect of each factor were analyzed to determine the optimal value of each factor. Their coding values ​​and corresponding actual values ​​are shown in Table 4. For other parameters with insignificant effects, they were selected according to the positive and negative effects of the PB test. Design-Expert software was used for design and data analysis. Each group of experiments was repeated 3 times, and the results were averaged.

[0063] Table 4 Factor level coding table for response surface experimental design

[0064]

[0065] According to the results of the single factor test, an orthogonal test was designed for inoculation concentration, temperature, and initial pH. The number of viable bacteria was used as the evaluation index to determine the optimal culture conditions. From the results in Table 5, it can be seen that the primary and secondary factors affecting the number of viable bacteria are: temperature > inoculation amount > initial pH value. The optimal culture conditions determined by the test results are: A2B3C2, that is, inoculation amount 2%, initial pH 7.0, temperature 30℃, at which time the number of viable bacteria is 56×10 8 CFU / mL.

[0066] Table 5 Orthogonal test results for optimization of culture conditions of Lactobacillus plantarum YR07

[0067]

[0068]

[0069] Design Expert 8.0 software was used to perform the Plackett-Burman experimental design. In the above single factor analysis, 8 single factors were selected for analysis, including sucrose, beef soybean, sodium acetate, ammonium citrate, dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, and Tween 80. The experimental results showed that when the culture medium contained 10g / L sucrose, 25g / L beef powder + soybean peptone (1:4), 2g / L sodium acetate, 2g / L ammonium citrate, 1g / L dipotassium hydrogen phosphate, 0.025g / L manganese sulfate, 0.3g / L magnesium sulfate, and 3g / L Tween 80, the maximum number of viable bacteria was obtained in each single factor experiment. Therefore, the single factors with the above concentrations were selected to perform the Plackett-Burman experiment, and the experimental results are shown in Table 6.

[0070] Table 6 Plackett-Burmen test results

[0071]

[0072]

[0073] Plackett-Burman test design and results. Sucrose (A), beef powder + soy peptone (B), sodium acetate (C), triammonium citrate (D), dipotassium hydrogen phosphate (E), manganese sulfate (F), magnesium sulfate (G), Tween 80 (H) were added as factors of the Plackett-Burman test and three virtual factors (J, K, L). The factors that had a significant effect on the number of viable Lactobacillus plantarum YR07 were screened out according to the results.

[0074] Table 7 Significance analysis of Plackett-Burmen test results

[0075]

[0076] As can be seen from Table 7, the P value of the variance analysis model is less than 0.05, that is, the obtained regression equation is significant, indicating that the entire regression area of ​​the model fits well. 2 =0.9881, indicating a good correlation, and the corrected determination coefficient R 2 Adj =0.9563, indicating that 95.6% of the variability of the experimental data can be explained by this regression model; in general, the lower the coefficient of variation (CV), the higher the reliability and accuracy of the experiment. The CV value is equal to 2.16%, indicating that the reliability and accuracy of the PB experiment are good. Precision is the ratio of effective signal to noise. If it is greater than 4.0, it is considered reasonable. The precision of this experiment reached 20.143. In addition, it can be seen from Table 7 that the three factors that have the greatest impact on bacterial growth at the 95% confidence interval are beef powder + soy peptone (30g / L), sodium acetate (2g / L), and triammonium citrate (4g / L) (P<0.05), and the next step of optimization experiment can be carried out. According to the prediction of Design Expert software, the optimization results of non-significant factors are: sucrose: 15g / L; dipotassium hydrogen phosphate: 1.40g / L; manganese sulfate: 0.025g / L; magnesium sulfate: 0.30g / L; Tween 80: 3.00g / L

[0077] From the PB test, we can see that beef powder + soy peptone, sodium acetate, and ammonium citrate have significant effects on bacterial growth, among which beef powder + soy peptone and ammonium citrate have significant positive effects, and the concentration of the later experiments should be increased, while sodium acetate has a significant negative effect, and the concentration of the later experiments should be reduced. According to the ratio of the effect size of these three factors, the direction of change and the step length were set for the experimental design and the results are shown in Table 8.

