A direct-inoculation lactic acid bacteria starter culture, its preparation method and application

Abstract

This invention provides a direct-inoculation lactic acid bacteria starter culture, its preparation method, and its application. The method includes: inoculating activated lactic acid bacteria strains into a nucleotide medium for expansion culture to obtain a high-density lactic acid bacteria inoculum; centrifuging the high-density lactic acid bacteria inoculum, mixing the bacterial cells with a freeze-drying protectant, and freeze-drying to obtain the direct-inoculation lactic acid bacteria starter culture; wherein the nucleotide medium comprises: 0.3–0.8 g / L nucleotides, 2–10 g / L beef extract powder, 25–35 g / L soybean 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 at least one selected from guanine, adenine, xanthine, or uracil. The method provided by this invention can improve the activity and stress resistance of lactic acid bacteria strains, and enhance their adaptability to harsh environments, thereby obtaining a direct-inoculation lactic acid bacteria starter with high density and freeze-drying survival rate.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a direct-inoculation lactic acid bacteria starter culture, its preparation method, and its application. Background Technology

[0002] Optimizing the production of probiotic starter cultures, particularly for lactic acid bacteria (LAB) and other microorganisms with multiple health benefits, is a current research hotspot in food science and biotechnology. To maximize the probiotic benefits of LAB, researchers are constantly exploring and improving preparation technologies, with high-density fermentation and freeze-drying being two core technologies. High-density fermentation technology ensures a sufficient number of live bacteria by precisely controlling culture conditions such as temperature, pH, and nutrient supply, which is fundamental to the effectiveness of probiotic products. Freeze-drying technology, through rapid freezing and vacuum drying, effectively extends the shelf life of probiotics, facilitating transportation and storage while reducing the risk of bacteriophage contamination, and is a crucial step in maintaining probiotic activity.

[0003] However, probiotics are easily damaged in their physiological structure and function under extreme conditions such as low temperature and dryness. This can lead to reduced activity of key enzymes, impaired cell membrane integrity, and even bacterial death, directly affecting the quality and efficacy of probiotic products. Therefore, improving the survival rate of probiotics during freeze-drying has become an urgent problem to be solved. To address this challenge, researchers have proposed various strategies, including adjusting the culture conditions of strains, screening and optimizing freeze-drying protectants, and finely controlling the process parameters of freeze-drying. Among these, modifying the culture medium composition, particularly by adding synergistic factors crucial for probiotic growth and metabolism, is considered a highly promising method.

[0004] In view of the various limitations of existing technologies regarding lactic acid bacteria proliferation density methods and freeze-drying technologies, in order to effectively improve the activity of direct-inoculation lactic acid bacteria starter cultures, increase the survival rate of bacteria during freeze-drying and preservation, and optimize their application quality, this invention provides a method for improving the activity and freeze-drying survival rate of direct-inoculation lactic acid bacteria starter cultures. Summary of the Invention

[0005] To solve all or part of the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] One objective of this invention is to provide a method for preparing a direct-inoculation lactic acid bacteria starter culture, comprising:

[0007] Activated lactic acid bacteria strains are inoculated into nucleotide medium for large-scale culture to obtain high-density lactic acid bacteria inoculum.

[0008] After centrifuging the high-density lactic acid bacteria inoculum, the bacterial cells were mixed with a composite freeze-drying protectant and then freeze-dried to obtain a direct-inoculation lactic acid bacteria starter.

[0009] The nucleotide culture medium comprises: 0.3–0.8 g / L nucleotides, 2–10 g / L beef extract, 25–35 g / L soybean 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 more of guanine, adenine, xanthine, or uracil.

[0011] The method provided by this invention can enhance the activity and stress resistance of bacterial strains, improve the ability of lactic acid bacteria to adapt to harsh environments, and obtain a direct-inoculation lactic acid bacteria starter with high density and freeze-drying survival rate, achieving a lactic acid bacteria density of 102. 10 With a CFU / mL or higher, the freeze-dried survival rate increases to over 85.6%.

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

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

[0014] In some preferred embodiments, the nucleotide culture medium comprises uracil and guanine in a concentration ratio of (1–3):(1–3). Alternatively, the nucleotide culture medium comprises uracil and xanthine in a concentration ratio of (1–4):(1–4). Alternatively, the nucleotide culture medium comprises uracil:xanthine in a concentration ratio of (1–3):(1–3). Alternatively, the nucleotide culture medium comprises uracil:guanine:adenine:xanthine in a concentration ratio of (1–3):(1–3):(1–3):(1–3).

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

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

[0017] In some embodiments, the composite freeze-drying protectant comprises: 5-20 wt% sugar, 10-30 wt% skim milk powder, 8-25 wt% proline, and the remainder comprises 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 freeze-drying protectant includes: mixing and grinding the sugar, proline and distilled water for 5-15 minutes, stirring at 40-100℃ and 50-300 rpm for 8-12 hours to form a uniform, viscous, and transparent liquid eutectic solvent, and then mixing it with a skim milk powder solution to obtain the composite freeze-drying protectant.

