Pediococcus acidilactici rm1 lyophilized powder, method of preparation and use

Abstract

The present application relates to the technical field of microbial food fermentation, and specifically discloses a Pediococcus acidilactici RM1 freeze-dried powder, a preparation method and an application, the Pediococcus acidilactici RM1 freeze-dried powder comprises the following components in parts by weight: 0.5-3 parts of Pediococcus acidilactici RM1, 2-3 parts of skimmed milk powder, 1-2 parts of glycerol, 15-25 parts of trehalose and 25-35 parts of sucrose; the preservation number of the Pediococcus acidilactici RM1 is GDMCC NO.66765; the preservation unit of the Pediococcus acidilactici RM1 is Guangdong Microbial Culture Collection Center; the preservation name of the Pediococcus acidilactici RM1 is Pediococcus acidilactici RM1; and the preservation time of the Pediococcus acidilactici RM1 is July 28, 2025. The present application has the characteristics of bright red color, less deterioration during storage and ideal quality and flavor.

Description

Technical Field

[0001] This invention relates to the field of microbial food fermentation technology, and in particular to a freeze-dried powder of a strain of Pediococcus lactis RM1, its preparation method and application. Background Technology

[0002] In China, due to its large population and dietary preferences, there is a significant demand for cured meat products. Dry-cured sausages are a typical example of cured meat products, favored by consumers for their unique flavor. Dry-cured sausages are made by mincing pork (or beef, mutton, etc.), mixing it with animal fat, salt, sugar, and spices, stuffing it into casings, and then drying it. Over its long development, it has evolved into various categories with regional flavors, such as Sichuan sausages, Guangdong sausages, and Zhejiang sausages. Traditional Chinese dry-cured sausages are typically produced through natural fermentation. Their quality and flavor depend primarily on the endogenous enzyme activity of the raw materials and the metabolic activity of beneficial microorganisms in the environment. The maturation process usually takes a long time, making it difficult to guarantee product safety and homogeneity. With industrialization, the sausage production cycle has been significantly shortened, but the resulting lack of flavor has become particularly prominent. Ensuring the acceptable flavor of dry-cured sausages under industrial production conditions is becoming increasingly important. In recent years, the use of fermenting agents has become a feasible and promising solution for the industrial production of cured meat products. A good fermenting agent is one of the main factors in ensuring the flavor of cured meat products. Microbial starter cultures have been widely used in the production of cured meat products worldwide. Practice has shown that using appropriate starter cultures can enable producers to effectively manage the fermentation process, meet industry standards, and improve the quality and safety of sausages during production.

[0003] Lactic acid bacteria (LAB) are the dominant bacterial group in dry-cured sausages and an important component of meat product starter cultures, playing a crucial role in sausage fermentation. In Europe, *Lactobacillus sakei* is the primary starter culture, while in the United States, *Pediococcus acidilactici* is the primary starter culture. These have been successfully developed as commercial starter cultures for sausage production. Studies have shown that adding potential fermentation strains such as *Lactobacillus* and *Pediococcus* to meat products can not only inhibit the growth of spoilage bacteria and stabilize shelf life, but also promote the formation of flavor and sensory quality in fermented meat products. Some studies also suggest that meat products fermented with the potential probiotics *Lactobacillus* and *Pediococcus* can be considered functional foods, offering health benefits after consumption. The preparation of high-activity direct-inoculation lactic acid bacteria starter cultures has become a research hotspot both domestically and internationally. Currently, the research and development of lactic acid bacteria starter cultures for meat products in my country is still in its initial stage, and there is a lack of commercial starter cultures adapted to local conditions. Furthermore, the corresponding fermentation processes and research and development of fermented meat products are still relatively weak.

[0004] Pediococcus acidilactici is a Gram-positive facultative anaerobic bacterium with antibacterial and probiotic properties. It is an important fermentation agent for meat products and has been included in my country's "List of Edible Fungi". Studies have shown that Pediococcus acidilactici has a strong acid-producing capacity, which can rapidly lower the pH of meat, facilitating the decomposition of NO2 into NO. NO then combines with myoglobin to form nitrosomyoglobin, giving meat products a bright red color. Pediococcus acidilactici can significantly inhibit the growth of Listeria monocytogenes, Clostridium perfringens, and Enterobacteriaceae in fermented sausages by producing bacteriocins, thus acting as a natural biological preservative. Furthermore, Pediococcus acidilactici also has strong antioxidant properties, significantly inhibiting lipid peroxidation in meat products and improving the sensory quality of fermented meat products during storage. Moreover, potential fermentation strains of Pediococcus acidilactici can also improve the flavor of fermented meat products, promote the production of free amino acids, reduce nitrite residues, and enhance the quality of fermented meat products. Currently, fermentation strains of Pediococcus lactis have been successfully applied to the production of Western-style fermented ham, sausages and other products. In my country, the uses of Pediococcus lactis are mainly concentrated on its probiotic properties and health effects, dairy product fermentation, animal health and feed additives, and the production of bacteriocins and extracellular polysaccharides. Research and application of Pediococcus lactis in meat products are still relatively limited.

[0005] Fermentation agents are a crucial factor influencing the quality of cured / fermented meat products. Currently, my country relies heavily on imported fermentation agents for meat products, while research and exploration into fermentation processes and the formation of meat quality are relatively weak. Studies have shown that imported commercial fermentation agents cannot always maintain an advantage in competition with local Chinese microbial populations, which may lead to the loss of ideal sensory characteristics in meat products. Furthermore, the long-term and widespread use of a single commercial fermentation agent can cause fermented meat products to become homogenized in flavor, lacking product distinctiveness. Therefore, screening fermentation agents with excellent fermentation performance from local microbial populations not only allows for better adaptation to the local meat processing environment but also makes them more competitive due to their unique metabolic capabilities, contributing to the creation of distinctive meat products with different flavors. The development of lactic acid bacteria fermentation agents and related fermentation processes adapted to the local processing environment can significantly enhance the added value of fermented meat products and promote the development of the intensive processing industry of livestock products.

[0006] Therefore, existing cured / fermented meat products have problems such as insufficient bright red color, easy spoilage during storage, and unsatisfactory quality. Summary of the Invention

[0007] In order to solve the above-mentioned technical problems existing in existing cured / fermented meat products, this invention provides a freeze-dried powder of Pediococcus lactis RM1, its preparation method and application, which has the characteristics of bright red color, not easy to deteriorate during storage and ideal quality and flavor.

[0008] The first technical solution of the present invention is: Pediococcus acidilactici RM1, wherein the preservation number of Pediococcus acidilactici RM1 is GDMCCNO.66765; the preservation unit of Pediococcus acidilactici RM1 is Guangdong Provincial Center for Microbial Culture Collection; the preservation name of Pediococcus acidilactici RM1 is Pediococcus acidilactici RM1; and the preservation date of Pediococcus acidilactici RM1 is July 28, 2025.

[0009] This invention utilizes an independently developed strain with strong metabolic activity, making it more adaptable to local processing environments and suitable for large-scale fermentation processes to develop high-quality dry-cured sausage products. This invention can be used as a fermenting agent in the fermentation process of meat products. During fermentation, the lactic acid bacteria produce lactic acid through metabolism, lowering the pH value and inhibiting the growth of harmful microorganisms. Simultaneously, it produces metabolites such as bacteriocins and catalase, further acting as a biological preservative. It also influences the state of myoglobin and the lipid oxidation process in meat products, ensuring color and flavor. Compared to other known strains or traditional processes, it produces superior overall effects in the same application scenario. This invention has high protease activity, significantly degrading milk protein and hydrolyzing proteins, promoting easy absorption by the human body while also enhancing flavor. It possesses good antioxidant capacity, producing lactic acid through fermentation, exhibiting suitable acid-producing ability. While lowering the pH, it also inhibits bacterial growth, significantly reducing the pH value of sausages, improving sausage color, increasing product redness, reducing nitrite residue, promoting rapid formation of sausage texture, promoting the flavor and free amino acid generation of fermented sausages, reducing the degree of lipid oxidation (TBARS value), and improving sausage safety.

[0010] Preferably, the nucleotide sequence of the *Pediococcus lactis* RM1 is shown in SEQ ID NO. 1.

[0011] The second technical solution of the present invention: a method for isolating Pediococcus lactis RM1, comprising the following steps,

[0012] (A01) Place the sausage in sterile water and shake.

[0013] (A02) Perform a gradient dilution of the shaking solution from step (A01);

[0014] (A03) Take the gradient dilutions from step (A02) and spread them onto MRS medium, then incubate them statically until single colonies grow.

[0015] (A04) Single colonies with different morphologies from step (A03) were picked and streaked onto MRS medium. After purification, the physicochemical properties of the single colonies with different morphologies were identified, yielding *Pediococcus lactis* RM1. This invention effectively and uniformly elutes microorganisms from sausages into a liquid to prepare an initial bacterial suspension. The mixed bacterial suspension is then dispersed on a solid medium to obtain pickable single colonies. Appropriate dilution ensures the growth of discrete single colonies. The composition of the MRS medium provides optimal nutrition for lactic acid bacteria growth, successfully isolating culturable single colonies of lactic acid bacteria from sausage samples. This invention serves as a sieve-like evaluation system for the targeted screening of target strains possessing multiple ideal characteristics from a large number of isolates. It establishes a highly efficient screening model for rapidly and targetedly screening candidate strains of starter cultures with excellent comprehensive performance from a large number of wild-type strains, directly isolating *Pediococcus lactis* RM1. M1 also provides a repeatable screening blueprint for solving similar technical problems. This invention provides a complete and customized screening index system. In the prior art, there is no identical identification method for screening fermented lactic acid bacteria in meat products that includes all these specific indicators and their specific threshold standards. This invention is not simply about isolating strains, but rather provides a targeted screening solution. By designing a set of multi-level physiological and biochemical screening standards closely related to the final application goal, it significantly improves the efficiency and success rate of obtaining high-performance strains from complex samples, avoiding blind random screening. The technical solution of the isolation method is highly reasonable and efficient in scientific logic.

[0016] Preferably, the sausage used in step (A01) is a sausage produced locally in Hangzhou. Separating from locally produced Hangzhou sausage reveals that cured meat products naturally contain a large number of adaptable lactic acid bacteria, from which indigenous strains more suitable for meat product fermentation can be obtained through screening.

[0017] Preferably, the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 10 to 1 / 8. More preferably, the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 9.

[0018] Preferably, the amount of sausage used in step (A01) is 20g to 30g. More preferably, the amount of sausage used in step (A01) is 25g.

[0019] Preferably, the amount of sterile water used in step (A01) is 200ml to 250ml. More preferably, the amount of sterile water used in step (A01) is 225ml.

[0020] Preferably, the oscillation in step (A01) is performed using a tapping oscillator.

[0021] Preferably, the oscillation time in step (A01) is 10 min to 15 min. More preferably, the oscillation time in step (A01) is 10 min.

[0022] Preferably, the dilution in step (A02) is performed in a clean bench. A suitable feed-to-liquid ratio and oscillation intensity maximize microbial elution efficiency while avoiding excessive cell damage. A beat shaker provides uniform and vigorous oscillation, which facilitates sample disruption and microbial release, resulting in a representative and highly active initial bacterial suspension, laying the foundation for subsequent dilution and separation.

[0023] Preferably, in step (A02), the gradient dilution is performed to 10. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 .

[0024] Preferably, in step (A03), 10 μL of each sample is taken. -4 10 -5 and 10 -6 Dilute the solution multiple times into MRS medium.

[0025] Preferably, the amount taken in step (A03) is 0.1 ml.

[0026] Preferably, the coating in step (A03) is a uniform coating.

[0027] Preferably, the culture in step (A03) is carried out in an incubator.

[0028] Preferably, the culture temperature in step (A03) is 33°C to 37°C. More preferably, the culture temperature in step (A03) is 35°C.

[0029] Preferably, the culture time in step (A03) is 36h to 60h. More preferably, the culture time in step (A03) is 42h to 54h. Even more preferably, the culture time in step (A03) is 48h.

[0030] Preferably, the purification process in step (A04) is repeated 2 to 4 times. More preferably, the purification process in step (A04) is repeated 3 times.

[0031] Preferably, the physicochemical properties in step (A04) include protease activity, antioxidant activity, acid production capacity, temperature tolerance range, salt tolerance range, sodium nitrite tolerance range, colony morphology, and 16S rRNA gene sequence.

[0032] Preferably, the protease activity is identified by preparing a nutrient broth agar medium containing 1.5% to 3% skim milk powder, picking a single colony and inoculating it into the nutrient broth agar medium for anaerobic culture, and identifying the bacteria that can degrade milk protein and form a hydrolysis zone as Pediococcus lactis RM1.

