A lactic acid bacteria ferment for enhancing the heat sensitivity of microorganisms and its application in food preservation.

Abstract

This application discloses a method for preparing a lactic acid bacteria ferment that enhances the heat sensitivity of microorganisms and its application in food preservation. The preparation method involves: initial fermentation using *Lactococcus lactis* as the primary fermenting organism, followed by stepwise fermentation using *Lactobacillus* as the secondary fermenting organism; after fermentation, the resulting fermentation broth undergoes solid-liquid separation and ultrafiltration to enrich small molecule peptides of 500–1000 Da, and the ultrafiltration product is dried to obtain the lactic acid bacteria ferment. This ferment can significantly enhance the heat sensitivity of target microorganisms, effectively reducing the D value of *Staphylococcus aureus* and *Bacillus subtilis* under heat treatment conditions, thereby lowering the sterilization temperature or shortening the sterilization time in food processing. Applying this lactic acid bacteria ferment to food systems can reduce the adverse effects of high-temperature treatment on food quality and flavor while ensuring the microbial safety of food, making it suitable for typical heat-sterilized food systems such as braised foods and soy products.

Description

Technical Field

[0001] This application relates to the field of probiotic fermentation preparation technology, and more specifically, it relates to a lactic acid bacteria ferment for enhancing the heat sensitivity of microorganisms and its application in food preservation. Background Technology

[0002] Food spoilage and microbial contamination are core issues restricting the development of the food industry. Currently, food preservation and anti-corrosion mainly rely on two types of technologies, but both have significant drawbacks: one is heat sterilization, which can kill microorganisms, but high temperatures easily destroy nutrients such as vitamins and proteins in food, causing products such as braised tofu and meat sausages to lose their crispness and natural flavor, while low-temperature sterilization is difficult to meet the requirements for long-term preservation; the other is the addition of chemical preservatives, which are low-cost and fast-acting, but excessive use of preservatives can easily lead to regulatory risks and cannot meet the trend of healthy consumption.

[0003] Lactic acid bacteria fermentation products, due to their high safety and good antibacterial effects, have become an important alternative to chemical preservatives. Existing technologies have disclosed methods for preparing preservation compositions using lactic acid bacteria fermentation, but key technical bottlenecks remain: First, the fermentation processes are mostly static and single-condition, without dynamic regulation based on the metabolic characteristics of lactic acid bacteria, resulting in low yields of active ingredients such as antimicrobial peptides and specific organic acids, and limited antibacterial effects. Second, the functions are singular, focusing only on antibacterial effects without combining with food sterilization processes or considering food taste. Therefore, developing a method for preparing lactic acid bacteria fermentation products that can improve microbial heat sensitivity, enhance product stability, and offer functional versatility has become a crucial area for breakthroughs in the food preservation field. Summary of the Invention

[0004] To address the technical problems mentioned in the background art, this application provides a lactic acid bacteria ferment for enhancing the heat sensitivity of microorganisms and its application in food preservation.

[0005] This application provides a method for preparing lactic acid bacteria fermentation products, using the following technical solution:

[0006] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0007] Step 1: Inoculate the first fermentation bacteria into the fermentation substrate, and use dynamic pH gradient control to anaerobic ferment for 10-14 hours to obtain the first fermentation broth;

[0008] Step 2: Inoculate the second fermentation bacteria into the first fermentation broth, maintain the tank temperature at 28±2℃, the tank pressure at 0.03-0.05MPa, and the pH value at 5.6±0.2, and continue anaerobic fermentation for 12-18h to obtain the second fermentation broth;

[0009] Step 3: After ultrafiltration, the second fermentation broth is freeze-dried under inert gas protection to obtain lactic acid bacteria fermentation product.

[0010] Preferably, the dynamic pH gradient control parameters in step 1 are as follows: under the conditions of tank temperature of 30±2℃ and tank pressure of 0.02-0.04MPa, the pH value is maintained at 6.5±0.2 for 0-6h, and the pH value is reduced to 6.0±0.2 for 6-12h.

[0011] Preferably, the fermentation base material in step 1, by weight percentage, includes: inulin 3-8%, pea protein peptide 2-5%, yeast extract 2-5%, sodium selenite 0.01-0.03%, vitamin E succinate 0.05-0.1%, and the balance being deionized water.

[0012] Preferably, in step 1, the first fermentation bacteria is Lactococcus lactis subsp. lactis, and the inoculation amount of the first fermentation bacteria is 1.5-3% of the mass of the fermentation substrate.

