Salt-tolerant lactic acid bacteria for increasing lactic acid content in soy sauce and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses salt-tolerant lactic acid bacteria capable of increasing the lactic acid content in soy sauce and application of the salt-tolerant lactic acid bacteria. According to the application, lactic acid content detection, salt tolerance screening and salt tolerance domestication are carried out on different lactic acid bacteria, a strain of salt-tolerant plantarum gxas-G with high lactic acid yield is finally screened out, soy sauce fermentation tests are carried out by utilizing the strain, and the strain is found to be capable of remarkably increasing the lactic acid content in soy sauce, so that the yield of the lactic acid in the soy sauce is increased, and the yield of the lactic acid in the soy sauce is increased. Therefore, the strain not only has excellent performance under laboratory conditions, but also has very high potential application value in actual production. Therefore, a new method is provided for increasing the lactic acid content in the soy sauce, and the method has important significance in the soy sauce brewing industry.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a salt-tolerant lactic acid bacterium for increasing the lactic acid content in soy sauce and its application. Background Art

[0002] The soy sauce fermentation process relies on three major bacteria: Aspergillus niger, yeast, and lactic acid bacteria. Aspergillus niger is a key strain in koji-making, yeast is a fungus that plays a major role in flavor formation in the later stages of mash fermentation, and lactic acid bacteria, as the primary flavor-enhancing bacteria, participate in the entire fermentation process. During the koji-making stage, the addition of lactic acid bacteria can significantly improve the quality of soy sauce and inhibit contamination by bacteria and other harmful substances. During the mash stage, the addition of salt-tolerant lactic acid bacteria can significantly promote yeast fermentation, inhibit browning, and improve the nutritional value of the soy sauce.

[0003] While exogenously added lactic acid bacteria offer numerous benefits, they also have potential drawbacks. For example, they may negatively impact the growth of Aspergillus niger during the koji-making and fermentation stages, and may be inhibited by yeast at the end of the mash fermentation process due to their limited salt and acid tolerance. Therefore, selecting the appropriate lactic acid strain is crucial in actual production. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a salt-tolerant lactic acid bacteria for increasing the lactic acid content in soy sauce and its application. The salt-tolerant lactic acid bacteria of this application can significantly increase the lactic acid content in soy sauce, opening up a new way to improve the quality of soy sauce.

[0005] To achieve the above objectives, the technical solutions adopted in this application are:

[0006] The present application provides a salt-tolerant lactic acid bacterium, which is Lactiplantibacillus plantarum gxas-G, which has been deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit number CGMCC No: 32924 and the deposit date of December 5, 2024.

[0007] In the technical solution of the present application, by testing the lactic acid content, screening for salt tolerance and acclimating different lactic acid bacteria, a strain of Lactobacillus plantarum gxas-G was finally screened out. This strain has the advantages of salt-tolerant growth and fermentation stability, and high lactic acid production. This strain can stably and significantly increase the lactic acid content of soy sauce, opening up a new way to improve the quality of soy sauce.

[0008] In some specific embodiments, the salt-tolerant lactic acid bacteria are derived from samples such as soy sauce, pickled foods, and commercially available probiotics.

[0009] In some specific embodiments, the method for screening salt-tolerant lactic acid bacteria comprises the following steps:

[0010] Samples of soy sauce, pickled foods, and commercially available probiotics were diluted in a gradient manner, applied to an acid-producing separation medium, and incubated at 30°C for 24 hours. Colonies with obvious calcium dissolution zones and white or milky white color were selected and streaked on MRS solid medium. After multiple purifications, the Lactobacillus plantarum gxas-G of the present application was obtained.

[0011] The acid-producing separation medium is prepared by adding 5 g of CaCO 3 and 18 g of agar powder to the MRS liquid medium.

[0012] MRS medium (g / L) contained: glucose 10 g, yeast powder 10 g, peptone 5 g, KH2PO4 0.25 g, K2HPO4 0.25 g, MgSO4·7H2O 0.4 g, MnSO4·5H2O 0.02 g, FeSO4·7H2O 0.02 g, and NaCl 0.02 g. The pH was adjusted to 6.8.