[0033] Preferably, during the protease activity identification process, four single colonies are inoculated on each plate.

[0034] Preferably, the culture temperature during the protease activity identification process is 33°C to 37°C. More preferably, the culture temperature is 35°C.

[0035] Preferably, the culture time for the protease activity identification process is 36h to 60h. More preferably, the culture time is 42h to 54h. Even more preferably, the culture time is 48h.

[0036] Preferably, the antioxidant activity is determined by picking a single colony, inoculating it into sterile MRS broth medium, incubating it statically, centrifuging it, collecting the supernatant, and using a DPPH and hydroxyl radical assay kit to detect the antioxidant capacity of the supernatant. When the OD of the bacterial culture... 600 When the value is 1.079, the bacteria with a hydroxyl radical scavenging capacity of 530.4 U / mL to 585.6 U / mL and a DPPH scavenging rate of 36.618% to 37.982% are Pediococcus lactis RM1.

[0037] Preferably, the culture temperature during the antioxidant activity identification process is 33℃ to 37℃. More preferably, the culture temperature is 35℃.

[0038] Preferably, the culture time during the antioxidant activity identification process is 36h to 60h. More preferably, the culture time is 42h to 54h. Even more preferably, the culture time is 48h.

[0039] Preferably, the acid-producing capacity is determined by picking a single colony and inoculating it into sterile MRS broth medium and allowing it to be cultured statically. The bacteria that cause the pH of the MRS broth medium to drop from 6-6.4 to 4.3-4.32 are identified as Pediococcus lactis RM1.

[0040] Preferably, the culture temperature during the acid production capacity assessment process is 33℃ to 37℃. More preferably, the culture temperature is 35℃.

[0041] Preferably, the culture time in the acid production capacity identification process is 36h to 60h. More preferably, the culture time is 42h to 54h. Even more preferably, the culture time is 48h.

[0042] Preferably, the temperature adaptability range is determined by picking a single colony and inoculating it into sterilized MRS broth medium, incubating it at a temperature range of 0℃ to 70℃ with gradual temperature increases, and measuring the OD of the bacterial solution at the corresponding temperature. 600 The bacteria that can grow stably in the temperature range of 15℃ to 50℃ are called Pediococcus lactis RM1.

[0043] Preferably, during the temperature adaptability range identification process, the incubation time at each temperature is 36h to 60h. More preferably, the incubation time at each temperature is 42h to 54h. Even more preferably, the incubation time at each temperature is 48h.

[0044] Preferably, the determination of the salt tolerance range is performed by picking a single colony and inoculating it into MRS broth medium with a salt content of 0% to 20%. The bacteria that can continue to grow under 12% salt conditions are identified as Pediococcus lactis RM1.

[0045] Preferably, the culture temperature during the salt tolerance range identification process is 33℃~37℃. More preferably, the culture temperature is 35℃.

[0046] Preferably, the determination of the sodium nitrite tolerance range is performed by picking single colonies and inoculating them into MRS broth medium with different sodium nitrite contents, and determining the OD at the same time point under different sodium nitrite contents. 600 Bacteria with no significant difference in values ​​are identified as Pediococcus lactis RM1.

[0047] Preferably, the sodium nitrite content in the sodium nitrite adaptability identification process is 50 mg / kg, 100 mg / kg, and 150 mg / kg.

[0048] Preferably, the culture temperature during the sodium nitrite suitability range identification process is 33℃~37℃. More preferably, the culture temperature is 35℃.

[0049] Preferably, the colony morphology is identified as follows: bacteria with a diameter of 0.19 cm to 0.31 cm, a milky white, round shape, neat edges, and a smooth, moist surface are identified as Pediococcus lactis RM1.

[0050] Preferably, the 16S rRNA gene sequence is identified by picking a single colony, inoculating it in MRS broth medium, centrifuging it to collect the precipitate and extracting the DNA, performing PCR on the 16S rRNA gene and sequencing it. The bacteria with the sequence shown in SEQ ID NO.1 are Pediococcus lactis RM1.

[0051] Preferably, the culture temperature during the 16S rRNA gene sequence identification process is 33℃ to 37℃. More preferably, the culture temperature is 35℃.

[0052] Preferably, the culture time during the 16S rRNA gene sequence identification process is 36h to 60h. More preferably, the culture time is 42h to 54h. Even more preferably, the culture time is 48h. This method decomposes complex final product quality issues into physiological and biochemical indicators that can be quantified and detected in the laboratory with high throughput; it possesses good antioxidant activity, inhibiting lipid and myoglobin oxidation and stabilizing meat product color; it has good acid-producing capacity, good adaptability to salt, temperature, and nitrite, enabling it to survive in harsh environments and dominate fermentation, thus playing a preservative role; it has good protease activity, indirectly affecting flavor and texture; specific quantitative standards ultimately accurately screen for the desired *Pediococcus lactis* RM1; for the first time, these indicators are creatively combined to form a synergistic screening funnel, thereby specifically screening for strains with excellent comprehensive performance like *Pediococcus lactis* RM1.

[0053] The third technical solution of the present invention: Lyophilized Peptococcus RM1, comprising the following components by weight:

[0054] RM10.5-3 parts of Pleurotus ostreatus, 2-3 parts of skim milk powder, 1-2 parts of glycerol, 15-25 parts of trehalose, and 25-35 parts of sucrose.

[0055] Preferably, the product comprises the following components in parts by weight.

[0056] Lactococcus RM11-2.5 parts, skim milk powder 2.2-2.8 parts, glycerol 1.2-1.8 parts, trehalose 17-23 parts, sucrose 27-33 parts.

[0057] Preferably, the product comprises the following components in parts by weight.

[0058] Lactococcus lactis RM11.5–2 parts, skim milk powder 2.4–2.6 parts, glycerol 1.4–1.6 parts, trehalose 19–21 parts, sucrose 29–31 parts.

[0059] Preferably, the product comprises the following components in parts by weight.

[0060] RM11.8 of Pleurotus ostreatus, 2.5 of skim milk powder, 1.5 of glycerin, 20 of trehalose, and 30 of sucrose.

[0061] The active ingredient of this invention, *Pediococcus lactis* RM1, provides core biological functions such as fermentation, preservation, and improvement of color and quality. Its unique genetic traits endow it with excellent acid production, antioxidant, and stress tolerance capabilities. Skim milk powder, as a dispersant and protein protectant, provides a non-reducing protein matrix, encapsulating the bacterial cells during freeze-drying, reducing mechanical damage to the cell membrane from ice crystals, and promoting cell membrane repair during rehydration. Glycerol, as a permeability protectant and a small-molecule polyol, can penetrate into cells, lower the intracellular freezing point, prevent intracellular ice crystal formation, and replace water molecules to maintain the stability of protein and membrane structures. Trehalose and sucrose, as non-permeability disaccharide protectants, form a glassy matrix outside the cell, stabilizing the cell membrane and protein structure, preventing phase transitions and structural collapse during dehydration and rehydration; the two work synergistically. This invention targets the specific strain *Pediococcus lactis* RM1, and to maximize its activity in solving meat product problems after freeze-drying, a large number of experiments were conducted to obtain the optimal balance of the required components. Formulating the formula; optimizing the proportions of each component to achieve the best balance between survival rate, stability, cost, and process feasibility directly affects the fermentation start-up speed of the freeze-dried bacterial powder in meat products; different protectants work synergistically, and the ratio of trehalose to sucrose determines the glass transition temperature and matrix strength; the amount of skim milk powder needs to be sufficient to coat the bacteria, but too much will affect rehydration and dispersion; too low a glycerol concentration will result in insufficient protection, while too high a concentration will lead to toxicity or excessive penetration; the *Pediococcus lactis* RM1 strain, due to its unique cell membrane composition and physiological characteristics, can respond specifically to the type and proportion of protectants; the RM1 freeze-dried powder prepared using this specific formula has extremely high freeze-drying survival rate and long-term storage stability, which directly affects the shelf life and efficacy as a commercial starter culture. It has high activity after rehydration, directly affecting the fermentation start-up speed. The final effect of fermenting meat products with this freeze-dried powder in terms of color, preservation, and quality is significantly better than the same RM1 strain protected with other conventional formulas, and also better than other lactic acid bacteria strains protected with the same formula.

[0062] The fourth technical solution of the present invention: a method for preparing lyophilized Pyrococcus RM1, comprising the following steps:

[0063] (B01) Take a single colony of Pyotrophic Lactococcus RM1 and inoculate it into MRS broth medium for culture;

[0064] (B02) Take the RM1 strain of Pyrococcus lactis cultured in step (B01) and inoculate it again into broth medium at an inoculation rate of 1% for a second activation. Use the activated bacterial solution as the seed solution.

[0065] (B03) Inoculate the seed culture from step (B02) into MRS broth medium at an inoculation rate of 1% for expansion culture, and then centrifuge to remove the supernatant;

[0066] (B04) After removing the supernatant in step (B03), add an appropriate amount of skim milk powder, glycerin, trehalose and sucrose to the bacterial sludge and vortex to dissolve the precipitate;

[0067] (B05) The dissolved substance from step (B04) is first frozen and then freeze-dried to obtain lyophilized Pediococcus lactis RM1 powder. This invention utilizes two activation and expansion cultures to restore the high activity of the strain from its stored state and obtain sufficient bacterial biomass. The first activation adapts the strain to the liquid environment, while the second activation further expands the strain and ensures the seed culture is in the logarithmic growth phase, resulting in the most robust seed culture and a highly active and concentrated seed culture and fermentation broth. The bacterial sludge is collected by centrifugation, and a protectant is added to concentrate the bacterial cells. The bacterial cells are then mixed evenly with the freeze-drying protectant to obtain a homogeneous suspension of bacterial cells and protectant, preparing for freeze-drying. Freezing and freeze-drying, through low-temperature dehydration, induce a dormant state in the bacterial cells, achieving long-term stable preservation. First, ice crystals are formed by freezing, then sublimation and dehydration are performed under vacuum to obtain a solid freeze-dried powder with extremely low water content.

[0068] Preferably, the amount of MRS broth culture medium used in step (B01) is 40 mL to 60 mL. More preferably, the amount of MRS broth culture medium used in step (B01) is 50 mL.

[0069] Preferably, the culture temperature in step (B01) is 33°C to 37°C. More preferably, the culture temperature in step (B01) is 35°C.

[0070] Preferably, the culture time in step (B01) is 36h to 60h. More preferably, the culture time in step (B01) is 42h to 54h. Even more preferably, the culture time in step (B01) is 48h.

[0071] Preferably, the amount of MRS broth culture medium used in step (B02) is 50 mL.

[0072] Preferably, the amount of MRS broth culture medium used in step (B03) is 250 mL.

[0073] Preferably, the expansion culture temperature in step (B03) is 33°C to 37°C. More preferably, the expansion culture temperature in step (B03) is 35°C.

[0074] Preferably, the expansion culture in step (B03) is a static culture or a shaker culture.

[0075] Preferably, the expansion culture time in step (B03) is 36h to 60h. More preferably, the expansion culture time in step (B03) is 42h to 54h. Even more preferably, the expansion culture time in step (B03) is 48h.

[0076] Preferably, the centrifugation temperature in step (B03) is 3°C to 5°C. More preferably, the centrifugation temperature in step (B03) is 4°C.

[0077] Preferably, the freezing temperature in step (B05) is -25°C to -15°C. More preferably, the freezing temperature in step (B05) is -20°C.

[0078] Preferably, the freezing time in step (B05) is 18h to 30h. More preferably, the freezing time in step (B05) is 24h.

[0079] Preferably, the freeze-drying in step (B05) is carried out in a freeze dryer.

[0080] The fifth technical solution of this invention: the application of Pediococcus lactis RM1 freeze-dried powder in cured and preserved meat products or cured / fermented meat products. This invention is based on the unique metabolic characteristics of Pediococcus lactis RM1 strain, such as strong acid production, high antioxidant capacity, and stress tolerance, and its application in meat product fermentation can specifically improve color, preservation, and quality.