[0020] In some embodiments, the freeze-drying specifically includes: centrifuging the high-density lactic acid bacteria inoculum, taking the bacterial cells, and adding an equal volume of freeze-drying protectant to form a mixture. The mixture is then pre-frozen at -50°C for 2–10 hours, and then sequentially heated to -40°C for 1–5 hours, -30°C for 1–5 hours, -20°C for 1–4 hours, -10°C for 1–3 hours, 0°C for 0–3 hours, and 10°C for 5–20 hours to obtain the direct-inoculation lactic acid bacteria starter.

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

[0022] The second objective of this invention is to provide a direct-inoculation lactic acid bacteria starter culture, which is prepared by any one of the methods described above.

[0023] The third objective of this invention is to provide the application of the aforementioned direct-inoculation lactic acid bacteria starter in fermented sausages.

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

[0025] In some embodiments, the minced pork also contains excipients, including salt, glucose, nitrite, garlic powder, fennel powder, black pepper powder, and sausage flavoring.

[0026] The direct-inoculation starter culture, prepared by synergistic use of a specially formulated compound nucleotide culture medium and a compound freeze-drying protectant, exhibits excellent fermentation characteristics in the production of fermented sausages. Its growth in fermented sausages is extremely good, with a significantly higher number of viable bacteria than other groups, exhibiting strong activity and rapid acid production, enabling faster and larger-volume production of flavor compounds. It demonstrates significant advantages in the production of fermented sausages. The microorganisms in this starter culture can continue to thrive and metabolize efficiently in a high-salt environment, endowing the sausages with a unique aroma and flavor.

[0027] Compared with traditional starter cultures, the direct-inoculation starter culture of this invention can significantly shorten the fermentation time of meat products, completing the fermentation process in as little as 9 days, thus increasing fermentation efficiency by 25%. Therefore, sausages fermented using the direct-inoculation starter culture of this invention not only have a unique flavor and superior quality, but also receive the highest scores in sensory evaluations, fully demonstrating the superior performance and practical value of this invention.

[0028] Compared with existing technologies, 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 cells, reduce damage to their cell walls and cell membranes, enhance the activity of LDH and ATPases in lactic acid bacteria, improve the sugar metabolism capacity of bacteria, and reduce the damage to the secondary structure of proteins after freeze-drying. Furthermore, it can increase the relative content of long-chain unsaturated fatty acids in the cell membrane of lactic acid bacteria, improve the compactness of the cell membrane, and make the cell membrane structure of the strain more stable, thus enabling it to better cope with environmental stress and stress damage during the freeze-drying stage. This results in obtaining a direct-inoculation lactic acid bacteria starter with high density and freeze-drying survival rate. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0031] Figure 2 , Figure 3 The images show the response surface plot and contour plot of the effects of beef meal, soy peptone, and triammonium citrate on bacterial growth.

[0032] Figure 4 , Figure 5 The images show the response surface plot and contour plot of the effects of beef meal, soy peptone, and sodium acetate on bacterial growth.

[0033] Figure 6 , Figure 7 The images show the response surface plot and contour plot of the effects of sodium acetate and triammonium citrate on bacterial cell growth, respectively.

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

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

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

[0037] Figure 12 The effects of MRS medium, optimized medium, and uracil medium on the ATPase activity (Na+) of Lactobacillus plantarum YR07. + K + The effect of α-ATPase;

[0038] Figure 13 The effects of MRS medium, optimized medium, and uracil medium on the ATPase activity (Ca) of Lactobacillus plantarum YR07. 2+ Mg 2- The effect of ATPase;

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

[0040] Figure 15 The effects of MRS medium, optimized medium, and uracil medium on the cell morphology of Lactobacillus plantarum YR07 after lyophilization;

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

[0042] Figure 17 The effects of MRS medium and compound nucleotide medium on the freeze-dried survival rates of Lactobacillus plantarum YR07, Lactobacillus sakei L48, and Lactobacillus curvature D2. Detailed Implementation

[0043] The technical solutions of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention. The specific functional details disclosed herein should not be construed as limiting, but are merely intended to form the basis of the claims and to teach those skilled in the art to employ the representative basis of the invention in different ways in any suitable detailed embodiment.

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

[0045] Biological Preservation Notes: The *Lactobacillus plantarum* YR07, *Lactobacillus sakei* L.48, and *Lactobacillus curvatus* D2 used in the embodiments of this invention are deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The depositary institution is abbreviated as CCTCC. The deposit date for *Lactobacillus plantarum* YR07 is August 18, 2022, with the biological accession number CCTCC NO: M20221303; the deposit date for *Lactobacillus sakei* L.48 is August 18, 2022, with the biological accession number CCTCC NO: M20221306; and the deposit date for *Lactobacillus curvatus* D2 is February 21, 2023, with the biological accession number CCTCC NO: M2023162.

[0046] In the specific embodiments of this invention, the percentage of inoculation concentration "%" refers to volume concentration.