[0078] Table 8 Steepest climbing test results

[0079]

[0080] As can be seen from Table 8, the highest number of viable bacteria in test No. 3 is 73×10 8 CFU / mL, which is the inflection point of the steepest climbing test, so the third group of data was selected as the center point of the subsequent central composite test design, that is, beef powder + soy peptone 30g / L, sodium acetate 2g / L, and ammonium citrate 4g / L. The three factors of beef powder + soy peptone, sodium acetate, and ammonium citrate were selected, and each factor took low (-1), medium (0), and high (+1) to establish a three-factor three-level mathematical model. The experimental results are shown in Tables 9 and 10.

[0081] Table 9 Response surface test results

[0082]

[0083]

[0084] Table 10 Analysis of variance of regression model

[0085]

[0086] As shown in Table 10, the F value of the model is 15.77517, the P value of the fitted model is 0.0007 < 0.05, the F value of the lack-of-fit term is 1.381215, and the P value is 0.3696, which is greater than 0.05, indicating that the lack-of-fit term is not significant and the model fits the actual situation well. Model R 2 =0.9530, indicating that the equation fits the actual situation well. The experimental results were fitted with a quadratic multivariate fitting software Design-Expert 8.0.6, and the equation obtained was Y=69.80+4.67×A+0.83×B+5.58×C-6.92×A×B+6.08×A×C+1.92×B×C-4.61×A 2 -4.78×B 2 +2.56×C 2 . From the table we can see: A, C, AB, AC, B 2 , A 2 The effect was extremely significant (P<0.01), the BC effect was significant (P<0.05); the model effect was extremely significant (P<0.01); the lack of fit term was not significant (P>0.05).

[0087] The three factors interact with each other. Figure 2 , 3 , as shown in Figure 4. The first-order partial derivative of the second-order equation is obtained. When the response value Y (viable bacteria count) is at its maximum, the factor levels are beef powder + soy peptone added at 40 g / L, triammonium citrate added at 4 g / L, and sodium acetate added at 3 g / L. At this time, the predicted viable bacteria count is 8.4×10 9 CFU / mL. Under this condition, the validation test was carried out and the number of viable bacteria was 8.3×10 9 CFU / mL, and the fitting rate with the model prediction value was 98.8%, indicating that the model can well predict the effect of culture medium composition on the number of viable bacteria.

[0088] Figure 1 Pareto Chart for different cultivation optimization factors. Figure 2 , Figure 3 These are the response surface diagram and contour diagram of the effects of beef powder + soy peptone and triammonium citrate on bacterial growth. Figure 4 , Figure 5 They are the response surface diagram and contour diagram of the effects of beef meal + soy peptone and sodium acetate on bacterial growth. Figure 6 , Figure 7They are the response surface diagram and contour diagram of the effects of sodium acetate and triammonium citrate on bacterial growth.

[0089] Therefore, the final optimized formula of the basic culture medium was as follows: beef extract powder: 8 g / L, soy peptone: 32 g / L, sodium acetate: 3 g / L, triammonium citrate: 4 g / L, sucrose: 15 g / L, dipotassium hydrogen phosphate: 1.40 g / L; manganese sulfate: 0.025 g / L; magnesium sulfate: 0.30 g / L; Tween 80: 3.00 g / L. Compared with MRS, the viable count of Lactobacillus plantarum YR07 in this culture medium increased from 3.0 × 10 9 CFU / mL increased to 8.3×10 9 CFU / mL increased by 2.76 times.

[0090] 2. Effects of different types of nucleotides on the viable count of Lactobacillus plantarum YR07

[0091] 500 mg / L of guanine, adenine, xanthine and uracil were added to the optimized culture medium obtained by the above optimization, respectively, to obtain four culture mediums containing guanine, adenine, xanthine and uracil, respectively, which were recorded as guanine culture medium, adenine culture medium, xanthine culture medium and uracil culture medium, and the optimized culture medium without nucleotides was used as a control. The above five culture mediums were respectively used to culture plant lactobacillus YR07, and the prepared freeze-drying protective agent was used to prepare the lactic acid bacteria direct-throwing starter, which specifically comprises the following steps:

[0092] The Lactobacillus plantarum YR07 strain was inoculated into the MRS medium at a concentration of 2% for activation culture, and static culture was carried out at 37° C. for 18 hours to obtain an activated lactic acid bacteria strain.