[0013] The present application also provides the use of the above-mentioned salt-tolerant lactic acid bacteria in condiment fermentation.

[0014] As a preferred embodiment of the application described in this application, the condiment includes soy sauce.

[0015] The present application also provides the use of the above-mentioned salt-tolerant lactic acid bacteria in increasing the lactic acid content in soy sauce.

[0016] This application uses Lactobacillus plantarum gxas-G in soy sauce fermentation experiments and finds that this strain can significantly increase the lactic acid content in soy sauce. This shows that Lactobacillus plantarum gxas-G not only performs well under laboratory conditions but also has high potential application value in actual production.

[0017] As a preferred embodiment of the application described in this application, the viable count of the salt-tolerant lactic acid bacteria is greater than 2×10 8 CFU / mL.

[0018] The present application also provides the use of the above-mentioned salt-tolerant lactic acid bacteria in the preparation of a soy sauce fermentation regulator.

[0019] The salt-tolerant lactic acid bacteria screened in this application can be used as a soy sauce fermentation regulator to further regulate the lactic acid content in soy sauce.

[0020] The present application also provides a soy sauce fermentation regulator, which includes the above-mentioned salt-tolerant lactic acid bacteria.

[0021] The present application also provides a method for increasing the lactic acid content in soy sauce, comprising inoculating the above-mentioned salt-tolerant lactic acid bacteria into the soy sauce for fermentation.

[0022] The present application used the above-mentioned salt-tolerant lactic acid bacteria to conduct soy sauce fermentation experiments and found that this strain of bacteria can significantly increase the lactic acid content in soy sauce. This application provides a new method for increasing the lactic acid content in soy sauce, which is of great significance to the soy sauce brewing industry.

[0023] As a preferred embodiment of the method for increasing the lactic acid content in soy sauce described in the present application, the viable cell count of the salt-tolerant lactic acid bacteria is greater than 2×10 8 CFU / mL.

[0024] Compared with the prior art, this application has the following beneficial effects:

[0025] The present application provides a salt-tolerant lactic acid bacterium for increasing the lactic acid content in soy sauce and its application. The present application performs lactic acid content detection, salt tolerance screening and salt tolerance acclimation on different lactic acid bacteria, and finally screens out a salt-tolerant Lactobacillus plantarum gxas-G with high lactic acid production. Soy sauce fermentation experiments are carried out using this strain, and it is found that this strain can significantly increase the lactic acid content in soy sauce, indicating that this strain not only performs well under laboratory conditions, but also has high potential application value in actual production. Therefore, the present application provides a new method for increasing the lactic acid content in soy sauce, which is of great significance to the soy sauce brewing industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth curves of each strain are shown;

[0027] Figure 2 This is the lactic acid content of each strain using soy sauce as the culture medium;

[0028] Figure 3 This is a schematic diagram of koji making and fermentation;

[0029] Figure 4 This is the lactic acid content of each strain using soy sauce koji as culture medium;

[0030] Figure 5 This is the lactic acid content of each strain using soy sauce koji as culture medium;

[0031] Figure 6 This is the spot plate result diagram of 24h salt tolerance screening;

[0032] Figure 7This is the spot plate result diagram of the 48h salt tolerance screening;

[0033] Figure 8 This is the spot plate result diagram of the 72h salt tolerance screening;

[0034] Figure 9 This is the spot plate result diagram of the 96-hour salt tolerance screening;

[0035] Figure 10 This is the spot plate result diagram of salt tolerance screening for 7 days;

[0036] Figure 11 is the OD of Lactiplantibacillus plantarum gxas-G under different NaCl concentrations 600 and pH result graph;

[0037] Figure 12 This is the lactic acid content of Lactiplantibacillus plantarum gxas-G under different NaCl concentrations;

[0038] Figure 13 OD of Lactiplantibacillus plantarum gxas-G under 10% NaCl concentration 600 , pH, lactic acid content and viable bacteria count result graph;

[0039] Figure 14 This is the result of applying Lactiplantibacillus plantarum gxas-G to the soy sauce fermentation system to increase the lactic acid content of soy sauce. DETAILED DESCRIPTION

[0040] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below with reference to the accompanying drawings and specific embodiments.