[0013] Preferably, in step 1, the lactococcus lactis subsp. Lactis is the lactococcus lactis subsp. Lactis, with accession number CICC 6242.

[0014] Preferably, in step 2, the second fermentation bacteria consists of Lactobacillus rhamnosus and Lactobacillus paracasei with an effective viable count ratio of 1-2:2-3.

[0015] Preferably, the Lactobacillus rhamnosus is Lactobacillus rhamnosus, with accession number CGMCC 1.2467.

[0016] Preferably, the Lactobacillus paracasei is Lactaseibacillus paracasei, with accession number CGMCC 1.2468.

[0017] Preferably, in step 2, the inoculation amount of the second fermentation bacteria is 2-5% of the mass of the first fermentation broth.

[0018] Preferably, the molecular weight of the ultrafiltration membrane used in step 3 is 500-1000 Da.

[0019] Application of a lactic acid bacteria ferment prepared by the above method in food preservation.

[0020] Preferably, compared to the control, the lactic acid bacteria ferment can increase the D of Staphylococcus aureus. 85 The value decreased by 45-55%, and the D of Bacillus subtilis was reduced. 85 The value decreased by 35-45%.

[0021] Preferably, the lactic acid bacteria ferment can lower the sterilization temperature of braised tofu, reducing it from 121°C and 20 min to 85°C and 20 min, thereby improving the taste of braised tofu while maintaining the original shelf life.

[0022] In summary, this application has the following beneficial effects:

[0023] This application employs a precise selection of *Lactococcus lactis* subsp. lactis (CICC 6242) as the primary fermentation strain for initial fermentation, combined with a secondary fermentation strain composed of *Lactobacillus rhamnosus* (CGMCC 1.2467) and *Lactobacillus paracasei* (CGMCC 1.2468) at a specific effective viable count ratio for deep fermentation. Simultaneously, dynamic pH gradient control technology is used. *Lactococcus lactis* subsp. lactis, as the primary fermentation strain, initiates fermentation first, rapidly utilizing nutrients such as inulin and pea protein peptides in the fermentation substrate. This efficiently produces acid and creates a stable, weakly acidic microenvironment, eliminating environmental compatibility barriers for the subsequent inoculation of the secondary fermentation strain. Furthermore, it pre-decomposes complex nutrients into small-molecule metabolites, providing easily absorbed nutrient substrates for the growth and reproduction of *Lactobacillus rhamnosus* and *Lactobacillus paracasei*. This solves the problems of unsuitable initial environment and slow growth encountered during direct fermentation of compound lactobacilli. The subsequent introduction of *Lactobacillus rhamnosus* and *Lactobacillus paracasei*, in an optimized ratio, resulted in a synergistic effect, with their metabolic pathways complementing each other. *Lactobacillus rhamnosus* excels at synthesizing bacteriocins with broad-spectrum antibacterial activity, while *Lactobacillus paracasei* efficiently produces organic acids such as lactic acid and acetic acid. The synergistic effect of these two antibacterial substances covers the inhibitory targets of various microorganisms, including *Staphylococcus aureus* and *Bacillus*, resulting in a broader antibacterial spectrum and significantly enhanced antibacterial strength of the ferment. Simultaneously, maintaining the fixed order of "primary fermentation by the first fermentation strain followed by deep fermentation by the second fermentation strain" avoids growth competition and metabolic inhibition between different microbial species, ensuring that the activities of all three strains are maintained at optimal levels. This significantly increases the yield of metabolites and substantially improves the antibacterial activity and application suitability of lactic acid bacteria ferments used to enhance the heat sensitivity of microorganisms.

[0024] This application utilizes a stepwise anaerobic fermentation process with a specific combination of microbial strains, coupled with dynamic pH gradient control and precise control of tank temperature and pressure parameters. This, combined with subsequent ultrafiltration membrane concentration with specific molecular weight and nitrogen-protected freeze-drying technology, enables the efficient enrichment of 500-1000 Da small molecule peptides and various active metabolites in the fermentation product. Among these, the small molecule peptides effectively disrupt the cell membrane structure of representative target microorganisms such as Staphylococcus aureus and Bacillus subtilis, altering cell membrane permeability and significantly reducing their heat tolerance, thus greatly enhancing their heat sensitivity. This characteristic allows for a significant reduction in sterilization intensity during food processing. Foods that originally required 121℃ autoclaving to maintain their shelf life can achieve the same shelf life with 85℃ pasteurization after adding this fermented product. This not only reduces the damage to nutritional components caused by high temperatures but also avoids the deterioration in taste caused by high temperatures. For example, products such as braised tofu can maintain excellent taste and flavor while ensuring undetectable total bacterial count for 30 days. In addition, as a natural fermentation product, its safety is superior to that of the chemical preservative potassium sorbate, providing a natural and efficient new solution for food preservation, and has broad application prospects in multiple food fields such as soy products and meat products. Attached Figure Description

[0025] Figure 1 The graphs show the heat sterilization curves of Examples 1-3, blank, and potassium sorbate against Staphylococcus aureus at 85°C.