[0093] The activated bacteria were inoculated into guanine medium, adenine medium, xanthine medium, uracil medium and optimized medium without nucleotides under sterile conditions at 2%, and then incubated in an incubator shaker at 30°C for 18 hours at a speed of 150 rpm / min until the growth reached the stable phase; the bacterial cells were harvested by centrifugation at 8000 rpm at 4°C for 10 minutes, and then washed 3 times with sterile saline.

[0094] Preparation of composite freeze-drying protective agent: glucose and proline were weighed, and distilled water was added, and the mixture was fully ground in a mortar for 10 minutes, the mixture was transferred to a beaker and sealed with a double layer of aluminum foil, and magnetically stirred for 10 hours at 90°C and 100rpm until the solid mixed system formed a uniform, viscous, transparent liquid to form a eutectic solvent, distilled water was added to adjust the concentration, and then mixed with skim milk powder solution to obtain a composite freeze-drying protective agent. The composition of the composite freeze-drying protective agent finally prepared was: 15wt% sorbitol, 20wt% skim milk powder, 13wt% proline, and the balance distilled water.

[0095] An equal volume of composite lyophilization protective agent was added to the above bacterial cells, mixed thoroughly, and distributed into vials, and freeze-dried using a SCIENTZ-50F / a freeze dryer. The freeze-drying process was as follows: the sample was initially pre-frozen at -80°C for 4 hours, then gradually heated, and then freeze-dried at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, 0°C for 2 hours, and finally 10°C for 16 hours. After the freeze-drying process was completed, the vials were sealed with rubber stoppers before being taken out. The lactic acid bacteria direct-injection starter was then stored at 4°C for further analysis.

[0096] Figure 8 The results show the effects of different nucleotides on the viable count of Lactobacillus plantarum YR07, indicating that different types of nucleotides have different growth-promoting effects on Lactobacillus plantarum YR07, among which uracil has the best promoting effect. 500 mg / L uracil culture medium can make the viable count reach 133×10 8 CFU / mL increased by 4.03 times compared with MRS optimized culture medium.

[0097] 3. The number of viable bacteria and freeze-dried survival rate of Lactobacillus plantarum cultured using MRS medium, optimized medium and uracil medium

[0098] MRS medium, optimized medium and uracil medium (500 mg / L uracil was added to the optimized medium) were respectively used to culture plant lactobacillus YR07 and prepare a lactic acid bacteria direct-injection starter, which specifically includes the following specific steps:

[0099] The MRS medium, the optimized medium and the uracil medium were sterilized at 121°C for 15 minutes, cooled to room temperature, and the activated Lactobacillus plantarum YR07 was inoculated into the corresponding medium at a concentration of 2%; they were then incubated in an incubator shaker at 30°C for 18 hours at a speed of 150 rpm / min. When the growth reached a stable phase, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C; the bacterial cells were then washed three times with sterile saline;

[0100] An equal volume of composite lyophilization protective agent was added to the above bacterial cells, mixed thoroughly, and distributed into vials, and freeze-dried using a SCIENTZ-50F / a freeze dryer. The freeze-drying process was as follows: the sample was initially pre-frozen at -80°C for 4 hours, then gradually heated, and then freeze-dried at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, 0°C for 2 hours, and finally 10°C for 16 hours. After the freeze-drying process was completed, the vials were sealed with rubber stoppers before being taken out. The lactic acid bacteria direct-injection starter was then stored at 4°C for further analysis.

[0101] Fig. 9 The viable count and freeze-dried survival rate of Lactobacillus plantarum YR07 were obtained by culturing them with MRS medium, optimized medium, and uracil medium. Fig. 9 It can be seen that the number of viable bacteria of Lactobacillus plantarum YR07 in the culture medium supplemented with uracil is 1.33×10 10 CFU / mL, the survival rate after freeze-drying was 75.5%. The number of viable bacteria in the optimized culture medium was 7.9×10 9 CFU / mL, the survival rate after freeze-drying was 65.1%. The number of viable bacteria in MRS medium was 4.6×10 9 CFU / mL, and the survival rate after freeze-drying was 37.9%.