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

[0048] The sensory evaluation criteria for sausages are as follows:

[0049] Sensory evaluation form

[0050]

[0051] 1. Culture medium optimization

[0052] This invention optimizes MRS culture medium to obtain a more suitable medium for Lactobacillus culture. Specifically, it optimizes the medium formulation using a combination of single-factor and response surface methodology based on MRS culture medium.

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

[0054] Based on the results of the single-factor experiments, inoculum size, temperature, and initial pH were selected as influencing factors, and viable cell concentration was used as the evaluation index to design an orthogonal experiment, as shown in Table 1.

[0055] Table 1. Factor Levels of Orthogonal Experiment for Fermentation Conditions

[0056]

[0057] Based on the optimization of culture conditions, single-factor optimization of components was conducted, specifically including: optimization of carbon source type and content, nitrogen source type and content, potassium dihydrogen phosphate concentration, ammonium citrate concentration, sodium acetate concentration, manganese sulfate concentration, magnesium sulfate concentration, and Tween 80 concentration. Based on the component optimization, PB experiments, steep slope experiments, and response surface methodology experiments were performed. The experimental design table is as follows:

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

[0059]

[0060] Table 3 Optimal Experimental Design for Climbing Steep Slopes

[0061]

[0062] Based on the optimal steep slope climbing test results and the experimental design principles of RSM response surface methodology, three factors were selected as research objects: beef meal plus soybean peptone, sodium acetate, and triammonium citrate. The viable bacterial count was used as the response value. Experiments were designed, and quadratic regression fitting was performed on the experimental data to analyze the main effects and interaction effects of each factor, determining the optimal values ​​for each factor. Their coded values ​​and corresponding actual values ​​are shown in Table 4. For other parameters with insignificant effects, the positive and negative effects of the PB experiment were used. Design-Expert software was used for both design and data analysis. Each experiment was repeated three times, and the results were averaged.

[0063] Table 4 Factor Level Coding Table for Response Surface Experimental Design

[0064]

[0065] Based on the results of single-factor experiments, an orthogonal experiment was designed to evaluate inoculum concentration, temperature, and initial pH. The optimal culture conditions were determined using viable cell count as the evaluation index. Table 5 shows that the primary and secondary factors affecting viable cell count were: temperature > inoculum size > initial pH. The optimal culture conditions determined by the experiments were: A2B3C2, i.e., inoculum size 2%, initial pH 7.0, and temperature 30℃, at which the viable cell count was 56 × 10⁶. 8 CFU / mL.

[0066] Table 5 Results of the orthogonal experiment for optimizing culture conditions of Lactobacillus plantarum YR07

[0067]

[0068]

[0069] Plackett-Burman experiments were conducted using Design Expert 8.0 software. In the single-factor analyses described above, eight single-factors were selected: sucrose, beef and soybean, sodium acetate, triammonium citrate, dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, and Tween 80. The results showed that the highest viable cell counts were obtained in each single-factor experiment when the culture medium contained 10 g / L sucrose, 25 g / L beef meal + soybean peptone (1:4), 2 g / L sodium acetate, 2 g / L triammonium citrate, 1 g / L dipotassium hydrogen phosphate, 0.025 g / L manganese sulfate, 0.3 g / L magnesium sulfate, and 3 g / L Tween 80, respectively. Therefore, Plackett-Burman experiments were conducted using the above concentrations of single-factors, and the results are shown in Table 6.

[0070] Table 6. Plackett-Burmen Experimental Results

[0071]

[0072]

[0073] The Plackett-Burman experimental design and results show that the amounts of sucrose (A), beef meal + soybean peptone (B), sodium acetate (C), triammonium citrate (D), dipotassium hydrogen phosphate (E), manganese sulfate (F), magnesium sulfate (G), and Tween 80 (H) added were used as factors in the Plackett-Burman experiment, along with three virtual factors (J, K, L). Based on the results, factors that significantly affected the viable count of *Lactobacillus plantarum* YR07 were screened.

[0074] Table 7. Significance Analysis of Plackett-Burmen Experiment Results

[0075]

[0076] As shown in Table 7, the P-value of the ANOVA model is <0.05, indicating that the obtained regression equation is significant and that the model fits well across the entire regression region. (Correlation coefficient R) 2 =0.9881, indicating a good correlation, and the adjusted coefficient of determination R0 2 Adj =0.9563, indicating that 95.6% of the experimental data variability can be explained by this regression model. Generally, the lower the coefficient of variation (CV), the higher the reliability and precision of the experiment. A CV value of 2.16% indicates that the reliability and precision of the PB experiment are good. Precision is the ratio of effective signal to noise; a value greater than 4.0 is considered reasonable. The precision of this experiment reached 20.143. Furthermore, Table 7 shows that the three factors with the greatest impact on cell growth at the 95% confidence interval are beef meal + soybean peptone (30 g / L), sodium acetate (2 g / L), and triammonium citrate (4 g / L) (P < 0.05). Further optimization experiments can be conducted. According to the Design Expert software prediction, the optimization results for non-significant factors are: 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.