[0026] Figure 2 This is a heat sterilization curve of comparative examples 1-6 Staphylococcus aureus at 85℃.

[0027] Figure 3 The graphs show the heat sterilization curves of Bacillus subtilis in Examples 1-3, the blank, and potassium sorbate at 85°C.

[0028] Figure 4 The graphs show the heat sterilization curves of Bacillus subtilis pairs 1-6 at 85℃. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the embodiments.

[0030] The *Lactococcus lactis* subsp. *Lactis* (accession number CICC 6242) and *Lactococcus lactis* subsp. *Lactis* (accession number HYCC 51619) used in the embodiments and comparative examples of this application were all purchased from Beijing Huayan Shijia Quality Inspection Technology Co., Ltd.; *Lactobacillus rhamnosus* (accession number CGMCC 1.2467), *Lactobacillus rhamnosus* (accession number CGMCC 1.2568), *Lactobacillus paracasei* (accession number CGMCC 1.2468), and *Lactobacillus paracasei* (accession number CGMCC 51619) were all purchased from Beijing Huayan Shijia Quality Inspection Technology Co., Ltd. 1.2744) were all purchased from the China General Microbiological Culture Collection Center; inulin was purchased from Sichuan Huanxu Biotechnology Co., Ltd.; pea protein peptides were purchased from Shandong Pingju Biotechnology Co., Ltd.; yeast extract was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.; sodium selenite was purchased from Hebei Rencan Biotechnology Co., Ltd.; and vitamin E succinate was purchased from Zhengzhou Yukong Biotechnology Co., Ltd.

[0031] Examples 1-3 provide a method for preparing lactic acid bacteria fermentation products.

[0032] Example 1

[0033] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0034] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH value at 6.3 for 0-6 hours and decrease it to 5.8 for 6-12 hours under the conditions of tank temperature of 28℃ and tank pressure of 0.02MPa. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptide 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, and the balance being deionized water. The first fermentation bacteria is Lactococcus lactis subsp. Lactis, with preservation number CICC 6242. The inoculation amount of the first fermentation bacteria is 1.5% of the fermentation substrate mass.

[0035] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26℃, the tank pressure at 0.03MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2467) and Lactobacillus paracasei (CGMCC 1.2468) with an effective viable count ratio of 1:2.

[0036] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0037] Example 2

[0038] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0039] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH at 6.5 for 0-6 hours at a tank temperature of 30℃ and a tank pressure of 0.03MPa, then decrease the pH to 6.0 for 6-12 hours. Perform anaerobic fermentation for 12 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 5%, pea protein peptides 4%, yeast extract 4%, sodium selenite 0.02%, vitamin E succinate 0.08%, and the remainder is deionized water. The first fermentation bacteria is *Lactococcus lactis* subsp. *Lactis*, with accession number CICC 6242. The inoculation amount of the first fermentation bacteria is 2.2% of the fermentation substrate mass.

[0040] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 3.5% of the mass of the first fermentation broth. Maintain the tank temperature at 28℃, the tank pressure at 0.04MPa, and the pH at 5.6. Continue anaerobic fermentation for 15 hours to obtain the second fermentation broth. The second fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2467) and Lactobacillus paracasei (CGMCC 1.2468) with an effective viable count ratio of 1.5:2.5.

[0041] Step 3: The second fermentation broth was concentrated to half its original volume using an ultrafiltration membrane with a molecular weight of 700 DaL at an operating pressure of 0.25 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -45°C for 11 hours to obtain the lactic acid bacteria fermentation product.

[0042] Example 3

[0043] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0044] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH at 6.7 for 0-6 hours at a tank temperature of 32℃ and a tank pressure of 0.04MPa, then decrease the pH to 6.2 for 6-12 hours. Perform anaerobic fermentation for 14 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 8%, pea protein peptides 5%, yeast extract 5%, sodium selenite 0.03%, vitamin E succinate 0.1%, and the remainder is deionized water. The first fermentation bacteria is *Lactococcus lactis* subsp. *Lactis*, with accession number CICC 6242. The inoculation amount of the first fermentation bacteria is 3% of the fermentation substrate mass.