[0102] The same method as above was used to test the viable bacterial count and post-lyophilization survival rate of the methods using 500 mg / L guanine medium, 500 mg / L adenine medium, and 500 mg / L xanthine medium. The viable bacterial count in the lactic acid bacteria direct-injection starter obtained with 500 mg / L guanine medium was 0.87×10 10 CFU / mL, the survival rate after freeze-drying was 65.7%; the number of live bacteria in the lactic acid bacteria direct-throw fermentation medium obtained with 500 mg / L adenine was 0.73×10 10 CFU / mL, the survival rate after freeze-drying was 62.3%; the number of live bacteria in the lactic acid bacteria direct-injection fermentation medium obtained with 500 mg / L xanthine was 0.75×10 10 CFU / mL, and the survival rate after freeze-drying was 58.8%.

[0103] The present invention further verifies the effects of the above-mentioned MRS medium, optimized medium and uracil medium on reducing Lactobacillus plantarum cell damage, increasing the activity of Lactobacillus plantarum and improving its ability to resist harsh environments, specifically from the following aspects:

[0104] (1) Lysozyme and sodium chloride sensitivity of Lactobacillus plantarum YR07

[0105] Fig.10The effect of MRS medium, optimized medium, and uracil medium on the lysozyme sensitivity of Lactobacillus plantarum YR07 is shown; Fig.11 The figure shows the effect of MRS medium, optimized medium and uracil medium on the sodium chloride sensitivity of Lactobacillus plantarum YR07. Fig.10 , Fig.11 It can be seen that bacterial cells are very sensitive to lysozyme. When the bacterial cell wall is damaged, it cannot grow on the selective medium containing lysozyme. By comparing the growth of the strain on the medium containing lysozyme and MRS medium, the damage to the cell wall of the strain after freeze-drying can be evaluated. Fig.10 , Fig.11 The figure shows the sensitivity of bacteria cultured in different media to lysozyme (survival rate %). It can be seen that the cell wall damage is lower when uracil is added during the culture and freeze-drying stages. The integrity of the cell membrane will affect the growth and reproduction of the strain. The sensitivity of the strain to NaCl can be used to evaluate the integrity of its cell membrane. + and Cl - It can enter bacteria through the cell membrane. If the bacterial cell membrane is destroyed, the balance of the bacterial cell will be broken. In terms of sodium chloride sensitivity (survival rate), the group with added uracil had lower cell membrane damage during the culture and freeze-drying stages. The above results show that adding uracil can reduce cell wall damage, better maintain the integrity of the cell wall and cell membrane, and thus improve the survival rate after freeze-drying.

[0106] (2) ATPase activity of Lactobacillus plantarum YR07

[0107] Fig.12 The effects of MRS medium, optimized medium and uracil medium on the ATPase activity (Na + K + -ATPase); Fig.13 The effects of MRS medium, optimized medium and uracil medium on the ATPase activity (Ca 2+ Mg 2- ATPase) is a membrane-bound enzyme that catalyzes the hydrolysis of ATP into ADP and phosphate ions and releases energy. It is directly related to the life activities of biological cells. ATPases in cells include Na + K + -ATPase, Mg 2+ -ATPase, Ca 2+ -ATPase and Ca 2+ Mg 2+ -ATPase, in which Na + K + -ATPase and Ca 2+ Mg2+ -ATPase is the two most critical enzymes. + K + -ATPase catalyzes the hydrolysis of ATP to provide energy, maintain the membrane potential on both sides of the cell membrane, and regulate cell osmotic pressure. 2+ Mg 2+ -ATPase can regulate the transfer of ions inside and outside the membrane, maintain cell osmotic pressure, and transmit cell signals. Fig.12 , Fig.13 It can be seen that the ATPase activity (Na + K + -ATPase, Ca 2+ Mg 2- ATPase) was higher than that of the bacteria in MRS medium, and the Na + K + -ATPase, Ca 2+ Mg 2- The ATPase activity was the highest. The results showed that after the strain metabolized uracil, the activity of Lactobacillus plantarum was improved, the number of viable bacteria increased, and the ATPase activity of the strain was maintained after freeze-drying, so that the strain had more energy to complete the transportation of intracellular and extracellular substances, thereby increasing the number of viable bacteria and freeze-drying survival rate of the strain.