[0077] The PB experiment showed that beef meal + soybean peptone, sodium acetate, and triammonium citrate had significant effects on cell growth. Beef meal + soybean peptone and triammonium citrate had significant positive effects, and their concentrations should be increased in later stages of the experiment. Sodium acetate had a significant negative effect, and its concentration should be decreased in later stages. The experimental design and results, based on the proportions of these three factors' effects, are shown in Table 8.

[0078] Table 8 Results of the steepest climb experiment

[0079]

[0080] As shown in Table 8, the highest viable count was 73 × 10⁻⁶ in Experiment 3. 8 CFU / mL, representing the steepest inflection point of the uphill test, was used as the central point for the subsequent central composite design, specifically beef meal + soybean peptone 30 g / L, sodium acetate 2 g / L, and triammonium citrate 4 g / L. A three-factor, three-level mathematical model was established using three factors: beef meal + soybean peptone, sodium acetate, and triammonium citrate, with each factor rated as low (-1), medium (0), and high (+1). The experimental results are shown in Tables 9 and 10.

[0081] Table 9 Results of Response Surface Experiment

[0082]

[0083]

[0084] Table 10 Analysis of Variance for the Regression Model

[0085]

[0086] Table 10 shows that 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-fit term is 1.381215, and the P-value is 0.3696, which is greater than 0.05, indicating that the lack-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. Using Design-Expert 8.0.6 software, a quadratic multivariate fitting was performed on the experimental results, yielding the equation 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 that: A, C, AB, AC, B 2 A 2 The effect was extremely significant (P < 0.01), the effect of BC was significant (P < 0.05), the model effect was extremely significant (P < 0.01), and the lack of fit term was not significant (P > 0.05).

[0087] The three factors interact in pairs, such as Figure 2 , 3 As shown in Figure 4. Taking the first-order partial derivative of the second-order equation, when the response value Y (viable bacteria count) reaches its maximum, the levels of each factor are: beef meal + soy peptone addition 40 g / L, triammonium citrate addition 4 g / L, and sodium acetate addition 3 g / L. At this point, the predicted viable bacteria count is 8.4 × 10⁻⁶. 9 CFU / mL. A validation test was conducted under these conditions, at which point the viable count was 8.3 × 10⁻⁶. 9 The CFU / mL value showed a 98.8% fit with the model predictions, indicating that the model can effectively predict the impact of culture medium composition on viable cell count.

[0088] Figure 1 Pareto Chart for different cultivation optimization factors. Figure 2 , Figure 3 The images show the response surface plot and contour plot of the effects of beef meal, soy peptone, and triammonium citrate on bacterial growth. Figure 4 , Figure 5 The images show the response surface plot and contour plot of the effects of beef meal, soy peptone, and sodium acetate on bacterial growth. Figure 6 , Figure 7The images show the response surface plot and contour plot of the effects of sodium acetate and triammonium citrate on bacterial cell growth, respectively.

[0089] Therefore, the final optimized formulation of the basal culture medium is as follows: beef extract powder: 8 g / L, soybean 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. In this medium, the viable count of *Lactobacillus plantarum* YR07 increased from 3.0 × 10⁻⁶ to that of MRS. 9 CFU / mL increased to 8.3 × 10⁻⁶ 9 The CFU / mL was increased by 2.76 times.

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

[0091] Add 500 mg / L of guanine, adenine, xanthine, and uracil to the optimized culture media obtained above, respectively, to obtain four culture media containing guanine, adenine, xanthine, and uracil, denoted as guanine medium, adenine medium, xanthine medium, and uracil medium, respectively. The optimized culture medium without nucleotides was used as a control. *Lactobacillus plantarum* YR07 was cultured using the above five culture media, and a prepared lyophilization protectant was used to prepare a direct-inoculation lactic acid bacteria starter. The specific steps include:

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

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

[0094] Preparation of the composite lyophilization protectant: Weigh glucose and proline, add distilled water, and grind thoroughly in a mortar for 10 minutes. Transfer the mixture to a beaker and seal it with double-layered aluminum foil. Stir magnetically at 90℃ and 100 rpm for 10 hours until the solid mixture forms a homogeneous, viscous, and transparent liquid to form a eutectic solvent. Add distilled water to adjust the concentration, and then mix with a skim milk powder solution to obtain the composite lyophilization protectant. The final composite lyophilization protectant consists of: 15 wt% sorbitol, 20 wt% skim milk powder, 13 wt% proline, and the remainder distilled water.

[0095] An equal volume of the compound lyophilization protectant was added to the bacterial cells described above, mixed thoroughly, and dispensed into vials. The samples were then freeze-dried using a SCIENTZ-50F / a lyophilizer. The freeze-drying process was as follows: the samples were initially pre-frozen at -80°C for 4 hours, then gradually heated, successively at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, and 0°C for 2 hours, finally freeze-dried at 10°C for 16 hours. After the freeze-drying process, the vials were sealed with rubber stoppers before removal. The lactic acid bacteria direct-inoculation starter culture was then stored at 4°C for further analysis.