[0081] As a preferred option, the application of Pediococcus lactis RM1 freeze-dried powder in cured meat products includes the following steps:

[0082] (C01) Dissolve an appropriate amount of Pediococcus lactis RM1 freeze-dried powder in edible water, then add sausage curing agent and mix to form a suspension;

[0083] (C02) Add the suspension from step (C01) to the minced meat product and stir until the concentration of the *Pediococcus lactis* RM1 inoculum is ≥1.0 × 10⁻⁶. 8 CFU / g;

[0084] (C03) The mixture from step (C02) is stuffed into sausages to form sausage meat products, and the sausage meat products are fermented until the water activity drops below 0.9. This invention uses RM1 freeze-dried powder with a high viable bacterial count, which is rehydrated and inoculated in suspension form to ensure that the concentration of the bacterial agent in the final product is ≥1.0 × 10⁻⁶. 8CFU / g ensures a sufficient number of highly active functional strains colonize the meat matrix to rapidly initiate fermentation, occupy ecological niche advantages, and ensure sufficient initial bacterial count for rapid acid production, inhibiting contaminating bacteria. This creates the most suitable environmental conditions for the fermentation of *Pediococcus lactis* RM1 and defines the product quality endpoint. The lean-to-fat ratio affects flavor, texture, and fat oxidation; RM1's antioxidant activity is most effective in inhibiting rancidity at this specific ratio. Low-temperature fermentation promotes the slow formation of flavor compounds and inhibits harmful bacteria; RM1's growth and metabolic activity at 15°C are its strengths. Low humidity promotes surface drying and dehydration, which is part of the sausage and cured meat process. Aw < 0.9 is the safe water activity threshold for inhibiting the growth of most bacteria. Post-inoculation resting allows the bacterial solution to penetrate and initially adapt to the internal environment of the meat block. The perfect match and synergy between the entire parameter combination and the characteristics of the RM1 strain are precisely what allows this specific process condition to maximize the stimulation and utilization of the unique genetic potential of the RM1 strain. The accompanying sausage curing agent provides a basic flavor and osmotic pressure environment, which helps extract myofibrillar proteins and inhibit contaminating bacteria.

[0085] Preferably, the viable count of the Pediococcus lactis RM1 lyophilized powder in step (C01) is greater than 2.0 × 10⁻⁶. 11 CFU / g.

[0086] Preferably, the mass-to-volume ratio of the lyophilized P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:90-110. More preferably, the mass-to-volume ratio of the lyophilized P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:100.

[0087] Preferably, the sausage curd preparation in step (C01) comprises the following components by weight:

[0088] 1-3 parts NaCl, 1-3 parts sucrose, 4-6 parts purified water.

[0089] Preferably, the sausage curd preparation in step (C01) comprises the following components by weight:

[0090] 2 parts NaCl, 2 parts sucrose, 5 parts purified water.

[0091] Preferably, the weight of the minced meat product in step (CO2) is 20 to 40 times the weight of the suspension. More preferably, the weight of the minced meat product in step (CO2) is 25 to 35 times the weight of the suspension. Even more preferably, the weight of the minced meat product in step (CO2) is 30 times the weight of the suspension.

[0092] Preferably, the mass ratio of fat to lean meat in the minced meat product of step (CO2) is 1:6 to 12. More preferably, the mass ratio of fat to lean meat in the minced meat product of step (CO2) is 1:7 to 11. More preferably, the mass ratio of fat to lean meat in the minced meat product of step (CO2) is 1:8 to 10. More preferably, the mass ratio of fat to lean meat in the minced meat product of step (CO2) is 1:9.

[0093] Preferably, the fermentation temperature in step (CO3) is 12°C to 18°C. More preferably, the fermentation temperature in step (CO3) is 13°C to 17°C. More preferably, the fermentation temperature in step (CO3) is 14°C to 16°C. More preferably, the fermentation temperature in step (CO3) is 15°C.

[0094] Preferably, the fermentation humidity in step (C03) is ≤65%.

[0095] Preferably, the sausage meat products in step (C03) include livestock and poultry sausages and cured sausages.

[0096] As a preferred option, the application of Pediococcus lactis RM1 freeze-dried powder in cured / fermented meat products includes the following steps:

[0097] (D01) Take an appropriate amount of Pediococcus lactis RM1 lyophilized powder, dissolve it in the first drinking water, and then dissolve it in the second drinking water;

[0098] (D02) Inoculate the final solution from step (D01) onto the whole meat product and then carry out hanging fermentation.

[0099] Preferably, the viable count of the Pediococcus lactis RM1 lyophilized powder in step (D01) is greater than 2.0 × 10⁻⁶. 11 CFU / g.

[0100] Preferably, the mass-to-volume ratio of the lyophilized *Pediococcus lactis* RM1 powder to the first edible water in step (D01) is 1:90-110. More preferably, the mass-to-volume ratio of the lyophilized *Pediococcus lactis* RM1 powder to the first edible water in step (D01) is 1:100.

[0101] Preferably, the mass of the second drinking water in step (D01) is 8 to 12 times the mass of the previously dissolved solution. More preferably, the mass of the second drinking water in step (D01) is 9 to 11 times the mass of the previously dissolved solution. Even more preferably, the mass of the second drinking water in step (D01) is 10 times the mass of the previously dissolved solution.

[0102] Preferably, the whole meat product in step (D02) includes salted meat, cured meat, cured chicken, and cured duck.

[0103] Preferably, the inoculation temperature in step (D02) is 3°C to 5°C. More preferably, the inoculation temperature in step (D02) is 4°C.

[0104] Preferably, in step (DO2), inoculation is performed 18-30 hours before hanging fermentation. More preferably, in step (DO2), inoculation is performed 24 hours before hanging fermentation.

[0105] The present invention has the following beneficial effects:

[0106] (1) The strains developed independently have strong metabolic activity and are more adapted to the local processing environment, making them suitable for large-scale fermentation processes and the development of high-quality sausage products.

[0107] (2) It can be used as a fermentation agent and can be used in the fermentation process of meat products. During the fermentation process, lactic acid bacteria produce lactic acid through metabolism, which lowers the pH value and inhibits the growth of harmful microorganisms. At the same time, it can produce metabolites such as bacteriocins and catalase, which further play a role in biological preservation. It can also affect the state of myoglobin and the lipid oxidation process in meat products, ensuring color and flavor. Compared with other known strains or traditional processes, it can produce better comprehensive effects in the same application scenario.

[0108] (3) It has high protease activity, which can significantly degrade milk protein, hydrolyze protein, promote human absorption, and also enhance flavor. It has good antioxidant capacity, produces lactic acid through fermentation, has a suitable acid production capacity, lowers pH and inhibits bacterial growth, can significantly reduce sausage pH, improve sausage color, increase product redness value, reduce nitrite residue, promote rapid formation of sausage texture, promote fermented sausage flavor and free amino acid generation, reduce fat oxidation degree TBARS value, and improve sausage safety.

[0109] (4) The active ingredient, Pediococcus lactis RM1, provides the core biological functions of fermentation, preservation, and improvement of color and quality. Its unique genetic traits endow it with excellent acid production, antioxidant and stress tolerance capabilities. Skim milk powder, as a dispersant and protein protectant, provides a non-reducing protein matrix, which encapsulates the bacteria during freeze-drying, reduces the mechanical damage of ice crystals to the cell membrane, and promotes cell membrane repair during rehydration. Glycerol, as a permeable protectant and a small molecule polyol, can penetrate into the cell, lower the intracellular freezing point, prevent the formation of intracellular ice crystals, and replace water molecules to maintain the stability of protein and membrane structures. Trehalose and sucrose, as non-permeable disaccharide protectants, form a glassy matrix outside the cell, stabilize the cell membrane and protein structure, and prevent phase transition and structural collapse during dehydration and rehydration. The two work synergistically.

[0110] (5) For the specific strain of Pediococcus lactis RM1, in order to maximize its activity in solving meat product problems after freeze-drying, a large number of experiments were conducted to obtain the required golden ratio of each component; optimizing the ratio of each component to achieve the best balance between survival rate, stability and cost, and process feasibility can directly affect the fermentation start-up speed of freeze-dried bacterial powder in meat products; different protective agents work synergistically, and the ratio of trehalose and sucrose determines the glass transition temperature and matrix strength; the amount of skim milk powder should be sufficient to coat the bacterial cells, but too much will affect rehydration and dispersion; too low a glycerol concentration will result in insufficient protection. Excessive concentration can lead to toxicity or excessive penetration. Due to its unique cell membrane composition and physiological characteristics, the RM1 strain of *Pediococcus lactis* can respond specifically to the type and ratio of protectants. The RM1 freeze-dried powder prepared using this specific ratio has extremely high freeze-drying survival rate and long-term storage stability, which can directly affect the shelf life and efficacy as a commercial fermentation agent. It has high activity after rehydration, which directly affects the fermentation start-up speed. The final effect of fermenting meat products with this freeze-dried powder in terms of color, preservation and quality is significantly better than the same RM1 strain protected with other conventional ratios, and also better than other lactic acid bacteria strains protected with the same ratio.

[0111] (6) Based on the unique metabolic characteristics of Pediococcus lactis RM1 strain, such as strong acid production, high antioxidant and stress resistance, its application in meat product fermentation can specifically improve color, preservation and quality. Attached Figure Description

[0112] Figure 1 This is a diagram showing the results of the present invention regarding the protease activity of *Pediococcus lactis* RM1.

[0113] Figure 2 This invention includes a colony morphology diagram of *Pediococcus lactis* RM1; and a method and system for end-to-end verification of cross-system file transfer based on semantic fingerprinting.

[0114] Figure 3 This is the BLAST alignment result of the 16S rRNA gene sequence of Pediococcus lactis RM1 in this invention;

[0115] Figure 4 This is a picture of fermented sausage made with Pediococcus lactis RM1 in the experimental example of the present invention. From left to right, it is the control group, the lactic acid bacteria group, the lactic acid bacteria + sodium nitrite group, and the sodium nitrite group.

[0116] Figure 5 This is a standard curve diagram of the nitrite residue determination process in sausages in the experimental examples of this invention;

[0117] Figure 6 This is a diagram showing the growth of *Pediococcus lactis* RM1 at different temperatures according to the present invention;

[0118] Figure 7This is a diagram showing the growth of *Pediococcus lactis* RM1 under different concentrations of nitrite.

[0119] Figure 8 This is a graph showing the effect of different treatments on the pH of fermented sausage in the experimental examples of this invention. In the graph, A: control; B: nitrite; C: lactic acid bacteria; D: lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0120] Figure 9 This is a graph showing the effect of different treatments on the TBARS value of fermented sausage in the experimental examples of this invention. In the graph: A: control; B: nitrite; C: lactic acid bacteria; D: lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0121] Figure 10 This is a graph showing the effect of different treatments on the residual amount of nitrite in fermented sausages in the experimental examples of this invention;

[0122] Figure 11 This is a fingerprint of the volatile flavor compounds in sausages according to the present invention. Detailed Implementation

[0123] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0124] Pediococcus acidilactici RM1, with accession number GDMCC NO. 66765, is deposited at the Guangdong Provincial Microbial Culture Collection Center. Its accession name is Pediococcus acidilacticiRM1, and its accession date is July 28, 2025. The nucleotide sequence of RM1 is shown in SEQ ID NO. 1. MR1 exhibits high protease activity and antioxidant capacity, with a protease clear zone diameter of 1.05 ± 0.19 cm. Inoculation with P. acidilactici MR1 strain for 48 hours can lower the pH of the culture medium from 5.32 to 4.31. Adding MR1 can significantly reduce the pH of sausages, improve sausage color, reduce nitrite residue, promote rapid texture formation in sausages, enhance the flavor and free amino acid production of fermented sausages, and reduce lipid oxidation. It can be used as a starter culture agent in the production of fermented meat products.

[0125] The method for isolating Pediococcus lactis RM1 includes the following steps:

[0126] (A01) Place the sausage in sterile water and shake; the sausage in step (A01) is a sausage produced locally in Hangzhou; the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 10 to 1 / 8; the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 9; the amount of sausage used in step (A01) is 20g to 30g; the amount of sausage used in step (A01) is 25g; the amount of sterile water used in step (A01) is 200ml to 250ml; the amount of sterile water used in step (A01) is 225ml; the shaking in step (A01) is performed using a tapping shaker; the shaking time in step (A01) is 10min to 15min; the shaking time in step (A01) is 10min.

[0127] (A02) The shaking solution in step (A01) is serially diluted; the dilution in step (A02) is performed in a clean bench; the serial dilution in step (A02) is carried out to 10. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 ;

[0128] (A03) Take the serially diluted solutions from step (A02) and spread them onto MRS medium, then incubate statically until single colonies appear; in step (A03), take 10... -4 10 -5 and 10 -6 Dilute the buffer to MRS medium several times; the volume taken in step (A03) is 0.1 ml; the spreading in step (A03) is uniform; the incubation in step (A03) is carried out in an incubator; the incubation temperature in step (A03) is 33℃~37℃; the incubation temperature in step (A03) is 35℃; the incubation time in step (A03) is 36h~60h; the incubation time in step (A03) is 42h~54h; the incubation time in step (A03) is 48h.