[0108] (3) Protein structure of Lactobacillus plantarum YR07

[0109] The secondary structure of proteins can be calculated using the amide band, which mainly includes α-helices (1650-1660 cm -1 ), irregular curl (1640-1650cm -1 ), β-fold (1600-1640cm -1 ) and β-turn (1660-1700cm -1 ). Fig.14 The effects of MRS medium, optimized medium and uracil medium on the protein structure of Lactobacillus plantarum YR07. Fig.14 As shown in the figure, during culture, the secondary structure of proteins in each group was similar and not much different; in the secondary structure of bacterial proteins in different culture media, β-folding accounted for the highest proportion in the culture stage; in the freeze-drying stage, it can be seen that β-turns accounted for the highest proportion, indicating that during the freeze-drying process, the structure of bacterial proteins changed from ordered to disordered, indicating that the proteins aggregated. After freeze-drying, Lactobacillus plantarum cultured in uracil culture medium had the highest content of ordered α-helices and β-folding structures, indicating that its protein secondary structure changed the least compared with other groups, thereby improving the survival rate after freeze-drying.

[0110] (4) Cell morphology of Lactobacillus plantarum YR07 after freeze-drying

[0111] Fig.15 The effects of MRS medium, optimized medium and uracil medium on the freeze-dried cell morphology of Lactobacillus plantarum YR07. Fig.15 As shown in the figure, the bacterial morphology of the three culture media after freeze-drying is that the bacterial morphology in the MRS medium becomes longer and more damaged, while the bacterial morphology in the optimized culture and uracil medium is generally short and round. The bacteria in this state can better withstand the harsh external environment, and these changes can reduce the damage to the cell membrane caused by freezing and dehydration during the freeze-drying process, thereby improving the freeze-drying survival rate of the strains in the experimental group.

[0112] (5) Fatty acid composition of freeze-dried Lactobacillus plantarum YR07

[0113] As shown in Table 11, the bacterial cell membranes in the three culture media are mainly composed of 7 fatty acids. It can be seen that after freeze-drying, the content of unsaturated fatty acids in the bacteria is increasing. By increasing the relative content of long-chain unsaturated fatty acids, the compactness of the cell membrane of the strain is improved, making the cell membrane structure of the strain more stable. These changes can reduce the damage to the cell membrane caused by freezing and dehydration during the freeze-drying process, thereby improving the freeze-drying survival rate of the strains in the experimental group.

[0114] Table 11 Effect of different culture media on the fatty acid composition of freeze-dried Lactobacillus plantarum YR07

[0115]

[0116] 4. Effects of different concentrations of nucleotide addition on lactic acid bacteria

[0117] Different concentrations of nucleotides were added to the optimized culture medium to verify the effects of different concentrations of nucleotides on lactic acid bacteria. The added concentrations and results are shown in Table 12. The specific steps are as follows:

[0118] The activated Lactobacillus plantarum YR07 was inoculated into the corresponding culture medium at a concentration of 2%; they were then incubated in an incubator shaker at 30°C for 18 hours at a rotation speed of 150 rpm / min. When the growth reached a stable phase, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C; the bacterial cells were then washed three times with sterile saline;

[0119] Add an equal volume of composite freeze-drying protective agent (composite freeze-drying protective agent is composed of 10wt% sorbitol, 30wt% skim milk powder, 8wt% proline, and the remaining distilled water) to the above-mentioned bacterial cells, mix thoroughly, and distribute into a vial, freeze-dry using a SCIENTZ-50F / a freeze dryer, and the freeze-drying process is as follows: the sample is initially pre-frozen at -80°C for 4 hours, then gradually warmed, successively at -40°C for 2h, at -30°C for 2h, at -20°C for 2h, at -10°C for 2h, at 0°C for 2h, and finally at 10°C for 16h for freeze-drying. After the freeze-drying process is completed, the vial is sealed with a rubber stopper before being taken out. The lactic acid bacteria direct-injection starter is then stored at 4°C for further analysis.

[0120] Table 12 Effect of different nucleotide concentrations on viable bacterial count and freeze-dried survival rate

[0121]

[0122] 5. Effects of different nucleotide complexes on Lactobacillus plantarum

[0123] Nucleotides of different compound ratios were added to the optimized culture medium to obtain compound nucleotide culture medium A, B, C, and D, as follows:

[0124] Group A: uracil:guanine = 1:1, total nucleotide concentration is 500 mg / L;

[0125] Group B: uracil: adenine = 1:1, total nucleotide concentration 500 mg / L;

[0126] Group C: Uracil:xanthine = 1:1, total nucleotide concentration is 500 mg / L

[0127] Group D: uracil: guanine: adenine: xanthine = 1:1:1:1, the total nucleotide concentration is 500 mg / L.