[0096] Figure 8 The effects of different nucleotides on the viable count of *Lactobacillus plantarum* YR07 were shown, indicating that different types of nucleotides had inconsistent growth-promoting effects on *Lactobacillus plantarum* YR07. Uracil showed the best promoting effect, with 500 mg / L uracil medium enabling a viable count of 133 × 10⁻⁶. 8 The CFU / mL concentration was increased by 4.03 times compared to the MRS-optimized medium.

[0097] 3. Viable cell count and freeze-drying survival rate of *Lactobacillus plantarum* cultured using MRS medium, optimized medium, and uracil medium.

[0098] Lactobacillus plantarum YR07 was cultured in MRS medium, optimized medium, and uracil medium (500 mg / L uracil was added to the optimized medium) to prepare a direct-inoculation lactic acid bacteria starter. The specific steps are as follows:

[0099] MRS medium, optimized medium, and uracil medium were sterilized at 121°C for 15 min, cooled to room temperature, and activated Lactobacillus plantarum YR07 was inoculated into the corresponding medium at a concentration of 2%. They were then incubated in a shaker at 30°C for 18 hours at a speed of 150 rpm / min. When the growth reached the stationary 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 physiological saline.

[0100] An equal volume of the compound lyophilization protectant was added to the bacterial cells described above, mixed thoroughly, and dispensed into vials. The samples were then freeze-dried using a SCIENTZ-50F / a lyophilizer. The freeze-drying process was as follows: the samples were initially pre-frozen at -80°C for 4 hours, then gradually heated, successively at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, and 0°C for 2 hours, finally freeze-dried at 10°C for 16 hours. After the freeze-drying process, the vials were sealed with rubber stoppers before removal. The lactic acid bacteria direct-inoculation starter culture was then stored at 4°C for further analysis.

[0101] Figure 9 This refers to the viable cell count and freeze-dried survival rate of *Lactobacillus plantarum* YR07 cultured using MRS medium, optimized medium, and uracil medium. Figure 9 The viable count of *Lactobacillus plantarum* YR07 in the culture medium supplemented with uracil was found to be 1.33 × 10⁻⁶. 10 The viable count was 75.5% after lyophilization (CFU / mL). The optimized culture medium contained 7.9 × 10⁻⁶ viable cells. 9 The CFU / mL concentration yielded a survival rate of 65.1% after lyophilization. The viable count in MRS medium was 4.6 × 10⁻⁶. 9 CFU / mL, survival rate after lyophilization was 37.9%.

[0102] The same method was used to test the viable bacterial count and lyophilization survival rate using 500 mg / L guanine, 500 mg / L adenine, and 500 mg / L xanthine media. The viable bacterial count in the direct-inoculation lactic acid bacteria starter culture obtained from 500 mg / L guanine media was 0.87 × 10⁻⁶. 10 The viable count of CFU / mL lyophilized lactic acid bacteria was 65.7%; the viable count of the direct-inoculation starter culture obtained from 500 mg / L adenine medium was 0.73 × 10⁻⁶. 10 The CFU / mL concentration was 62.3% after lyophilization; the viable count of the lactic acid bacteria in the direct-inoculation starter culture obtained from 500 mg / L xanthine medium was 0.75 × 10⁻⁶. 10 CFU / mL, survival rate after lyophilization was 58.8%.

[0103] This invention further verified the effects of the above-mentioned MRS medium, optimized medium, and uracil medium on reducing Lactobacillus plantarum cell damage, improving Lactobacillus plantarum activity, and enhancing its ability to withstand harsh environments. Specifically, the verification was conducted in the following aspects:

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

[0105] Figure 10The effects of MRS medium, optimized medium, and uracil medium on the lysozyme sensitivity of Lactobacillus plantarum YR07 are shown. Figure 11 The effects of MRS medium, optimized medium, and uracil medium on the sodium chloride sensitivity of *Lactobacillus plantarum* YR07 are shown. Figure 10 , Figure 11 It is known that bacterial cells are highly sensitive to lysozyme; when the bacterial cell wall is damaged, they cannot grow on selective media containing lysozyme. By comparing the growth of bacterial strains on lysozyme-containing media and MRS media, the degree of cell wall damage after lyophilization can be assessed. Figure 10 , Figure 11 The results show the sensitivity (survival rate %) of bacterial cells cultured in different media to lysozyme. During the culturing and freeze-drying stages, it was observed that the addition of uracil resulted in less cell wall damage. Cell membrane integrity affects the growth and reproduction of the strain. The strain's sensitivity to NaCl can be used to assess its cell membrane integrity. + and Cl - Uracil can cross the cell membrane and enter bacteria; if the bacterial cell membrane is damaged, the balance of the bacterial cell will be disrupted. Regarding sodium chloride sensitivity (survival rate), the group with added uracil showed lower cell membrane damage during both the culturing and lyophilization stages. These results indicate 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 lyophilization.