[0045] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 5% of the mass of the first fermentation broth. Maintain the tank temperature at 30℃, the tank pressure at 0.05MPa, and the pH at 5.8. Continue anaerobic fermentation for 18 hours to obtain the second fermentation broth. The second fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2467) and Lactobacillus paracasei (CGMCC 1.2468) with an effective viable count ratio of 2:3.

[0046] Step 3: The second fermentation broth was concentrated to half its original volume using an ultrafiltration membrane with a molecular weight of 1000 DaL at an operating pressure of 0.3 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -50°C for 12 hours to obtain the lactic acid bacteria fermentation product.

[0047] Comparative Example 1

[0048] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0049] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH value at 6.3 for 0-6 hours and decrease it to 5.8 for 6-12 hours under the conditions of tank temperature of 28℃ and tank pressure of 0.02MPa. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptide 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, and the balance being deionized water. The first fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2467) and Lactobacillus paracasei (CGMCC 1.2468) with an effective viable count ratio of 1:2. The inoculation amount of the first fermentation bacteria is 1.5% of the mass of the fermentation substrate.

[0050] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26℃, the tank pressure at 0.03MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria is Lactococcus lactis subsp. Lactis, with the preservation number CICC 6242.

[0051] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0052] Comparative Example 2

[0053] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0054] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH at 6.3 for 0-6 hours at a tank temperature of 28℃ and a tank pressure of 0.02MPa, then decrease the pH to 5.8 for 6-12 hours. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptides 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, with the remainder being deionized water. The first fermentation bacteria is *Lactococcus lactis* subsp. *Lactis*, with accession number CICC 6242. The inoculation amount of the first fermentation bacteria is 1.5% of the fermentation substrate mass.

[0055] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26°C, the tank pressure at 0.03 MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria is Lactobacillus paracasei, with the preservation number CGMCC1.2468.

[0056] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0057] Comparative Example 3

[0058] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0059] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH value at 6.3 for 0-6 hours and decrease it to 5.8 for 6-12 hours under the conditions of tank temperature of 28℃ and tank pressure of 0.02MPa. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptide 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, and the balance being deionized water. The first fermentation bacteria is Lactococcus lactis subsp. Lactis, with preservation number CICC 6242. The inoculation amount of the first fermentation bacteria is 1.5% of the fermentation substrate mass.

[0060] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26°C, the tank pressure at 0.03 MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria is Lactobacillus rhamnosus, with the preservation number CGMCC 1.2467.

[0061] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0062] Comparative Example 4

[0063] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0064] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH at 6.3 for 0-6 hours at a tank temperature of 28℃ and a tank pressure of 0.02MPa, then decrease the pH to 5.8 for 6-12 hours. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptides 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, with the remainder being deionized water. The first fermentation bacteria is *Lactococcus lactis* subsp. *Lactis*, preservation number HYCC 51619, and the inoculation amount of the first fermentation bacteria is 1.5% of the fermentation substrate mass.

[0065] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26℃, the tank pressure at 0.03MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2568) and Lactobacillus paracasei (CGMCC 1.2744) with an effective viable count ratio of 1:2.

[0066] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0067] Comparative Example 5

[0068] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0069] Step 1: Inoculate the fermentation substrate with the first fermentation bacteria. Using dynamic pH gradient control, maintain the pH value at 6.3 for 0-6 hours and decrease it to 5.8 for 6-12 hours under the conditions of tank temperature of 28℃ and tank pressure of 0.02MPa. Perform anaerobic fermentation for 10 hours to obtain the first fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptide 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, and the balance being deionized water. The first fermentation bacteria is Lactococcus lactis subsp. Lactis, with preservation number CICC 6242. The inoculation amount of the first fermentation bacteria is 1.5% of the fermentation substrate mass.

[0070] Step 2: Inoculate the first fermentation broth with the second fermentation bacteria at a rate of 2% of the mass of the first fermentation broth. Maintain the tank temperature at 26℃, the tank pressure at 0.03MPa, and the pH at 5.4. Continue anaerobic fermentation for 12 hours to obtain the second fermentation broth. The second fermentation bacteria consist of Lactobacillus rhamnosus (CGMCC 1.2467) and Lactobacillus paracasei (CGMCC 1.2468) with an effective viable count ratio of 2:1.