[0128] The activated Lactobacillus plantarum YR07 was inoculated into the corresponding culture medium at a concentration of 2%; they were then incubated in an incubator shaker at 30°C for 18 hours at a rotation speed of 150 rpm / min. When the growth reached a stable phase, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C; the bacterial cells were then washed three times with sterile saline;

[0129] Add an equal volume of composite freeze-drying protective agent (composite freeze-drying protective agent is composed of 20wt% sorbitol, 10wt% skim milk powder, 25wt% proline, and the remaining distilled water) to the above-mentioned bacterial cells, mix thoroughly, and distribute into a vial, freeze-dry using a SCIENTZ-50F / a freeze dryer, and the freeze-drying process is as follows: the sample is initially pre-frozen at -80°C for 4 hours, then gradually warmed, successively at -40°C for 2h, at -30°C for 2h, at -20°C for 2h, at -10°C for 2h, at 0°C for 2h, and finally at 10°C for 16h for freeze-drying. After the freeze-drying process is completed, the vial is sealed with a rubber stopper before being taken out. The lactic acid bacteria direct-injection starter is then stored at 4°C for further analysis.

[0130] Table 13 Effects of different nucleotide complexes on viable counts and freeze-dried survival rates of Lactobacillus plantarum

[0131]

[0132] 6. Effects of different freeze-drying protective agents on Lactobacillus plantarum

[0133] The activated Lactobacillus plantarum YR07 was inoculated into MRS medium and composite nucleotide medium (uracil: guanine: adenine: xanthine = 1:1:1:1) at a concentration of 2%, respectively; they were then incubated in an incubator shaker at 30°C for 18 hours at a rotation speed of 150 rpm / min. When the growth reached a stable phase, the bacterial cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C; the bacterial cells were then washed three times with sterile saline; equal volumes of lyophilization protectants A, B, C, D, and E were added to the two groups of bacterial cells obtained by the above centrifugation. The compositions of the five composite lyophilization protectants are shown in Table 14.

[0134] Table 14 Composition of different lyophilization protective agents

[0135]

[0136]

[0137] After the thalline and the above-mentioned lyophilization protective agent are fully mixed, and distributed in a cillin bottle, a SCIENTZ-50F / a freeze dryer is used for freeze drying, and the freeze drying process is as follows: the sample is initially pre-frozen at -80°C for 4 hours, and then gradually warmed up, successively at -40°C for 2h, at -30°C for 2h, at -20°C for 2h, at -10°C for 2h, at 0°C for 2h, and finally at 10°C for 16h for freeze drying. After the freeze drying process is completed, the cillin bottle is sealed with a rubber stopper before taking out. Subsequently, the lactic acid bacteria direct injection fermentation agent is stored at 4°C for further analysis. Table 15 shows the effects of different lyophilization protective agents on the freeze-dried survival rate of plant lactobacillus.

[0138] Table 15 Effect of different freeze-drying protective agents on the freeze-drying survival rate of Lactobacillus plantarum

[0139]

[0140] 7. Cultivation of different bacterial species

[0141] Lactic acid bacteria of different species (Lactobacillus plantarum YR07, Lactobacillus sakei L48, and Lactobacillus curvatum D2) were selected for fermentation culture, and MRS medium and composite nucleotide medium (based on the optimized medium, uracil: guanine: adenine: xanthine with a total concentration of 500 mg / L and a mass ratio of 1:1:1:1) were added to the three lactic acid bacteria, specifically including the following steps:

[0142] The activated Lactobacillus plantarum YR07, Lactobacillus sakei L48, and Lactobacillus curvatum D2 were inoculated into MRS medium and composite nucleotide medium at a concentration of 2%, and then they were incubated in an incubator shaker at 30°C for 18 hours at a rotation speed of 150 rpm / min, and the live bacteria were counted when the growth reached a stable phase; the bacterial liquid was collected and centrifuged at 8000 rpm for 10 minutes at 4°C to harvest the bacterial cells, and then the bacterial cells were washed three times with sterile saline;

[0143] An equal volume of composite freeze-drying protectant (15wt% sorbitol, 20wt% skim milk powder, 13wt% proline, and the remainder of distilled water) was added to the bacterial cells after washing with saline, mixed thoroughly, and dispensed into vials. Freeze drying was performed using a SCIENTZ-50F / a freeze dryer, and the freeze drying process was as follows: the sample was initially pre-frozen at -80°C for 4 hours, then gradually heated up, at -40°C for 2h, at -30°C for 2h, at -20°C for 2h, at -10°C for 2h, at 0°C for 2h, and finally at 10°C for 16h for freeze drying. After the freeze drying process was completed, the vials were sealed with rubber stoppers before being taken out. The samples were then stored at 4°C for further analysis.