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

[0107] Figure 12 The effects of MRS medium, optimized medium, and uracil medium on the ATPase activity (Na+) of Lactobacillus plantarum YR07. + K + The effect of α-ATPase; Figure 13 The effects of MRS medium, optimized medium, and uracil medium on the ATPase activity (Ca) of Lactobacillus plantarum YR07. 2+ Mg 2- The effects of ATPase. ATPase is a membrane-bound enzyme that catalyzes the hydrolysis of ATP into ADP and phosphate ions, releasing energy, and is directly related to the life activities of biological cells. Cellular ATPases 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 one of the two most critical enzymes. Na + K + -ATPase catalyzes the hydrolysis of ATP to provide energy, maintain the membrane potential across the cell membrane, and regulate cell osmotic pressure. Ca... 2+ Mg 2+ -ATPases regulate ion transfer across the membrane, maintain cell osmotic pressure, and transmit cell signals. Figure 12 , Figure 13 It can be seen that the ATPase activity (Na+) of bacteria cultured in optimized culture medium and uracil medium is higher than that of bacteria cultured in uracil medium. + K + -ATPase, Ca 2+ Mg 2- The ATPase content of the cells was higher than that of the cells in MRS medium, and the Na+ content of the cells in uracil medium was also higher. + K + -ATPase, Ca 2+ Mg 2- The ATPase activity was the highest. The results showed that after the strain metabolized uracil, it enhanced the activity of Lactobacillus plantarum, increased its viable cell count, maintained the ATPase activity of the strain after freeze-drying, and enabled the strain to have more energy to complete the transport of intracellular and extracellular substances, thereby improving the viable cell count and freeze-drying survival rate of the strain.

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

[0109] The secondary structure of a protein can be calculated using the amide band, which mainly includes α-helices (1650-1660 cm). -1 Irregular curls (1640-1650cm) -1 ), β-fold (1600-1640cm) -1 ) and β-turn (1660-1700cm) -1 ). Figure 14 The effects of MRS medium, optimized medium, and uracil medium on the protein structure of *Lactobacillus plantarum* YR07. Figure 14 As shown, during cultivation, the secondary structure composition of proteins in each group was similar with little difference. Among the secondary structures of bacterial proteins in different culture media, β-sheets accounted for the highest proportion during the cultivation stage. During the freeze-drying stage, β-turns showed the highest proportion, indicating that the bacterial protein structure transitioned from ordered to disordered during freeze-drying, suggesting protein aggregation. After freeze-drying, *Lactobacillus plantarum* cultured in uracil medium exhibited the highest content of ordered α-helices and β-sheets, indicating that its protein secondary structure underwent the least change compared to other groups, thus improving its survival rate after freeze-drying.

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

[0111] Figure 15 The effects of MRS medium, optimized medium, and uracil medium on the morphology of lyophilized Lactobacillus plantarum YR07 cells were investigated. Figure 15 As shown, the morphology of the bacteria in the three culture media after freeze-drying was as follows: the bacteria in MRS medium became longer and more damaged, while the bacteria in the optimized culture and uracil medium were generally short and round in a normal state. The bacteria in this state can better resist the harsh external environment, and these changes can reduce the damage to the cell membrane caused by freezing and dehydration during freeze-drying, thereby improving the freeze-drying survival rate of the experimental group strains.

[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 were mainly composed of seven fatty acids. It can be seen that the content of unsaturated fatty acids in the bacterial cells increased after freeze-drying. By increasing the relative content of long-chain unsaturated fatty acids, the cell membrane compactness of the strains was improved, making the cell membrane structure more stable. These changes can reduce the damage to the cell membrane caused by freezing and dehydration during freeze-drying, thereby improving the freeze-dried survival rate of the experimental group strains.

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

[0115]

[0116] 4. Effects of different concentrations of nucleotides added on lactic acid bacteria

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

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

[0119] An equal volume of a compound lyophilization protectant (consisting of 10 wt% sorbitol, 30 wt% skim milk powder, 8 wt% proline, and the remainder distilled water) was added to the bacterial cells described above. The mixture was thoroughly mixed and dispensed into vials. The samples were then freeze-dried using a SCIENTZ-50F / a lyophilizer. The freeze-drying process was as follows: the samples were initially pre-frozen at -80°C for 4 hours, then gradually heated, successively at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, and 0°C for 2 hours, finally freeze-dried at 10°C for 16 hours. After the freeze-drying process, the vials were sealed with rubber stoppers before removal. The lactic acid bacteria direct-inoculation starter was then stored at 4°C for further analysis.

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

[0121]

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

[0123] Different ratios of nucleotides were added to the optimized culture medium to obtain nucleotide complex culture media A, B, C, and D, as detailed below:

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

[0125] Group B: Uracil:Adenine = 1:1, total nucleotide concentration was 500 mg / L;

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

[0127] Group D: Uracil: Guanine: Adenine: Xanthine = 1:1:1:1, total nucleotide concentration is 500 mg / L.