[0071] Step 3: The second fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 DaL at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40℃ for 12 hours to obtain the lactic acid bacteria fermentation product.

[0072] Comparative Example 6

[0073] A method for preparing a lactic acid bacteria ferment includes the following preparation steps:

[0074] Step 1: Inoculate the primary and secondary fermentation bacteria into the fermentation substrate. Using dynamic pH gradient control, maintain the pH at 6.3 for 0-6 hours at a tank temperature of 28℃ and a tank pressure of 0.02MPa, then decrease the pH to 5.8 for 6-12 hours. Perform anaerobic fermentation for 10 hours to obtain the primary fermentation broth. The fermentation substrate, by weight percentage, includes: inulin 3%, pea protein peptides 2%, yeast extract 2%, sodium selenite 0.01%, vitamin E succinate 0.05%, and the remainder is deionized water. The primary fermentation bacteria is *Lactococcus lactis* subsp. *Lactis*, accession number CICC 6242, and the inoculation amount is 1.5% of the fermentation substrate mass. The secondary fermentation bacteria consist of *Lactobacillus rhamnosus* (accession number CGMCC 1.2467) and *Lactobacillus paracasei*, with an effective viable count ratio of 1:2. Paracasei, with accession number CGMCC 1.2468, was used as the inoculum for the second fermentation strain at a rate of 2% of the fermentation substrate mass.

[0075] Step 2: The first fermentation broth was concentrated to 1 / 4 of its original volume using an ultrafiltration membrane with a molecular weight of 500 Da at an operating pressure of 0.2 MPa. Then, it was freeze-dried under nitrogen protection at a temperature of -40°C for 12 hours to obtain the lactic acid bacteria fermentation product.

[0076] Performance testing

[0077] The first-order kinetic model represents a linear relationship between the logarithm of bacterial inactivation and treatment time, and its formula is:

[0078]

[0079] In the formula, N refers to the number of colonies in the sample after treatment time t (CFU / mL); N0 refers to the initial number of colonies in the control sample (CFU / mL); D refers to the treatment time (min) required to reduce microorganisms by 90%; and t refers to the treatment time (min).

[0080] The Dt value measures the heat resistance of microorganisms. The larger the Dt value, the more heat-resistant the microorganism is at that temperature and the more difficult it is to kill. The Dt value is strongly dependent on temperature.

[0081] The lactic acid bacteria fermentation products prepared in Examples 1-3 and Comparative Examples 1-6 of this application and the food preservative potassium sorbate were added to the sterilized nutrient broth culture medium to prepare a lactic acid bacteria fermentation product suspension with a concentration of 0.1 wt% and a potassium sorbate suspension with a concentration of 0.1 wt%.

[0082] Staphylococcus aureus and Bacillus subtilis were inoculated separately into nutrient broth medium and then incubated in a shaker at 37°C for 24 hours to obtain activated strain nutrient broth solutions. The activated strain nutrient broth solutions were then diluted with nutrient broth to an OD value. 600 The bacterial suspension with a value of 1, wherein Staphylococcus aureus (product number: B81854(A)) and Bacillus subtilis (product number: B98052) were purchased from Ningbo Mingzhou Biotechnology Co., Ltd., and the nutrient broth medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0083] Mix 0.9 mL of bacterial suspension with 8.1 mL of lactic acid bacteria fermentation product suspension or potassium sorbate suspension and place in an 85°C water bath; mix 0.9 mL of bacterial suspension with 8.1 mL of nutrient broth medium as a blank control group. Finally, the total bacterial count of each group was measured at 0, 4, 8, 12, 16, and 20 min to plot the heat sterilization curves of Staphylococcus aureus and Bacillus subtilis. The heat sterilization curves are shown in the appendix. Figure 1-4 To calculate D 85 Value, D 85 The values ​​are shown in Table 1.

[0084] The lactic acid bacteria fermentation products obtained in Examples 1-3 and Comparative Examples 1-6, along with food-grade preservative potassium sorbate, were added to the brine at a rate of 0.1% of the total weight of the brine and tofu embryos. After thorough mixing, the tofu embryos were added, and the mixture was braised at 90°C for 60 minutes. The braised tofu was then transferred to a clean cooling room and cooled to a core temperature of 25°C, taking care to prevent condensation from forming on the surface. After cooling, the tofu was vacuum-packed using food-grade composite vacuum packaging bags (barrier ≥90%, meeting the requirements of GB / T 21302-2007). After packaging, the tofu was pasteurized at 85°C for 20 minutes. The blank control group was also subjected to autoclaving at 121°C for 20 minutes. The total bacterial count was measured on day 0 and day 30, and sensory evaluation was performed.