[0144] Fig.16 The effect of MRS medium and compound nucleotide medium on the viable count of different lactic acid bacteria is shown. Fig.17 The figure shows the effect of MRS medium and compound nucleotide medium on the freeze-dried survival rate of different lactic acid bacteria. Fig.16 , 17 It was found that the number of live bacteria and freeze-dried survival rate of lactic acid bacteria of different species after being cultured in the composite nucleotide culture medium were higher than those of the MRS culture medium on the market.

[0145] 8. Application of lactic acid bacteria direct-injection starter in sausage fermentation

[0146] The plant lactobacillus YR07 direct-injection starter prepared with different culture media and different freeze-dried protective agents was applied to sausage fermentation. The test groups were the following four groups: A, B, C, and D, as shown in Table 16:

[0147] Table 16

[0148]

[0149] The plant lactobacillus YR07 direct-injection starter prepared by using the above culture medium and freeze-dried protective agent is applied to sausage fermentation, and the specific steps are as follows:

[0150] The pork hind leg meat was blended and mixed at a fat-to-lean ratio of 3:7, and the auxiliary materials were added in the following proportions: 2.5wt% salt, 0.7wt% glucose, 0.01wt% nitrite, 0.015wt% garlic powder, 0.05wt% fennel powder, 0.05wt% black pepper powder, 0.225wt% sausage flavor, and mixed evenly to obtain pork mince. 0.1g of plant lactobacillus YR07 direct-injection fermentation agent was added to 10mL of distilled water to obtain a bacterial suspension, which was evenly sprayed on 100g of minced meat and rolled for 15 minutes. The amount of fermentation agent was 1wt%. When enema was performed, the intestine was ensured to be full and free of bubbles. The operating temperature of the whole process was 4°C.

[0151] Then it was hung in a constant temperature and humidity box and fermented for 24 hours at 30°C and 85% relative humidity, and then air-dried for 15 days at 15°C and 45% relative humidity to obtain the finished product.

[0152] Table 17 Effects of different direct-injection starter cultures on the quality of sausages

[0153]

[0154] As can be seen from the above table, the sausage fermented by the direct-throw starter culture prepared by culturing the composite nucleotide culture medium of the present invention and the composite lyophilization protectant of the present invention obtained the highest score in the sensory evaluation, and it can be seen from the lactic acid bacteria count that due to the synergistic effect of the culture medium and the lyophilization protectant, the growth state of the lactic acid bacteria in the fermented sausage is good, the number of live bacteria is higher than that of other groups, and the activity is good, the acid production is fast, and the flavor substances are produced more quickly and in larger quantities, and the fermented sausage has good flavor and quality.

[0155] In summary, in combination with the above-mentioned culture results, it can be known that the optimized culture medium in the present invention synergizes with nucleotides and composite freeze-drying protective agents, while improving the viable count of the bacterium, also improves the stress resistance of the bacterium, reduces the cell wall and cell membrane damage of Lactobacillus plantarum YR07, improves its LDH enzyme and ATP enzyme activity, improves the sugar metabolism ability of bacteria, and reduces the damage of the secondary structure of protein after freeze-drying. Improve the relative content of long-chain unsaturated fatty acids in the cell membrane of Lactobacillus plantarum YR07, improve the compactness of the cell membrane of the strain, make the cell membrane structure of the strain more stable, and better cope with environmental stress and stress damage in the freeze-drying stage.

[0156] The various aspects, embodiments, features and examples of the present invention should be considered as illustrative in all aspects and are not intended to limit the present invention, the scope of the present invention is defined only by the claims. Other embodiments, modifications and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.