[0129] (A04) Select single colonies with different colony morphologies from step (A03) and streak them onto MRS medium. After purification, identify the physicochemical characteristics of the single colonies with different morphologies to obtain Pediococcus lactis RM1. The number of purification times in step (A04) is 2 to 4 times. The number of purification times in step (A04) is 3 times. The physicochemical characteristics in step (A04) include protease activity, antioxidant activity, acid production capacity, temperature tolerance range, salt tolerance range, sodium nitrite tolerance range, colony morphology, and 16S rRNA gene sequence.

[0130] The protease activity was determined by preparing a nutrient broth agar medium containing 1.5%–3% skim milk powder, picking a single colony and inoculating it into the nutrient broth agar medium for anaerobic culture. The bacteria capable of degrading milk protein and forming a hydrolysis zone were identified as *Pediococcus lactis* RM1. During the protease activity determination process, four single colonies were inoculated onto each plate. The culture temperature for the protease activity determination process was 33℃–37℃; the culture temperature was 35℃; the culture time for the protease activity determination process was 36h–60h; the culture time for the protease activity determination process was 42h–54h; and the culture time for the protease activity determination process was 48h.

[0131] The antioxidant activity was determined by inoculating a single colony into sterile MRS broth medium, incubating it statically, centrifuging it, and collecting the supernatant. The antioxidant capacity of the supernatant was detected using a DPPH and hydroxyl radical assay kit. When the OD value of the bacterial culture was... 600 When the value is 1.079, the bacteria with a hydroxyl radical scavenging capacity of 530.4 U / mL to 585.6 U / mL and a DPPH scavenging rate of 36.618% to 37.982% are identified as Pediococcus lactis RM1. The culture temperature for the antioxidant activity identification process is 33℃ to 37℃; the culture temperature is 35℃; the culture time for the antioxidant activity identification process is 36h to 60h; the culture time is 42h to 54h; and the culture time is 48h.

[0132] The acid-producing capacity was determined by inoculating a single colony into sterile MRS broth medium and incubating it statically. The bacteria whose pH decreased from 6–6.4 to 4.3–4.32 were identified as *Pediococcus lactis* RM1. The incubation temperatures for the acid-producing capacity determination were 33℃–37℃; the incubation temperatures were 35℃; the incubation times were 36h–60h; the incubation times were 42h–54h; and the incubation times were 48h.

[0133] The temperature adaptability range was determined by inoculating a single colony into sterile MRS broth medium and incubating it at a gradually increasing temperature range of 0℃ to 70℃, while measuring the OD of the bacterial culture at each temperature. 600 The bacteria that can stably grow in the temperature range of 15℃ to 50℃ are identified as Pediococcus lactis RM1. During the temperature adaptability identification process, the incubation time at each temperature was 36h to 60h; the incubation time at each temperature was 42h to 54h; and the incubation time at each temperature was 48h.

[0134] The determination of salt tolerance range was carried out by picking a single colony and inoculating it into MRS broth medium with a salt content of 0% to 20%. The bacteria that could continue to grow under 12% salt conditions were identified as Pediococcus lactis RM1. The culture temperature during the salt tolerance range determination process was 33℃ to 37℃. The culture temperature was 35℃.

[0135] The suitability range of sodium nitrite was determined by inoculating single colonies into MRS broth media with different sodium nitrite concentrations and culturing them. The OD values ​​at the same time point were compared under different sodium nitrite concentrations. 600 The bacteria with no significant difference in values ​​are identified as Pediococcus lactis RM1; the different sodium nitrite contents in the sodium nitrite tolerance identification process are 50 mg / kg, 100 mg / kg and 150 mg / kg; the culture temperature in the sodium nitrite tolerance identification process is 33℃~37℃; the culture temperature is 35℃;

[0136] The colony morphology is identified as follows: bacteria with a diameter of 0.19 cm to 0.31 cm, a milky white and round appearance, neat edges, and a smooth and moist surface are identified as Pediococcus lactis RM1.

[0137] The 16S rRNA gene sequence was identified by picking a single colony, inoculating it into MRS broth medium, centrifuging to collect the precipitate, extracting DNA, performing PCR on the 16S rRNA gene, and sequencing. The bacterial strain with the sequence shown in SEQ ID NO. 1 is Pediococcus lactis RM1. The culture temperature during the 16S rRNA gene sequence identification process was 33℃~37℃; the culture temperature was 35℃; the culture time was 36h~60h; the culture time was 42h~54h; and the culture time was 48h.

[0138] The lyophilized powder of Pediococcus lactis RM1 contains the following components by weight: 0.5 to 3 parts of Pediococcus lactis RM1, 2 to 3 parts of skim milk powder, 1 to 2 parts of glycerol, 15 to 25 parts of trehalose, and 25 to 35 parts of sucrose.

[0139] The lyophilized powder of Pediococcus lactis RM1 contains the following components by weight: 1-2.5 parts of Pediococcus lactis RM1, 2.2-2.8 parts of skim milk powder, 1.2-1.8 parts of glycerol, 17-23 parts of trehalose, and 27-33 parts of sucrose.

[0140] The lyophilized powder of Pediococcus lactis RM1 is characterized by comprising the following components by weight: 1.5 to 2 parts of Pediococcus lactis RM1, 2.4 to 2.6 parts of skim milk powder, 1.4 to 1.6 parts of glycerol, 19 to 21 parts of trehalose, and 29 to 31 parts of sucrose.

[0141] The lyophilized powder of Pediococcus lactis RM1 contains the following components by weight: 1.8 parts Pediococcus lactis RM1, 2.5 parts skim milk powder, 1.5 parts glycerin, 20 parts trehalose, and 30 parts sucrose.

[0142] The preparation method of Pediococcus lactis RM1 lyophilized powder includes the following steps:

[0143] (B01) Inoculate a single colony of *Pediococcus lactis* RM1 into MRS broth medium and culture it; the volume of MRS broth medium used in step (B01) is 40 mL to 60 mL; the volume of MRS broth medium used in step (B01) is 50 mL; the culture temperature in step (B01) is 33℃ to 37℃; the culture temperature in step (B01) is 35℃; the culture time in step (B01) is 36 h to 60 h; the culture time in step (B01) is 42 h to 54 h; the culture time in step (B01) is 48 h.

[0144] (B02) Take the RM1 strain of Pyrococcus lactis cultured in step (B01) and inoculate it again into broth medium at an inoculation rate of 1% for a second activation. Use the activated bacterial solution as the seed culture. The amount of MRS broth medium used in step (B02) is 50 mL.

[0145] (B03) Inoculate the seed culture from step (B02) into MRS broth medium at a 1% inoculum for expansion culture, then centrifuge and remove the supernatant; the volume of MRS broth medium used in step (B03) is 250 mL; the expansion culture temperature in step (B03) is 33℃~37℃; the expansion culture temperature in step (B03) is 35℃; the expansion culture in step (B03) is either static culture or shaking culture; the expansion culture time in step (B03) is 36h~60h; the expansion culture time in step (B03) is 42h~54h; the expansion culture time in step (B03) is 48h; the centrifugation temperature in step (B03) is 3℃~5℃; the centrifugation temperature in step (B03) is 4℃;

[0146] (B04) After removing the supernatant in step (B03), add an appropriate amount of skim milk powder, glycerin, trehalose and sucrose to the bacterial sludge and vortex to dissolve the precipitate;

[0147] (B05) Freeze and then freeze the dissolved substance from step (B04) to obtain lyophilized Pediococcus lactis RM1 lyophilized powder; the freezing temperature in step (B05) is -25℃ to -15℃; the freezing temperature in step (B05) is -20℃; the freezing time in step (B05) is 18h to 30h; the freezing time in step (B05) is 24h; the freeze-drying in step (B05) is carried out in a freeze dryer.

[0148] The application of Pediococcus lactis RM1 freeze-dried powder in cured and bacon minced meat products includes the following steps.

[0149] (C01) Dissolve an appropriate amount of Pediococcus lactis RM1 freeze-dried powder in edible water, then add sausage curing agent and mix to form a suspension; in step (C01), the viable count of Pediococcus lactis RM1 freeze-dried powder is greater than 2.0 × 10⁻⁶. 11CFU / g; The mass-to-volume ratio of lyophilized P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:90-110; The mass-to-volume ratio of lyophilized P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:100; The sausage curing agent in step (C01) includes the following components by weight: 1-3 parts NaCl, 1-3 parts sucrose, and 4-6 parts purified water; The sausage curing agent in step (C01) includes the following components by weight: 2 parts NaCl, 2 parts sucrose, and 5 parts purified water.

[0150] (C02) Add the suspension from step (C01) to the minced meat product and stir until the concentration of the *Pediococcus lactis* RM1 inoculum is ≥1.0 × 10⁻⁶. 8 CFU / g; The weight of the minced meat product in step (CO2) is 20 to 40 times the weight of the suspension; The weight of the minced meat product in step (CO2) is 25 to 35 times the weight of the suspension; The weight of the minced meat product in step (CO2) is 30 times the weight of the suspension; The mass ratio of fat to lean meat in the minced meat product in step (CO2) is 1:6 to 12; The mass ratio of fat to lean meat in the minced meat product in step (CO2) is 1:7 to 11; The mass ratio of fat to lean meat in the minced meat product in step (CO2) is 1:8 to 10; The mass ratio of fat to lean meat in the minced meat product in step (CO2) is 1:9;

[0151] (C03) Stuff the mixture after stirring in step (C02) into sausages to form sausage meat products, and ferment the sausage meat products until the water activity drops below 0.9; the fermentation temperature in step (C03) is 12℃~18℃; the fermentation temperature in step (C03) is 13℃~17℃; the fermentation temperature in step (C03) is 14℃~16℃; the fermentation temperature in step (C03) is 15℃; the fermentation humidity in step (C03) is ≤65%; the sausage meat products in step (C03) include livestock and poultry sausages and cured sausages.

[0152] The application of Pediococcus lactis RM1 freeze-dried powder in cured / fermented meat products includes the following steps.

[0153] (D01) Dissolve an appropriate amount of Pediococcus lactis RM1 lyophilized powder in the first edible water, and then dissolve it in the second edible water; the viable count in the Pediococcus lactis RM1 lyophilized powder in step (D01) is greater than 2.0 × 10⁻⁶. 11CFU / g; In step (D01), the mass-to-volume ratio of *Pediococcus lactis* RM1 lyophilized powder to the first edible water is 1:90-110; In step (D01), the mass-to-volume ratio of *Pediococcus lactis* RM1 lyophilized powder to the first edible water is 1:100; In step (D01), the mass of the second edible water is 8-12 times the mass of the previous solution; In step (D01), the mass of the second edible water is 9-11 times the mass of the previous solution; In step (D01), the mass of the second edible water is 10 times the mass of the previous solution.

[0154] (D02) The final solution from step (D01) is inoculated onto the whole meat product and then hung for fermentation; the whole meat product in step (D02) includes salted meat, cured meat, cured chicken and cured duck; the inoculation temperature in step (D02) is 3℃~5℃; the inoculation temperature in step (D02) is 4℃; hang for fermentation 18h~30h after inoculation in step (D02); hang for fermentation 24h after inoculation in step (D02).

[0155] Example 1: A method for isolating Pediococcus lactis RM1, comprising the following steps:

[0156] (A01) Place the sausage in sterile water and shake it; the sausage in step (A01) is a sausage produced locally in Hangzhou; the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 10; the shaking in step (A01) is performed using a tapping shaker; the shaking time in step (A01) is 10 minutes.

[0157] (A02) The shaking solution in step (A01) is serially diluted; the dilution in step (A02) is performed in a clean bench; the serial dilution in step (A02) is carried out to 10. -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 ;

[0158] (A03) Take the serially diluted solutions from step (A02) and spread them onto MRS medium, then incubate statically until single colonies appear; in step (A03), take 10... -4 10 -5 and 10 -6 Dilute the solution to MRS medium multiple times; the volume taken in step (A03) is 0.1 ml; the spreading in step (A03) is uniform spreading; the incubation in step (A03) is carried out in an incubator; the incubation temperature in step (A03) is 33℃; the incubation time in step (A03) is 60 h;

[0159] (A04) Single colonies with different morphologies from step (A03) were streaked onto MRS medium and purified. The physicochemical properties of the different morphologies of the single colonies were then identified to obtain *Pediococcus acidilactici* RM1. The purification process in step (A04) involved two purification cycles. The physicochemical properties in step (A04) included protease activity, antioxidant activity, acid production capacity, temperature tolerance range, salt tolerance range, sodium nitrite tolerance range, colony morphology, and 16S rRNA gene sequence. The accession number for *Pediococcus acidilactici* RM1 is GDMCC NO. 66765. The depository institution for *Pediococcus acidilactici* RM1 is Guangdong Provincial Microbial Culture Collection Center. The accession name for *Pediococcus acidilactici* RM1 is Pediococcus acidilactici RM1. The accession date for *Pediococcus acidilactici* RM1 is July 28, 2025. The nucleotide sequence of *Pediococcus acidilactici* RM1 is shown in SEQ ID NO. 1.