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

[0129] An equal volume of a compound lyophilization protectant (consisting of 20 wt% sorbitol, 10 wt% skim milk powder, 25 wt% proline, and the remainder distilled water) was added to the bacterial cells described above. The mixture was thoroughly mixed and dispensed into vials. The samples were then freeze-dried using a SCIENTZ-50F / a lyophilizer. The freeze-drying process was as follows: the samples were initially pre-frozen at -80°C for 4 hours, then gradually heated, successively at -40°C for 2 hours, -30°C for 2 hours, -20°C for 2 hours, -10°C for 2 hours, and 0°C for 2 hours, finally freeze-dried at 10°C for 16 hours. After the freeze-drying process, the vials were sealed with rubber stoppers before removal. The lactic acid bacteria direct-inoculation starter culture was then stored at 4°C for further analysis.

[0130] Table 13 Effects of different nucleotide combinations on the viable count and freeze-dried survival rate of Lactobacillus plantarum

[0131]

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

[0133] Activated *Lactobacillus plantarum* YR07 was inoculated at a concentration of 2% into MRS medium and compound nucleotide medium (uracil:guanine:adenine:xanthine = 1:1:1:1); then incubated in a shaker at 30°C for 18 hours at a speed of 150 rpm / min. When the growth reached the stationary 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 physiological saline; equal volumes of lyophilization protectants A, B, C, D, and E were added to the two groups of bacterial cells obtained by centrifugation. The composition of the five compound lyophilization protectants is shown in Table 14.

[0134] Table 14 Composition of different freeze-drying protectants

[0135]

[0136]

[0137] After thoroughly mixing the bacterial cells and the aforementioned lyophilization protectant, the mixture was dispensed into vials and freeze-dried using a SCIENTZ-50F / a lyophilizer. The freeze-drying process was as follows: the samples were initially pre-frozen at -80℃ for 4 hours, then gradually heated, successively at -40℃ for 2 hours, -30℃ for 2 hours, -20℃ for 2 hours, -10℃ for 2 hours, and 0℃ for 2 hours, finally freeze-dried at 10℃ for 16 hours. After the freeze-drying process, the vials were sealed with rubber stoppers before removal. The direct-inoculation lactic acid bacteria starter was then stored at 4℃ for further analysis. Table 15 shows the effect of different lyophilization protectants on the freeze-dried survival rate of *Lactobacillus plantarum*.

[0138] Table 15 Effects of different freeze-drying protectants on the freeze-dried survival rate of Lactobacillus plantarum

[0139]

[0140] 7. Cultivation of different bacterial strains

[0141] Different species of lactic acid bacteria (Lactobacillus plantarum YR07, Lactobacillus sakei L48, and Lactobacillus curvature D2) were selected for fermentation culture. MRS medium and a compound nucleotide medium (an optimized medium supplemented with 500 mg / L uracil:guanine:adenine:xanthine in a mass ratio of 1:1:1:1) were used for the three lactic acid bacteria. The specific steps included:

[0142] Activated *Lactobacillus plantarum* YR07, *Lactobacillus sakei* L48, and *Lactobacillus curvatureis* D2 were inoculated at a concentration of 2% into MRS medium and compound nucleotide medium, respectively. They were then incubated in a shaker at 30°C for 18 hours at a rotation speed of 150 rpm / min. When the growth reached the stationary phase, viable cell counts were performed. The bacterial suspension was collected and centrifuged at 8000 rpm for 10 minutes at 4°C to harvest bacterial cells. The bacterial cells were then washed three times with sterile physiological saline.

[0143] An equal volume of a compound lyophilization protectant (composed of 15 wt% sorbitol, 20 wt% skim milk powder, 13 wt% proline, and the remainder distilled water) was added to the bacterial cells after washing with saline, mixed thoroughly, and dispensed into vials. The samples were lyophilized using a SCIENTZ-50F / a lyophilizer as follows: the samples were initially pre-frozen at -80°C for 4 hours, then gradually heated to -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 at 10°C for 16 hours. After lyophilization, the vials were sealed with rubber stoppers before removal. The samples were then stored at 4°C for further analysis.

[0144] Figure 16 The effects of MRS medium and compound nucleotide medium on the viable counts of different lactic acid bacteria are shown. Figure 17 The effects of MRS medium and compound nucleotide medium on the freeze-dried survival rate of different lactic acid bacteria are shown. Figure 16 , 17 It was found that the viable count and freeze-drying survival rate of different species of lactic acid bacteria were higher than those of commercially available MRS culture medium after being cultured in a compound nucleotide medium.

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

[0146] Lactobacillus plantarum YR07 direct-inoculation starter culture prepared with different culture media and different freeze-drying protectants was applied to sausage fermentation. The test groups are A, B, C and D as shown in Table 16.

[0147] Table 16

[0148]

[0149] The Lactobacillus plantarum YR07 direct-inoculation starter culture prepared using the above-mentioned culture medium and freeze-drying protectant was applied to sausage fermentation. The specific steps are as follows:

[0150] Mince and mix pork hind leg meat at a lean-to-fat ratio of 3:7. Add the following auxiliary ingredients: 2.5 wt% salt, 0.7 wt% glucose, 0.01 wt% nitrite, 0.015 wt% garlic powder, 0.05 wt% fennel powder, 0.05 wt% black pepper powder, and 0.225 wt% sausage flavoring. Mix thoroughly to obtain minced pork. Add 0.1 g of *Lactobacillus plantarum* YR07 direct-inoculation starter culture to 10 mL of distilled water to obtain a bacterial suspension. Spray this suspension evenly onto 100 g of minced pork and tumble for 15 minutes. The starter culture dosage is 1 wt%. Ensure the sausages are full and free of air bubbles during stuffing. The entire process is carried out at a temperature of 4℃.