[0157] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0158] Although the present invention has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions and / or additions may be made without departing from the spirit and scope of the present invention and that the elements of the embodiments may be replaced by substantial equivalents. In addition, many modifications may be made without departing from the scope of the present invention to adapt specific circumstances or materials to the teachings of the present invention. Therefore, it is not intended herein to limit the present invention to the disclosed specific embodiments for performing the present invention, but it is intended that the present invention will include all embodiments within the scope of the appended claims. In addition, unless specifically stated, any use of the terms first, second, etc. does not indicate any order or importance, but rather uses the terms first, second, etc. to distinguish one element from another.

Claims

1. A method for preparing a lactic acid bacteria direct-injection starter, characterized in that: include: The activated lactic acid bacteria strains are inoculated into a nucleotide culture medium for expansion culture to obtain a high-density lactic acid bacteria agent; After centrifuging the high-density lactic acid bacteria agent, the bacterial bodies are mixed with a composite freeze-drying protective agent and freeze-dried to obtain a lactic acid bacteria direct-injection fermentation agent; The components of the nucleotide culture medium include: 0.3-0.8 g / L nucleotides, 2-10 g / L beef extract powder, 25-35 g / L soy peptone, 10-17 g / L sucrose, 1-6 g / L anhydrous sodium acetate, 2-8 g / L triammonium citrate, 1.0-1.6 g / L potassium dihydrogen phosphate, 0.1-0.8 g / L magnesium sulfate monohydrate, 0.023-0.029 g / L manganese sulfate and 2-6 g / L Tween 80; The nucleotides include one or a combination of multiple of guanine, adenine, xanthine or uracil.

2. The preparation method according to claim 1, characterized in that: The nucleotide medium includes uracil.

3. The preparation method according to claim 1, characterized in that: Specifically include: The activated lactic acid bacteria are inoculated into the nucleotide culture medium at a volume concentration of 1-5%, cultured at 25-37°C for 12-18 hours, centrifuged at a speed of 5000-9000 rpm for 10-15 minutes, the high-density lactobacillus agent obtained by centrifugation is washed with sterile physiological saline, and then freeze-dried.

4. The preparation method according to claim 1, characterized in that: The components of the composite freeze-drying protective agent include: 5-20wt% sugar, 10-30wt% skim milk powder, 8-25wt% proline, and the rest includes distilled water.

5. The preparation method according to claim 4, characterized in that: The sugar includes one or a combination of glucose, trehalose, sorbitol or sucrose.

6. The preparation method according to claim 4, characterized in that: The preparation method of the composite lyophilization protective agent comprises: The sugar, proline and distilled water are mixed and ground for 5-15 minutes, stirred at 40-100° C. and 50-300 rpm for 8-12 hours to form a uniform, viscous, transparent liquid eutectic solvent, and then mixed with a skimmed milk powder solution to obtain the composite lyophilization protective agent.

7. The preparation method according to claim 1, characterized in that: The freeze-drying specifically includes: taking out the bacterial bodies after centrifuging the high-density lactic acid bacteria agent, and adding a composite freeze-drying protective agent of the same volume as before centrifugation to form a mixture, pre-freezing the mixture at -50°C for 2 to 10 hours, and then gradually heating the mixture to -40°C for 1 to 5 hours, -30°C for 1 to 5 hours, -20°C for 1 to 4 hours, -10°C for 1 to 3 hours, 0°C for 0 to 3 hours, and 10°C for 5 to 20 hours, so as to obtain the lactic acid bacteria direct-injection fermentation agent.

8. The preparation method according to claim 1, characterized in that: The lactic acid bacteria are Lactobacillus plantarum YR07, Lactobacillus sakei L48 or Lactobacillus curvatum D2.

9. A lactic acid bacteria direct-injection starter, characterized in that: It is prepared by the method described in any one of claims 1 to 8.

10. Use of the lactic acid bacteria direct-injection starter culture according to claim 9 in fermented sausages; Preferably, the application includes: The lactic acid bacteria direct-injection starter is uniformly mixed with pork mince at a concentration of 1 to 5 wt %, and fermented for 12 to 48 hours in an environment of 10 to 40° C. and 30 to 90% humidity, and then air-dried to obtain sausages; Preferably, the minced pork further contains auxiliary materials, which include salt, glucose, nitrite, garlic powder, fennel powder, black pepper powder and sausage flavor.

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