[0160] The protease activity was determined by preparing a nutrient broth agar medium containing 1.5% skim milk powder, picking a single colony and inoculating it into the nutrient broth agar medium for anaerobic culture. The bacteria that could degrade milk protein and form a hydrolysis zone were identified as Pediococcus lactis RM1. During the protease activity determination process, four single colonies were inoculated onto each plate. The culture temperature during the protease activity determination process was 33℃, and the culture time was 60 hours.

[0161] The antioxidant activity was determined by inoculating a single colony into sterile MRS broth medium, incubating it statically, centrifuging it, and collecting the supernatant. The antioxidant capacity of the supernatant was detected using a DPPH and hydroxyl radical assay kit. When the OD value of the bacterial culture was... 600 When the value is 1.079, the bacteria with a hydroxyl radical scavenging capacity of 530.4 U / mL to 585.6 U / mL and a DPPH scavenging rate of 36.618% to 37.982% are identified as Pediococcus lactis RM1; the culture temperature during the antioxidant activity identification process is 33℃; the culture time during the antioxidant activity identification process is 60 h.

[0162] The acid-producing capacity was determined by picking a single colony and inoculating it into sterile MRS broth medium and incubating it statically. The bacteria that caused the pH of the MRS broth medium to drop from 6-6.4 to 4.3-4.32 were identified as Pediococcus lactis RM1. The culture temperature during the acid-producing capacity determination process was 33℃, and the culture time was 60h.

[0163] The temperature adaptability range was determined by inoculating a single colony into sterile MRS broth medium and incubating it at a gradually increasing temperature range of 0℃ to 70℃, while measuring the OD of the bacterial culture at each temperature. 600The bacteria that can grow stably in the temperature range of 15℃ to 50℃ are identified as Pediococcus lactis RM1; during the temperature adaptability identification process, the incubation time at each temperature was 60h.

[0164] The determination of salt tolerance range was carried out by picking a single colony and inoculating it into MRS broth medium with a salt content of 0% to 20%. The bacteria that could continue to grow under 12% salt conditions were identified as Pediococcus lactis RM1. The culture temperature during the salt tolerance range determination process was 33℃.

[0165] The suitability range of sodium nitrite was determined by inoculating single colonies into MRS broth media with different sodium nitrite concentrations and culturing them. The OD values ​​at the same time point were compared under different sodium nitrite concentrations. 600 The bacteria with no significant difference in values ​​are identified as Pediococcus lactis RM1; the different sodium nitrite contents used in the sodium nitrite tolerance identification process were 50 mg / kg, 100 mg / kg, and 150 mg / kg; the culture temperature used in the sodium nitrite tolerance identification process was 35℃;

[0166] The colony morphology is identified as follows: bacteria with a diameter of 0.19 cm to 0.31 cm, a milky white and round appearance, neat edges, and a smooth and moist surface are identified as Pediococcus lactis RM1.

[0167] The 16S rRNA gene sequence was identified by picking a single colony, inoculating it into MRS broth medium, centrifuging it, extracting the precipitate and DNA, performing PCR on the 16S rRNA gene and sequencing it. The bacterial strain with the sequence shown in SEQ ID NO.1 is Pediococcus lactis RM1. The culture temperature during the 16S rRNA gene sequence identification process was 33℃, and the culture time was 60h.

[0168] Example 2: This example is basically the same as Example 1, except that the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 8; the shaking time in step (A01) is 15 min; the culture temperature in step (A03) is 37℃; the culture time in step (A03) is 36 h; the purification times in step (A04) are 4; the protease activity is identified by preparing a nutrient broth agar medium containing 3% skim milk powder, picking a single colony and inoculating it into the nutrient broth agar medium for anaerobic culture, and identifying the bacteria that can degrade milk protein and form hydrolysis zones as Pediococcus lactis RM1; the culture temperature during the protease activity identification process is 37℃. The culture temperature for each of the following conditions was ℃; the culture time for protease activity identification was 36 h; the culture temperature for antioxidant activity identification was 37℃; the culture time for antioxidant activity identification was 36 h; the culture temperature for acid production capacity identification was 37℃; the culture time for acid production capacity identification was 36 h; the culture time for each temperature range identification was 36 h; the culture temperature for salt tolerance range identification was 37℃; the culture temperature for sodium nitrite tolerance range identification was 37℃; the culture temperature for 16S rRNA gene sequence identification was 37℃; the culture time for 16S rRNA gene sequence identification was 36 h.

[0169] Example 3: This example is basically the same as Example 1, except that the mass-to-volume ratio of sausage to sterile water in step (A01) is 1 / 9; the shaking time in step (A01) is 10 min; the culture temperature in step (A03) is 35℃; the culture time in step (A03) is 48 h; the purification times in step (A04) are 3 times; the protease activity is identified by preparing a nutrient broth agar medium containing 2.5% skim milk powder, picking a single colony and inoculating it into the nutrient broth agar medium for anaerobic culture, and identifying the bacteria that can degrade milk protein and form a hydrolysis zone as Pediococcus lactis RM1; the culture temperature during the protease activity identification process is 3℃. The culture temperature for each of the following conditions was 5℃; the culture time for protease activity identification was 48h; the culture temperature for antioxidant activity identification was 35℃; the culture time for antioxidant activity identification was 48h; the culture temperature for acid production capacity identification was 35℃; the culture time for acid production capacity identification was 48h; the culture time for each temperature range identification was 48h; the culture temperature for salt tolerance range identification was 35℃; the culture temperature for sodium nitrite tolerance range identification was 35℃; the culture temperature for 16S rRNA gene sequence identification was 35℃; the culture time for 16S rRNA gene sequence identification was 48h.

[0170] Example 4: Lyophilized Powder of Pediococcus lactis RM1, comprising the following components by weight: 0.5 parts of Pediococcus lactis RM1, 2 parts of skim milk powder, glycerol, 15 parts of trehalose, and 25 parts of sucrose.

[0171] The preparation method of Pediococcus lactis RM1 lyophilized powder includes the following steps:

[0172] (B01) Take a single colony of Pyrococcus RM1 and inoculate it into MRS broth medium for culture; the amount of MRS broth medium used in step (B01) is 40 mL; the culture temperature in step (B01) is 33℃; the culture time in step (B01) is 60 h.

[0173] (B02) Take the RM1 strain of Pyrococcus lactis cultured in step (B01) and inoculate it again into broth medium at an inoculation rate of 1% for a second activation. Use the activated bacterial solution as the seed culture. The amount of MRS broth medium used in step (B02) is 50 mL.

[0174] (B03) The seed culture from step (B02) was inoculated into MRS broth medium at an inoculum rate of 1% for expansion culture, followed by centrifugation to remove the supernatant; the volume of MRS broth medium used in step (B03) was 250 mL; the expansion culture temperature in step (B03) was 33℃; the expansion culture in step (B03) was a static culture; the expansion culture time in step (B03) was 60 h; the centrifugation temperature in step (B03) was 3℃.

[0175] (B04) After removing the supernatant in step (B03), add an appropriate amount of skim milk powder, glycerin, trehalose and sucrose to the bacterial sludge and vortex to dissolve the precipitate;

[0176] (B05) The solution from step (B04) is first frozen and then freeze-dried to obtain pyrococcus RM1 freeze-dried powder; the freezing temperature in step (B05) is -25℃; the freezing time in step (B05) is 18h; the freeze-drying in step (B05) is carried out in a freeze dryer.

[0177] Example 5: This example is basically the same as Example 4, except that the lyophilized powder of Pediococcus lactis RM1 includes the following components by weight: 3 parts Pediococcus lactis RM1, 3 parts skim milk powder, 2 parts glycerol, 25 parts trehalose, and 35 parts sucrose.

[0178] In step (B01), the amount of MRS broth medium used is 60 mL; the culture temperature in step (B01) is 37℃; the culture time in step (B01) is 36 h; the expansion culture temperature in step (B03) is 37℃; the expansion culture in step (B03) is shaker culture; the expansion culture time in step (B03) is 36 h; the centrifugation temperature in step (B03) is 5℃; the freezing temperature in step (B05) is -15℃; the freezing time in step (B05) is 30 h.

[0179] Example 6: This example is basically the same as Example 4, except that the lyophilized Pediococcus lactis RM1 powder, by weight, includes the following components: 1.8 parts Pediococcus lactis RM1, 2.5 parts skim milk powder, 1.5 parts glycerol, 20 parts trehalose, and 30 parts sucrose. The amount of MRS broth culture medium used in step (B01) is 50 mL; the culture temperature in step (B01) is 35℃; the culture time in step (B01) is 48 h; the expansion culture temperature in step (B03) is 35℃; the expansion culture time in step (B03) is 48 h; the centrifugation temperature in step (B03) is 4℃; the freezing temperature in step (B05) is -20℃; and the freezing time in step (B05) is 24 h.

[0180] Example 7: Application of Pediococcus lactis RM1 freeze-dried powder in cured meat products, including the following steps:

[0181] (C01) Dissolve an appropriate amount of Pediococcus lactis RM1 freeze-dried powder in edible water, then add sausage curing agent and mix to form a suspension; in step (C01), the viable count of Pediococcus lactis RM1 freeze-dried powder is greater than 2.0 × 10⁻⁶. 11 CFU / g; The mass-volume ratio of lyophilized P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:90; The sausage curd in step (C01) includes the following components by weight: 1 part NaCl, 1 part sucrose, and 4 parts purified water.

[0182] (C02) Add the suspension from step (C01) to the minced meat product and stir until the concentration of the *Pediococcus lactis* RM1 inoculum is ≥1.0 × 10⁻⁶. 8 CFU / g; The weight of the minced meat product in step (CO2) is 20 times the weight of the suspension; The mass ratio of fat to lean meat in the minced meat product in step (CO2) is 1:6;

[0183] (C03) Stuff the mixture after stirring in step (C02) into sausages to form sausage meat products, and ferment the sausage meat products until the water activity drops below 0.9; the fermentation temperature in step (C03) is 12°C; the fermentation humidity in step (C03) is ≤65%; the sausage meat products in step (C03) include livestock and poultry sausages and cured sausages.

[0184] Example 8: This example is basically the same as Example 7, except that the mass-volume ratio of the freeze-dried P. RM1 lactic acid bacteria powder to drinking water in step (C01) is 1:110; the sausage curing preparation in step (C01) includes the following components by weight: 3 parts NaCl, 3 parts sucrose, and 6 parts purified water; the weight of the minced meat product in step (C02) is 40 times the weight of the suspension; the mass ratio of fat to lean meat in the minced meat product in step (C02) is 1:12; and the fermentation temperature in step (C03) is 15°C.

[0185] Example 9: This example is basically the same as Example 7, except that in step (C01), the mass-volume ratio of the freeze-dried Pyrococcus RM1 lactic acid bacteria powder to the edible water is 1:100; the sausage curing preparation in step (C01) includes the following components by weight: 2 parts NaCl, 2 parts sucrose, and 5 parts purified water; the weight of the minced meat product in step (C02) is 30 times the weight of the suspension; the mass ratio of fat to lean meat in the minced meat product in step (C02) is 1:9; and the fermentation temperature in step (C03) is 15°C.

[0186] Example 10: Application of Pediococcus lactis RM1 freeze-dried powder in cured / fermented meat products, including the following steps:

[0187] (D01) Dissolve an appropriate amount of Pediococcus lactis RM1 lyophilized powder in the first edible water, and then dissolve it in the second edible water; the viable count in the Pediococcus lactis RM1 lyophilized powder in step (D01) is greater than 2.0 × 10⁻⁶. 11 CFU / g; In step (D01), the mass-to-volume ratio of the lyophilized P. RM1 lactic acid bacteria powder to the first edible water is 1:90; In step (D01), the mass of the second edible water is 8 times the mass of the previous solution;

[0188] (D02) The final solution from step (D01) is inoculated onto the whole meat product and then hung for fermentation; the whole meat product in step (D02) includes salted meat, cured meat, cured chicken and cured duck; the inoculation temperature in step (D02) is 3℃; after inoculation in step (D02) for 18 hours, it is hung for fermentation.

[0189] Example 11: This example is basically the same as Example 10, except that the mass-to-volume ratio of the lyophilized P. lactic acid cocci RM1 powder to the first edible water in step (D01) is 1:110; the mass of the second edible water in step (D01) is 12 times the mass of the previous solution; the inoculation temperature in step (D02) is 5°C; and the suspension fermentation is carried out 30 hours after inoculation in step (D02).