[0151] It was then suspended in a constant temperature and humidity chamber and fermented for 24 hours at 30°C and 85% relative humidity, followed by air drying for 15 days at 15°C and 45% relative humidity to obtain the finished product.

[0152] Table 17. Effects of different direct-inoculation starter cultures on sausage quality.

[0153]

[0154] As shown in the table above, the sausages fermented with the direct-inoculation starter culture prepared by the compound nucleotide culture medium of this invention and the compound freeze-drying protectant of this invention achieved the highest score in sensory evaluation. Furthermore, the lactic acid bacteria count shows that, due to the synergistic effect of this culture medium and the freeze-drying protectant, the lactic acid bacteria in the fermented sausages exhibited good growth, higher viable counts than other groups, better activity, faster acid production, and more rapid and abundant production of flavor substances. The fermented sausages thus possessed excellent flavor and quality.

[0155] In summary, based on the above culture results, the optimized culture medium, nucleotides, and compound freeze-drying protectant in this invention synergistically enhance the bacterial cell count, increase the cell's stress resistance, reduce damage to the cell wall and cell membrane of *Lactobacillus plantarum* YR07, increase its LDH and ATPase activities, improve the bacteria's sugar metabolism capacity, and reduce damage to the secondary structure of proteins after freeze-drying. Furthermore, increasing the relative content of long-chain unsaturated fatty acids in the *Lactobacillus plantarum* YR07 cell membrane improves the cell membrane compactness, making the cell membrane structure more stable and enabling the strain to better cope with environmental stress and stress damage during the freeze-drying stage.

[0156] All aspects, embodiments, features, and examples of this invention are to be regarded as illustrative in all respects and are not intended to limit the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

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

[0158] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated otherwise, any use of the terms first, second, etc., does not indicate any order or importance, but is used to distinguish one element from another.

Claims

1. A method for preparing a direct-inoculation lactic acid bacteria starter culture, characterized in that, include: The activated lactic acid bacteria strain was inoculated into a nucleotide medium for expansion culture to obtain a high-density lactic acid bacteria inoculum. The lactic acid bacteria was Lactobacillus plantarum YR07, and its biological preservation number was CCTCC NO: M20221303. After centrifuging the high-density lactic acid bacteria inoculum, the bacterial cells were mixed with a composite freeze-drying protectant and then freeze-dried to obtain a direct-inoculation lactic acid bacteria starter. The nucleotide culture medium is composed of 0.5 g / L nucleotides, 2-10 g / L beef extract, 25-35 g / L soybean 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 are composed of guanine, adenine, xanthine, and uracil in a ratio of 1:1:1:

1. The composite freeze-drying protectant comprises: 5-20 wt% sugar, 10-30 wt% skim milk powder, 8-25 wt% proline, and the remainder being distilled water. The sugar is one or a combination of glucose, trehalose, sorbitol, or sucrose. The preparation method includes: mixing and grinding the sugar, proline, and distilled water for 5-15 min, stirring at 40-100℃ and 50-300 rpm for 8-12 h to form a uniform, viscous, and transparent liquid eutectic solvent, and then mixing it with the skim milk powder solution to obtain the composite freeze-drying protectant.

2. The preparation method according to claim 1, characterized in that, Specifically, it includes: The activated lactic acid bacteria strains were inoculated into the nucleotide culture medium at a volume concentration of 1-5%, and cultured statically at 25-37°C for 12-18 hours. The high-density lactobacillus agent obtained by centrifugation was washed with sterile physiological saline and then freeze-dried.

3. The preparation method according to claim 1, characterized in that, The freeze-drying process specifically includes: centrifuging the high-density lactic acid bacteria inoculum, taking the bacterial cells, and adding an equal volume of composite freeze-drying protectant to form a mixture. The mixture is then pre-frozen at -50℃ for 2-10 hours, and then gradually heated to -40℃ for 1-5 hours, -30℃ for 1-5 hours, -20℃ for 1-4 hours, -10℃ for 1-3 hours, 0℃ for 0-3 hours, and 10℃ for 5-20 hours to obtain the direct-inoculation lactic acid bacteria starter.

4. A method for producing fermented sausage, characterized in that, include: Lactic acid bacteria direct-inoculation starter culture was prepared by any one of claims 1-3; The lactic acid bacteria direct-inoculation starter is mixed evenly with minced pork at a concentration of 1-5 wt%, and fermented for 12-48 hours at 10-40°C and 30-90% humidity, and then air-dried to obtain sausage.

5. The production method according to claim 4, characterized in that: The minced pork also contains auxiliary ingredients, including salt, glucose, nitrite, garlic powder, fennel powder, black pepper powder, and sausage flavoring.

Images