[0085] Sensory evaluation of braised tofu: The sensory evaluation was conducted in accordance with the sensory indicators of the industry standard SB / T 10632-2011 "Braised Tofu". The specific sensory evaluation indicators are shown in Table 2, and the test results are shown in Table 3.

[0086] Table 1. D values ​​of lactic acid bacteria ferments obtained in Examples 1-3 and Comparative Examples 1-6, and control samples. 85 Value measurement results

[0087] As shown in Table 1, the lactic acid bacteria fermentation product prepared in this application significantly reduced the D of Staphylococcus aureus and Bacillus subtilis. 85 The values ​​decreased by more than 50% and 40%, respectively. This indicates that the lactic acid bacteria ferment can significantly enhance the heat sensitivity of Staphylococcus aureus and Bacillus subtilis, and its synergistic effect is significantly better than that of the comparative ratio and the conventional preservative potassium sorbate.

[0088] Table 2 Sensory evaluation indicators for braised tofu

[0089] Table 3. Effects of lactic acid bacteria fermentation products prepared in Examples 1-3 and Comparative Examples 1-6 on braised tofu.

[0090] As shown in Table 3, the lactic acid bacteria fermentation product prepared in this application can lower the sterilization temperature of braised tofu, allowing tofu that originally required high-temperature sterilization to extend its shelf life to achieve the same shelf life in days under pasteurization. This solves the problem of high-temperature sterilization damaging the taste of tofu and effectively improves the food consumption experience. It provides a natural and efficient new solution for food preservation with broad application prospects.

[0091] The specific embodiments described herein are merely illustrative of this application and are not intended to limit it. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing a lactic acid bacteria ferment, characterized in that, The preparation steps include the following: Step 1: Inoculate the first fermentation bacteria into the fermentation substrate, and use dynamic pH gradient control to perform anaerobic fermentation for 10-12 hours to obtain the first fermentation broth; Step 2: Inoculate the second fermentation bacteria into the first fermentation broth, maintain the tank temperature at 28±2℃, the tank pressure at 0.03-0.05MPa, and the pH value at 5.6±0.2, and continue anaerobic fermentation for 12-18h to obtain the second fermentation broth; Step 3: After ultrafiltration, the second fermentation broth is freeze-dried under inert gas protection to obtain lactic acid bacteria fermentation product; The dynamic pH gradient control parameters in step 1 are as follows: under the conditions of tank temperature of 30±2℃ and tank pressure of 0.02-0.04MPa, the pH value is maintained at 6.5±0.2 for 0-6h, and the pH value is reduced to 6.0±0.2 for 6-12h. In step 1, the first fermentation bacteria is *Lactococcus lactis* subsp. *lactococcus* (… Lactococcus lactis subsp. Lactis The preservation number is CICC 6242, and the inoculum size for the first fermentation strain is 1.5-3% of the fermentation substrate mass. In step 2, the second fermentation bacteria consists of *Lactobacillus rhamnosus* with an effective viable cell ratio of 1-2:2-3. Lactobacillus rhamnosus ), with accession number CGMCC 1.2467 and Lactobacillus paracasei ( Lacticaseibacillus paracasei The composition is as follows: (CGMCC 1.2468); the inoculum amount of the second fermentation bacteria in step 2 is 2-5% of the mass of the first fermentation broth; The ultrafiltration membrane used in step 3 has a molecular weight of 500-1000 Da.

2. The method for preparing lactic acid bacteria fermentation products according to claim 1, characterized in that, The fermentation base material in step 1, by weight percentage, includes: inulin 3-8%, pea protein peptide 2-5%, yeast extract 2-5%, sodium selenite 0.01-0.03%, vitamin E succinate 0.05-0.1%, and the balance is deionized water.

3. The application of a lactic acid bacteria ferment prepared by the method described in any one of claims 1-2 in food preservation.

4. The application of the lactic acid bacteria fermentation product according to claim 3 in food preservation, characterized in that, The lactic acid bacteria ferment can lower the sterilization temperature of braised tofu, reducing it from 121℃ and 20min to 85℃ and 20min, thereby improving the taste of braised tofu while maintaining the original shelf life.

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