[0190] Example 12: This example is basically the same as Example 10, except that the mass-volume ratio of the lyophilized P. lactic acid cocci RM1 powder to the first edible water in step (D01) is 1:100; the mass of the second edible water in step (D01) is 10 times the mass of the previous solution; the inoculation temperature in step (D02) is 4°C; and the suspension fermentation is carried out 24 hours after inoculation in step (D02).

[0191] Example 13: Isolation of Pediococcus lactis RM1

[0192] Take 25g of locally produced Hangzhou sausage and add it to 225ml of sterile water. Process the mixture using a shaker for 10 minutes, then serially dilute the mixture to 10 in a laminar flow hood. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 were absorbed respectively -4 10 -5 10 -6 Add 0.1 ml of the multiplied mixture to MRS medium, spread it evenly, and incubate it in a 35°C incubator for 48 hours. After single colonies grow, pick single colonies with different morphologies and streak them on MRS medium. After two purifications, identify the physicochemical characteristics of single colonies with different morphologies.

[0193] Physicochemical properties include protease activity, antioxidant activity, acid production capacity, temperature tolerance range, salt tolerance range, sodium nitrite tolerance range, colony morphology, and 16S rRNA gene sequence.

[0194] To identify protease activity, a 3% skim milk broth agar medium was prepared. Single colonies were picked and inoculated into this medium, with four single colonies inoculated per plate. The plates were incubated at 35°C for 48 hours, and the hydrolysis zones were observed. The results were as follows: Figure 1 As shown, Pediococcus lactis RM1 can significantly degrade milk protein, forming obvious hydrolysis zones;

[0195] The antioxidant activity was determined by picking a single colony of RM1 and incubating it in sterile MRS broth at 35°C for 48 hours. The supernatant was then collected by centrifugation, and its antioxidant capacity was assessed using a DPPH and hydroxyl radical assay kit. The results showed that the OD500 of the RM1 bacterial culture was significantly higher than that of the supernatant. 600 When the value was 1.079, its supernatant hydroxyl radical scavenging capacity was 558±27.6 U / mL, and the DPPH scavenging rate was 37.3±0.682%;

[0196] The acid-producing capacity was determined by picking a single colony and inoculating it into MRS broth medium. After static incubation for 48 hours, the results showed that RM1 could reduce the pH of the medium from 6.2±0.2 to 4.31±0.01, indicating that it had a suitable acid-producing capacity. Strains with excessive acid-producing capacity would cause meat products to become severely acidic, affecting the final quality.

[0197] The temperature adaptability range was determined by picking a single colony, inoculating it into MRS broth, and culturing it at different temperatures, then measuring the OD of the bacterial culture. 600 The results showed that RM1 is adaptable to a temperature range from 50℃ to 15℃, with an optimal temperature of 35℃. It can grow at 10℃, but growth is very slow, and use at this temperature is not recommended.

[0198] The determination of salinity tolerance range was carried out by picking single colonies and inoculating them into MRS broth media with different salinity contents, and incubating them at 35°C. The results showed that the growth of RM1 slowed down with increasing salinity, and it could grow slowly under 12% salinity conditions, with OD on the third day. 600 The value can increase threefold;

[0199] The suitability range of sodium nitrite was determined by inoculating single colonies into MRS broth media with different sodium nitrite concentrations and culturing them at 35°C. The results showed that sodium nitrite concentration had no effect on the growth rate of RM1. Under different sodium nitrite conditions (50 mg / kg, 100 mg / kg, 150 mg / kg), the OD values ​​of RM1 at the same time point were... 600 The differences in values ​​were not significant.

[0200] The identification of colony morphology is as follows: Figure 2 As shown, the colony diameter of Pyocortis RM1 is 0.25±0.06cm, it is milky white and round, with neat edges and a smooth and moist surface;

[0201] The 16S rRNA gene sequence was identified by picking a single colony, inoculating it into MRS broth medium, incubating it at 35°C for 48 hours, centrifuging to collect the precipitate, extracting DNA, and performing PCR and sequencing on the 16S rRNA gene. The sequence is as follows:

[0202]

[0203] Upon comparison, the bacterium was identified as *Pediococcus lactis*, with a gene sequence similarity of 99.72% with its closest related species, *Pediococcus lactis*. Figure 3 As shown, this bacterium was therefore named Pediococcus lactis RM1.

[0204] Example 14: Preparation of lyophilized Pediococcus lactis RM1 powder. A single colony of RM1 was picked and inoculated into 50 ml LMR broth and cultured at 35°C for 48 hours. Then, a second activation was performed by inoculating 1% of the colony into 50 ml LMR broth. The activated bacterial solution was used as a seed culture for subsequent scale-up culture. Scale-up culture: The RM1 seed culture was inoculated into 250 ml LMR broth at a 1% inoculation rate for scale-up culture, and cultured at 35°C in a static / shaking incubator for 48 hours. The supernatant was then removed by centrifugation at 4°C. A lyophilization protectant was added to the bacterial sludge, with the following formula: 2.5% skim milk powder, 1.5% glycerol, 20% trehalose, and 30% sucrose. The precipitate was vortexed to fully dissolve, then frozen at -20°C for 24 hours, followed by lyophilization. The collected powder was the lyophilized bacterial agent powder, which can be used for subsequent applications.

[0205] Example 15: Application of Pediococcus lactis RM1 freeze-dried powder in cured meat products, with a live bacteria count of 2.0 × 10⁻⁶. 11 First, dissolve the bacterial powder with a concentration of CFU / g or higher in 100 times its weight of drinking water. Then, mix it with other sausage curing agents in a fixed proportion to form a suspension. Add this solution to 20-40 times its weight of minced meat products to ensure that the final concentration of the bacterial agent in the fermented minced meat products is greater than or equal to 1.0 × 10⁻⁶. 8 CFU / g; The minced meat with added pickling agents and bacterial agents is thoroughly mixed and stuffed into sausages to form sausage products. These sausage products are fermented at 15±3℃ and 65% or lower relative humidity until the final product's water activity drops below 0.9. This method is suitable for preparing various fermented minced meat products, including but not limited to livestock and poultry sausages and cured sausages. It indicates that *Pediococcus lactis* RM1 can significantly improve the color of sausages, such as... Figure 4 As shown, it can increase the redness value of the product, and also reduce sodium nitrite residue and enhance the product flavor.

[0206] Example 16: Application of Pediococcus lactis RM1 freeze-dried powder in cured / fermented meat products, with a viable count of 2.0 × 10⁻⁶. 11First, dissolve the bacterial powder with a concentration of CFU / g or higher in 100 times its weight of drinking water. After it is fully dissolved, dissolve it in 10 times its weight of drinking water. This bacterial solution can be used to inoculate whole meat products such as salted meat and cured meat. After inoculation at 4°C for 24 hours, hang it for fermentation to produce various fermented meat products. This method is suitable for preparing various whole meat products, including but not limited to salted meat, cured meat, cured chicken, cured duck and other livestock and poultry meat products. It can significantly increase the flavor of the products, inhibit fat peroxidation, and improve the redness value of meat products.

[0207] Experimental Example: 1.1 Detection of relevant characteristics of Pediococcus lactis RM1

[0208] 1.1.1 RM1 protease activity

[0209] Prepare protease medium, i.e. MRS medium containing 1.5% skim milk powder. Inoculate with an inoculation needle, poke holes in the medium, and anaerobic culture for 2 days. The appearance of a clear zone indicates the presence of protease activity. Measure the clear zone and the diameter of the colony.

[0210] 1.1.2 RM1 nitrate reductase activity

[0211] RM1 was cultured in MRS medium containing potassium nitrate. After 48 hours of culture, the bacterial culture was centrifuged, and the supernatant was collected to prepare Griess solutions A and B: 0.5g p-aminobenzenesulfonic acid + 150mL 20% hydrochloric acid = solution A, 0.2g naphthylethylenediamine hydrochloride + 100mL distilled water = solution B. 2mL of 4g / L p-aminobenzenesulfonic acid solution was added, mixed well, and allowed to stand for 3 to 5 minutes. Then, 1mL of 2g / L naphthylethylenediamine hydrochloride solution was added, and the mixture was allowed to stand for 15 minutes. A reagent blank was prepared at the same time, and the absorbance was measured at 538nm.

[0212] 1.1.3RM1 is resistant to high temperatures, low temperatures, and salt.

[0213] The strain was subcultured twice in liquid medium to activate it. It was then inoculated into MRS liquid medium at a 1 mL inoculum and cultured for 24 h. The cultures were then placed at 10℃ and 50℃, and the OD600 values ​​were measured at 24 h and 72 h to investigate the lactic acid bacteria's adaptability to different temperatures. Additionally, the strain was inoculated into MRS liquid medium containing 50 mg / kg, 100 mg / kg, and 150 mg / kg nitrite at a 1 mL inoculum and cultured. The OD600 values ​​were measured at 24 h and 72 h to investigate the lactic acid bacteria's adaptability to different concentrations of nitrite.

[0214] RM1 bacterial culture was cultured under different temperatures and nitrite concentrations, with a control group set up. The survival rate was detected by OD value at certain intervals to determine the adaptability of RM1 under different temperatures and salt concentrations.

[0215] 1.2 Preparation of Lyophilized Powder of Pediococcus lactis RM1

[0216] 1.2.1 Selection of Protective Agent

[0217] There are many types of freeze-drying protectants, each with different protective mechanisms. Traditionally, they are divided into three main categories: permeable protectants such as glycerol, which can penetrate the cell membrane to alleviate cell dehydration; semi-permeable protectants such as monosaccharides, sucrose, and amino acids, which can pass through the cell wall but not the cell membrane, inducing plasmolysis and inhibiting ice crystal formation; and non-permeable protectants such as polysaccharides, trehalose, and skim milk powder, which cannot enter the cell but exert a protective effect around the cell, forming a mucus layer that stabilizes the phospholipid bilayer and membrane protein structure of the cell membrane, preventing cell wall damage and cytoplasmic leakage that can lead to cell death due to freeze-drying. Therefore, this experiment selected glycerol, sucrose, trehalose, and skim milk powder as single-factor experiments. Since the protective mechanisms of freeze-drying protectants differ, a single protectant can only provide one protective effect and cannot offer the best protection for cells. Therefore, an orthogonal experiment needs to be designed based on the results of the single-factor experiments to find the optimal protectant formulation.

[0218] 1.2.2 Preparation of bacterial powder

[0219] The RM1 strain was purified and activated by inoculation twice into 50 mL LMRS liquid medium. Then, 1 mL of the bacterial culture was inoculated into 250 mL of medium for expansion culture for 48 hours. The culture was then evenly distributed into six 50 mL centrifuge tubes, ensuring the weight of each tube was accurate to 0.1 g. Centrifugation was performed at 10,000 rpm at 4°C. The supernatant was discarded, leaving the bacterial sludge. Five concentration gradients were set up for single-factor experiments, and nine groups were set up for orthogonal experiments. The cryoprotectant was added to each centrifuge tube, which was then labeled. The sludge and cryoprotectant were thoroughly mixed using a pipette. Two concentration dilutions were selected for plating as controls for colony count before freeze-drying. The thoroughly mixed bacterial cells were transferred to petri dishes, sealed with plastic wrap, and tied tightly with rubber bands. The dishes were frozen overnight at -20°C, then freeze-dried. After removal, the cells were reconstituted with an equal volume of sterile water. Each concentration was plated into three plates, and colonies were observed and counted after two days of anaerobic incubation.

[0220] 1.3 Sausage preparation

[0221] The sausages are made with a fat-to-lean meat ratio of 1:9. The curing agent formula is: 2% NaCl, 2% sucrose, and 5% purified water. Four groups were set up: Group A (control group), Group B (nitrite group), Group C (lactic acid bacteria group), and Group D (lactic acid + nitrite group). Fermentation was carried out at 25℃ for one day or at 15℃ for seven days.

[0222] 1.4 Testing of the physicochemical properties of sausages

[0223] 1.4.1 Determination of pH, moisture content, water activity, and color difference of sausage

[0224] After completing three-point calibration, the handheld pH meter was inserted into the fermented sausage to measure the pH value. For moisture content and water activity measurements, 2.0g of sausage sample (with casing removed and chopped) was quickly placed into a sample dish, the measuring chamber was sealed, and measurements were taken. After calibration, the colorimeter was used to measure the L*, a*, and b* values ​​of the fermented sausage slices. The a* value represents the redness value; a higher a* value indicates better product color and higher freshness, while the b* value has the opposite effect. Three parallel measurements were performed, and the average value was taken.

[0225] 1.4.2 Determination of nitrite residue in sausages

[0226] Preparation of the nitrite standard curve: Pipette 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, 1.00 mL, and 1.50 mL of sodium nitrite standard working solution (equivalent to 0.0 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, and 7.5 μg sodium nitrite, respectively) into 25 mL stoppered colorimetric tubes. Add 1 mL of p-aminobenzenesulfonic acid solution to the stoppered colorimetric tube, mix well, let stand for 3-5 min, then add 0.5 mL of naphthylethylenediamine hydrochloride solution, dilute to the mark with water, mix well, let stand for 15 min, pipette 200 μL, measure OD538, and plot the standard curve. The x-axis represents sodium nitrite content (μg), and the y-axis represents OD538. 538 Standard curve: Y = 0.5132x + 0.0166, as shown... Figure 5 As shown, R 2 =0.9999, indicating a good linear relationship.

[0227] Determination of nitrite in samples,

[0228]

[0229] In the formula: X1 represents the sodium nitrite content in the sample (mg / kg); m2 represents the mass of sodium nitrite in the sample solution (μg); m3 represents the mass of the sample (g); V1 represents the volume of the sample solution (mL); V0 represents the total volume of the sample treatment solution (mL). The result is retained to two significant figures.

[0230] 1.4.3 Determination of TBARS value of sausage

[0231] Mince the fermented sausage sample, weigh 5g, and place it in a 50mL centrifuge tube. Add 50mL of a 7.5% trichloroacetic acid mixture (containing 75g / L trichloroacetic acid and 1g / L disodium ethylenediaminetetraacetate). Place the tube in a shaker and shake at 150rpm / min for 30min. Filter the solution through filter paper, repeating the filtration once. Add an equal volume of 20mmol / L thiobarbituric acid solution (TBA) to 5mL of the filtrate, mix well, incubate in a water bath for 40min, then at 90℃ for 40min. Remove and cool for 1h. Transfer the solution to a centrifuge tube, centrifuge at 1600rpm for 5min, pour the supernatant into the tube, add 5mL of chloroform, shake well, and allow to separate into layers. Transfer 200μL of the supernatant to a 96-well plate and measure the absorbance at 532nm and 600nm wavelengths using a microplate reader. Record the absorbance values ​​and calculate the TBA value. Calculate using the formula:

[0232] TBARS value (mg / 100g) =

[0233] 1.4.4 Determination of protein carbonyl and total sulfhydryl groups in sausage

[0234] The detection of protein carbonyl and total sulfhydryl groups follows the following reaction principle: carbonyl groups react with 2,4-dinitrophenylhydrazine to generate 2,4-dinitrophenylhydrazone, which has a characteristic absorption peak at 370 nm.

[0235] The thiol content of proteins can be indirectly determined by measuring the total thiol content and GSH content. Thiol groups react with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) to form a yellow compound with a maximum absorption peak at 412 nm.

[0236] Protein carbonyl groups are formed by the oxidation of myofibrillar proteins and can reflect the degree of protein oxidation. A decrease in sulfhydryl group content is related to the formation of disulfide bonds through oxidation; the greater the decrease in sulfhydryl group content, the more severe the protein oxidation.

[0237] 1.4.5 Determination of Protein Content in Sausage

[0238] Each sample group was tested in triplicate. The final calculation formula is as follows:

[0239]

[0240] In the formula, X is the protein content in the sample, in g / 100g; C is the concentration of the hydrochloric acid standard titration solution, in moles per liter (mol / L).

[0241] F is the coefficient for converting nitrogen to protein; m is the sample mass in grams (g).

[0242] 1.5 Microbiological index determination

[0243] Determination of total lactic acid bacteria in sausage: Take 10g sausage sample for each group, add 90mL of sterile water, homogenize at high speed for 10min, select an appropriate dilution factor for coating, and incubate under anaerobic conditions for 48h before counting.

[0244] 1.6 Determination of Sausage Flavor Compounds

[0245] Three 2g parallel samples from each of the four groups were placed in headspace vials and sent for testing. Flavor detection was performed at the test site using gas chromatography-mass spectrometry (GC-IMS).

[0246] Conditions: First, the headspace vial containing the sample was pre-incubated at 80℃ for 10 min. Then, it was extracted at 80℃ for 30 min using a 50 / 30μm DVB / CAR / PEMS extraction needle. Finally, the extraction needle was desorbed at 230℃ for 2 min at the injection port.

[0247] Volatile flavor compounds were qualitatively analyzed using VOCal software, and their odor characteristics were analyzed according to Fan Xinyang's odor analysis.

[0248] 1.7 Data Processing

[0249] Experimental results are expressed as mean ± standard deviation. Graphs were created using Excel, and data analysis was performed using IBM SPSS Statistics 27.

[0250] 2 Results and Analysis

[0251] 2.1 Measurement of lactic acid bacteria protease activity

[0252] As shown in Table 1, Pediococcus lactis RM1 produced a clear zone when grown on MRS medium containing skim milk powder. This is because lactic acid bacteria contain proteases that can hydrolyze the proteins in skim milk powder, thus producing a clear zone around the colony. The larger the zone, the greater the protease activity.

[0253] Table 1. Transparent zone of protease and colony diameter of bacterial strains

[0254] strain Colony diameter (d) / cm Diameter of transparent ring (D) / cm RM1 0.28±0.07 1.05±0.19

[0255] 2.2 Nitrate reductase activity of lactic acid bacteria

[0256] As shown in Table 2, there was no significant difference in OD538 values ​​between the control group and the experimental group (P > 0.05), therefore, *Pediococcus lactis* RM1 did not have nitrate reductase activity.

[0257] Table 2 Nitrate reductase activity of strains

[0258] Group OD538 value control group <![CDATA[0.192±0.003 a ]]> experimental group <![CDATA[0.192±0.023 a ]]>

[0259] Note: Different lowercase letters indicate significant differences (p<0.05).

[0260] 2.3 Analysis of the high and low temperature tolerance and salt tolerance of lactic acid bacteria

[0261] OD 600 The value represents the concentration of the bacterial solution; the higher the OD value, the higher the bacterial concentration. The OD value of lactic acid bacteria at 48 hours is as follows: Figure 6 As shown, the control group and the high-temperature group grew rapidly in the first 24 hours. The control group grew slowly in the later period, while the high-temperature group grew slowly (P<0.05), indicating that lactic acid bacteria have a certain tolerance to high temperature. Compared with the control group, the low-temperature group grew very slowly (P<0.05), indicating that lactic acid bacteria are not adapted to low-temperature growth.

[0262] like Figure 7 As shown, the overall trend of the four groups of lactic acid bacteria was initially rapid growth followed by a plateau. With the increase of nitrite concentration, the density of the three experimental groups decreased compared with the control. Salt conditions inhibited bacterial growth. The density of lactic acid bacteria at 50 mg / kg and 100 mg / kg was significantly higher (P < 0.05) than that at 150 mg / kg. This indicates that lactic acid bacteria have good nitrite tolerance.

[0263] 2.4 Experimental formulation of Pediococcus lactis RM1 lyophilized powder

[0264] 2.4.1 Screening of Single Protective Agents

[0265] Single-factor experiments were designed, with 0.5%, 1.0%, 1.5%, 2.0%, 2.5% skim milk powder, 1.0%, 1.5%, 2.0%, 2.5%, 3.0% glycerol, 5%, 10%, 15%, 20%, 25% sucrose, and 5%, 10%, 15%, 20%, 25% trehalose as the concentrations to be selected to screen for the highest survival rate in each group.

[0266] 2.4.2 Orthogonal Experiment of Protective Agent

[0267] Based on the results of the single-factor experiment, the experiment was designed using an orthogonal array, and the results are shown in the table below. As shown, the survival rate is calculated according to the formula.

[0268] Freeze-dried survival rate / % = (3-1)

[0269] In the formula: NA is the number of viable bacteria after freeze-drying, CFU / mL; NB is the number of viable bacteria before freeze-drying, CFU / mL.

[0270] surface Analysis table of orthogonal experiment results

[0271] Experiment No. A Skim Milk Powder β-glycerin C Trehalose D Sucrose Survival rate (%) 1 1（1.5%） 1（1.0%） 1（20%） 1（20%） 1.27 2 1 2（1.5%） 2（25%） 2（25%） 8.82 3 1 3（2.0%） 3（30%） 3（30%） 7.54 4 2（2.0%） 1 2 3 5.25 5 2 2 3 1 7.44 6 2 3 1 2 7.90 7 3（2.5%） 1 3 2 21.51 8 3 2 1 3 52.45 9 3 3 2 1 44.50 K1 17.63 28.03 61.62 53.21 K2 20.59 68.71 58.57 38.23 K3 118.46 59.94 36.49 65.24 k1 5.877 9.343 20.540 17.737 k2 6.863 22.903 19.523 12.743 k3 39.487 19.980 12.163 21.747 R 33.610 13.560 8.377 9.003 Factor ranking A > B > D > C Horizontal sorting k3>k2>k1 k2>k3>k1 k1>k2>k3 k3>k1>k2 Excellent level A3 B2 C1 D3 Excellent combination A3B2C1D3

[0272] As shown in the table, the range of the four factors is RA > RB > RD > RC. The importance of the four protectants to the freeze-drying protection of lactic acid bacteria is skim milk powder > glycerin > trehalose > sucrose. The optimal combination of the four protectants is A3B2C1D3, which is 2.5% skim milk powder, 1.5% glycerin, 20% trehalose, and 30% sucrose.

[0273] 2.5 pH changes in fermented sausages

[0274] During sausage fermentation, the pH level affects the color and flavor of the sausage. Lactic acid bacteria utilize carbohydrates to produce large amounts of lactic acid and acetic acid, lowering the pH and thus impacting the fermentation process. Figure 8 As shown, the pH range of the four sausage groups was between 5.68 and 5.84. Group A showed a significant difference from the other three groups (P < 0.05). Among them, the lowest pH was 5.68 for the group with lactic acid bacteria added alone. This indicates that the addition of lactic acid bacteria powder, nitrite, lactic acid, and the combination of nitrite all have the effect of lowering the pH of the sausage, which can inhibit bacterial growth.

[0275] 2.6 Determination of moisture content and water activity of fermented sausages

[0276] As shown in Table 4, the moisture content of group C was significantly higher than that of the other three groups (P < 0.05), which is unfavorable for sausage storage. Compared with group B, the water activity of groups C and D was significantly lower (P < 0.05), indicating that the combination of lactic acid bacteria, lactic acid bacteria, and nitrite can reduce the water activity in sausages, promote sausage storage, and inhibit bacterial growth. Related studies have shown that an Aw value below 0.850 can inhibit bacterial growth and reproduction to some extent.

[0277] Table 4. Determination of Sausage Moisture Content and Water Activity

[0278] Group water activity Moisture content A <![CDATA[0.672±0.035 b ]]> <![CDATA[10.71±2.61 b ]]> B <![CDATA[0.764±0.022 a ]]> <![CDATA[13.71±0.94 b ]]> C <![CDATA[0.716±0.032 ab ]]> <![CDATA[18.81±3.19 a ]]> D <![CDATA[0.705±0.032 ab ]]> <![CDATA[12.75±0.98 b ]]>

[0279] Note: A: Control; B: Nitrite; C: Lactic acid bacteria; D: Lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0280] 2.7 Effects of different treatments on the color of fermented sausages

[0281] The color of sausages is one of the factors consumers consider when making a purchase decision; the redder the color, the more consumers prefer it. In food color testing, the α value represents the depth of red. As shown in Table 5, groups B and D showed significant differences from the other two groups (P < 0.05), indicating that nitrite has a significant effect on sausage color development. Group C showed no significant difference from group A (P > 0.05), indicating that lactic acid bacteria had a limited color-enhancing effect on sausages.

[0282] Table 5. Effects of different treatments on color difference in fermented sausages

[0283] index A B C D <![CDATA[L * ]]> <![CDATA[42.29±7.24 a ]]> <![CDATA[36.77±0.91 a ]]> <![CDATA[45.06±3.58 a ]]> <![CDATA[37.33±1.91 a ]]> <![CDATA[a * ]]> <![CDATA[5.34±0.12 b ]]> <![CDATA[8.68±1.35 a ]]> <![CDATA[4.51±0.80 b ]]> <![CDATA[8.01±1.57 a ]]> <![CDATA[b * ]]> <![CDATA[9.39±1.73 a ]]> <![CDATA[5.34±1.27 b ]]> <![CDATA[9.18±0.63 a ]]> <![CDATA[5.31±1.09 b ]]>

[0284] Note: A: Control; B: Nitrite; C: Lactic acid bacteria; D: Lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0285] 2.8 Effects of different treatments on the TBARS value of fermented sausage

[0286] TBARS represents the degree of lipid oxidation; a higher value indicates a greater degree of lipid oxidation. Lipid peroxidation has a significant impact on sausage quality. Figure 9 As shown, group A showed a significant difference from the other three groups (P < 0.05). This is because lactic acid bacteria themselves possess protease hydrolytic activity, inhibiting the production of malondialdehyde and preventing rancidity caused by lipid oxidation. Furthermore, a comparison between group B and group A shows that the addition of nitrite also had a certain inhibitory effect.

[0287] 2.9 Effects of different treatments on nitrite residues in fermented sausages

[0288] Nitrites have color-enhancing, antibacterial, antioxidant, and flavor-producing properties; however, they are also toxic to humans, with just a few grams potentially causing death. Currently, no other substance can replace the functions of nitrites, and their content in fermented sausages must be strictly controlled. Figure 10 As shown, there was a significant difference between group B and group D (P < 0.05), indicating that lactic acid bacteria have nitrite reductase activity, which can degrade nitrite and thus reduce the residual amount of nitrite in sausage.

[0289] 2.10 Determination of protein content, total sulfhydryl groups, and carbonyl groups in fermented sausages

[0290] As shown in Table 6, the protein content of groups C and D was significantly different from the other two groups (P < 0.05). The moisture content in the fermented sausage decreased continuously with the extension of fermentation and maturation time, leading to an increase in protein content. There was no significant difference in protein content between groups A and B (P > 0.05). Therefore, the amount of sodium nitrite added had no significant effect on the total protein content of the fermented sausage. The levels of total sulfhydryl content and protein carbonyl content represent the degree of protein oxidation; both showed no significant difference (P > 0.05), indicating that lactic acid bacteria and nitrite had no significant effect on counteracting protein oxidation.

[0291] Table 6. Determination of Sausage Protein Hydrolysis Index, Total Sulfhydryl Group and Carbonyl Group Content

[0292] Group Protein content (g / 100g) Total thiol content (μmol / g) Protein carbonyl content (μmol / g) A <![CDATA[4.01±0.71 b ]]> <![CDATA[5.97±0.03 a ]]> <![CDATA[0.16±0.03 a ]]> B <![CDATA[3.8±0.1 b ]]> <![CDATA[5.37±0.91 a ]]> <![CDATA[0.08±0.06 a ]]> C <![CDATA[4.75±0.75 ab ]]> <![CDATA[4.51±1.09 a ]]> <![CDATA[0.13±0.05 a ]]> D <![CDATA[5.13±0.21 a ]]> <![CDATA[6.5±2.88 a ]]> <![CDATA[0.09±0.02 a ]]>

[0293] Note: A: Control; B: Nitrite; C: Lactic acid bacteria; D: Lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0294] 2.11 Qualitative Analysis of Fermented Sausage Flavor

[0295] Table 7. Qualitative analysis results of volatile flavor compounds in different fermented sausages.

[0296]

[0297]

[0298]

[0299] Note: A: Control; B: Nitrite; C: Lactic acid bacteria; D: Lactic acid bacteria + nitrite. Different lowercase letters indicate significant differences (p<0.05).

[0300] The peak volumes of various substances are shown in Table 7, where the volume represents the amount of substance. To visually compare the four groups of volatile substances, three parallel spectra were selected for comparison, as shown below. Figure 11 As shown in the table and figure, the flavor produced by adding lactic acid bacteria alone is more pronounced than that of the control, while the combination of lactic acid bacteria and nitrite, and the nitrite group, are not as effective as those with lactic acid bacteria alone. A total of 46 volatile substances were detected in this sausage flavor analysis, including 9 ketones, 9 aldehydes, 13 lipids, 3 pyrazines, 3 alcohols, 2 acids, 2 furans, and 5 other substances.

[0301] Analysis based on substance classification shows that group C, inoculated with lactic acid bacteria, had higher levels of 3-methylbutyraldehyde, 2-hexanealdehyde monomer, 2-hexanealdehyde dimer, 2-methylpropionaldehyde, and 5-methylfurfural than the control group A, giving the sausage a fresh aroma.

[0302] Inoculation with lactic acid bacteria can promote the production of lipids. The levels of ethyl formate, butyl formate, ethyl 2-methylbutyrate monomer, ethyl 2-methylbutyrate dimer, isobutyl 2-butenoate, ethyl acetate monomer, ethyl acetate dimer, butyric acid, 3-methyl-butyrate, methyl 3-(methylthio)propionate, and propyl palmitate in group C were significantly higher than those in group A. These volatile lipids give the sausage a strong ester aroma.

[0303] Group C fermented sausages had higher levels of 2-methylpyrazine and 2-acetylpyrazine than Group A, imparting a chocolate aroma. Group C fermented sausages also had higher levels of n-propanol than the other three groups, giving them a strong alcoholic aroma. Adding lactic acid bacteria alone during sausage fermentation yielded better results, producing more flavor compounds and adding aromas such as freshness, fruitiness, fattiness, alcohol, and chocolate to the original flavor profile. While adding nitrite alone to Group B and the compound group D showed significant increases in a few individual cases compared to Group A, the overall increase in flavor compounds was not very significant. Therefore, adding lactic acid bacteria alone is the best choice.

[0304] 3.1 Summary

[0305] (1) Pietrococcus lactis has significant protease activity but no obvious nitrate reductase activity. It can tolerate certain high temperatures (50℃) but is not adapted to growth at low temperatures (10℃) and can tolerate a certain degree of nitrite.

[0306] (2) Based on the results of single-factor and orthogonal experiments, the suitable freeze-drying protectant formula for RM1 was obtained: 2.5% skim milk powder, 1.5% glycerol, 20% trehalose, and 30% sucrose, which can yield a bacterial powder with a survival rate of 52.45%.

[0307] (3) Through the study of the physicochemical properties and volatile flavor substances of several groups of sausages, the comprehensive evaluation concluded that adding lactic acid bacteria powder alone has a better effect, which can significantly reduce the pH, TBARS value and nitrite residue of sausages, and increase the content and types of volatile flavor substances.

[0308] Freeze-dried powder protectants can protect the bacteria cells from freezing and drying as much as possible, increasing the survival rate of lactic acid bacteria. These protectants are also chosen because they are inexpensive and readily available. Experiments showed that low concentrations of skim milk powder and glycerin were more effective than trehalose and sucrose. This is because, firstly, the amounts of trehalose and sucrose added are not low, at 20% and 30% respectively; secondly, both protectants, after freeze-drying, produce granular crystalline substances, especially sucrose, which is not suitable for room temperature storage and should be stored in a cool, dry place. Otherwise, the high sugar content will cause it to absorb water and melt.

[0309] Lactic acid bacteria powder plays a certain role in the production of dry-cured sausages: it ferments to produce lactic acid, lowering the pH and inhibiting bacterial growth; it possesses protease activity, hydrolyzing proteins, making them easier for the body to absorb and enhancing flavor; and it has nitrite reductase activity, breaking down nitrites and reducing the nitrite content in sausages. Lactic acid bacteria themselves have a certain degree of heat and salt tolerance, utilizing organic compounds to produce various flavor substances, especially aldehydes and acids, making them suitable as microbial starter cultures.

Claims

1. Lyophilized Powder of Pediococcus lactis RM1, characterized by: It includes the following components in parts by weight. 0.5-3 parts of Pediococcus lactis RM1, 2-3 parts of skim milk powder, 1-2 parts of glycerol, 15-25 parts of trehalose, and 25-35 parts of sucrose; The accession number of *Pediococcus acidilactici* RM1 is GDMCC NO.66765; the depository of *Pediococcus acidilactici* RM1 is Guangdong Provincial Center for Microbial Culture Collection; the accession name of *Pediococcus acidilactici* RM1 is Pediococcus acidilactici RM1; and the accession date of *Pediococcus acidilactici* RM1 is July 28, 2025.

2. The lyophilized Peptococcus lactis RM1 powder according to claim 1, characterized in that: It includes the following components in parts by weight. RM1 lactic acid bacteria 1-2.5 parts, skim milk powder 2.2-2.8 parts, glycerol 1.2-1.8 parts, trehalose 17-23 parts, sucrose 27-33 parts.

3. The lyophilized Peptococcus lactis RM1 powder according to claim 1, characterized in that: The nucleotide sequence of the *Pediococcus lactis* RM1 is shown in SEQ ID NO.

1.

4. The method for preparing Pediococcus lactis RM1 lyophilized powder according to claim 1, characterized in that: Including the following step, (B01) Take a single colony of Pyotrophic Lactococcus RM1 and inoculate it into MRS broth medium for culture; (B02) Take the RM1 strain of Pyrococcus lactis cultured in step (B01) and inoculate it again into broth medium at an inoculation rate of 1% for a second activation. Use the activated bacterial solution as the seed solution. (B03) Inoculate the seed culture from step (B02) into MRS broth medium at an inoculation rate of 1% for expansion culture, and then centrifuge to remove the supernatant; (B04) After removing the supernatant in step (B03), add an appropriate amount of skim milk powder, glycerin, trehalose and sucrose to the bacterial sludge and vortex to dissolve the precipitate; (B05) Freeze and freeze the solution from step (B04) to obtain lyophilized Pediococcus lactis RM1 lyophilized powder.

5. The method for preparing Pediococcus lactis RM1 lyophilized powder according to claim 1, characterized in that: In step (B01), the amount of MRS broth culture medium used is 40 mL to 60 mL; the culture temperature in step (B01) is 33℃ to 37℃; the culture time in step (B01) is 36 h to 60 h; the scale-up culture temperature in step (B03) is 33℃ to 37℃; the scale-up culture in step (B03) is either static culture or shaker culture; the scale-up culture time in step (B03) is 36 h to 60 h; the centrifugation temperature in step (B03) is 3℃ to 5℃; the freezing temperature in step (B05) is -25℃ to -15℃; the freezing time in step (B05) is 18 h to 30 h.

6. Application of Pediococcus lactis RM1 freeze-dried powder in cured and preserved meat products or cured / fermented meat products.

7. The application of the freeze-dried Peptococcus lactis RM1 powder according to claim 6 in cured meat products, characterized in that: Including the following step, (C01) Dissolve an appropriate amount of Pediococcus lactis RM1 freeze-dried powder in edible water, then add sausage curing agent and mix to form a suspension; (C02) Add the suspension from step (C01) to the minced meat product and stir until the concentration of the *Pediococcus lactis* RM1 inoculum is ≥1.0 × 10⁻⁶. 8 CFU / g; (C03) Stuff the mixture after stirring in step (C02) into sausage meat products, and ferment the sausage meat products until the water activity drops below 0.

9.

8. The application of the freeze-dried Peptococcus lactis RM1 powder according to claim 7 in cured meat products, characterized in that: In step (C01), the viable count of the Pediococcus lactis RM1 lyophilized powder is greater than 2.0 × 10⁻⁶. 11 CFU / g; The mass-to-volume ratio of the lyophilized P. lactic acid cocci RM1 powder to drinking water in step (C01) is 1:90-110; The sausage curd preparation in step (C01) comprises the following components by weight: 1-3 parts NaCl, 1-3 parts sucrose, 4-6 parts purified water. In step (CO2), the weight of the minced meat product is 20 to 40 times the weight of the suspension; in step (CO2), the mass ratio of fat to lean meat in the minced meat product is 1:6 to 12; and in step (CO3), the fermentation humidity is ≤65%.

9. The application of the lyophilized P. RM1 lactic acid bacteria powder according to claim 6 in cured / fermented meat products, characterized in that: Includes the following steps, (D01) Dissolve an appropriate amount of Pediococcus lactis RM1 freeze-dried powder in a first edible water solution, and then dissolve it in a second edible water solution; the viable count in the Pediococcus lactis RM1 freeze-dried powder in step (D01) is greater than 2.0 × 10⁻⁶. 11 CFU / g; (D02) Inoculate the final solution from step (D01) onto the whole meat product and then carry out hanging fermentation.

10. The application of the lyophilized P. RM1 lactic acid bacteria powder according to claim 9 in cured / fermented meat products, characterized in that: In step (D01), the mass-to-volume ratio of the lyophilized P. RM1 lactic acid bacteria powder to the first edible water is 1:90-110; in step (D01), the mass of the second edible water is 8-12 times the mass of the previous dissolving solution; the whole meat products in step (D02) include salted meat, cured meat, cured chicken, and cured duck; the inoculation temperature in step (D02) is 3℃-5℃; and in step (D02), hanging fermentation is carried out 18-30 hours after inoculation.

Images