[0041] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are all the same.

[0042] Lactic acid bacteria were isolated and purified from commercially available probiotics and fermented foods, and their growth curves were analyzed using MRS medium under natural oxygen conditions.

[0043] MRS medium (g / L): contains 10 g of glucose, 10 g of yeast powder, 5 g of peptone, 0.25 g of KH2PO4, 0.25 g of K2HPO4, 0.4 g of MgSO4·7H2O, 0.02 g of MnSO4·5H2O, 0.02 g of FeSO4·7H2O, and 0.02 g of NaCl. The pH was adjusted to 6.8.

[0044] Acid production separation medium (g / L): Add 5g of CaCO3 and 18g of agar powder to MRS liquid medium.

[0045] Example 1, strain screening

[0046] 1. Isolation and purification of lactic acid bacteria:

[0047] The samples of soy sauce, kimchi and compound probiotic powder were diluted with sterile saline (10 -1 to 10 -5 ) and then spread onto acid-producing isolation medium and incubate at 30°C for 24 hours. Colonies with a distinct calcium-dissolving zone and white or milky white color are selected, streaked onto MRS solid medium, and purified multiple times.

[0048] 2. Identification of lactic acid bacteria:

[0049] 2.1 Morphological observation:

[0050] Follow the standard method of Bergey's Manual of Bacterial Identification to observe the colony and bacterial morphology of the target strain.

[0051] 2.2 Molecular Biology Identification:

[0052] The target strain was inoculated into 10 mL of MRS liquid medium and cultured overnight at 30°C and 180 rpm with shaking. Genomic DNA was then extracted using the Ezup Column-Based Bacterial Genomic DNA Drawer Kit and used as a template for PCR amplification. PCR amplification was performed using the 16S rDNA universal primers 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 1) and 16S27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO: 2). The product was sent to Sangon Biotech Co., Ltd. for sequencing and verification.

[0053] 3. Determination of lactic acid bacteria growth curve:

[0054] Growth curves of different lactic acid bacteria were measured. 100 μL of culture medium was taken from glycerol tubes of Lactobacillus plantarum, Lactococcus lactis, Lactococcus cremoris, Lactobacillus fermentans, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus plantarum 2, Lactobacillus plantarum 3, and Weissella fusion, which were listed in the national standard "List of Strains Applicable to Foods, 2022, No. 14." The tubes were inoculated into 10 mL of MRS broth and incubated at 30°C and 180 rpm for 16 hours. A 1% inoculum was then transferred to MRS broth containing 0%, 8%, and 10% NaCl, respectively. The cultures were incubated at 30°C and 180 rpm, with samples taken every 6 hours until the strains showed signs of complete and stable decline.

[0055] The growth curves of each strain are shown in Figure 1 , three strains of Lactobacillus plantarum and Lactobacillus rhamnosus grew faster.

[0056] 4. Detection of lactic acid production:

[0057] Taking culture medium as the starting point, the base material and koji material in the soy sauce system were used as culture medium respectively, and fermentation experiments were carried out on the lactic acid bacteria initially screened out to detect the lactic acid production.

[0058] 4.1 Lactic acid fermentation experiment using soy sauce base as culture medium:

[0059] In the experiment, the mixing ratio of soybean flour to wheat flour was set at 7:3; the amount of water added was based on the solid-to-water mass ratio of 1:9 (i.e., 20 grams of solid components corresponded to 180 grams of water); and the inoculation ratio of bacterial liquid was 2%. During the experiment, samples were collected every 24 hours to determine the lactic acid content. Detailed results are shown in Figure 2 .

[0060] The study found that when soy sauce base was used as a culture medium for lactic acid fermentation, Lactobacillus plantarum, Lactobacillus plantarum 2, Lactobacillus plantarum 3 and Lactobacillus rhamnosus all showed excellent growth and lactic acid fermentation abilities.

[0061] Lactic acid content was determined by high performance liquid chromatography (HPLC). The specific parameters are as follows: instrument: Waters, USA; detector: UV detector; chromatographic column: Rezex TM ROA-Organic Acid H+ (8%), LC Column 300×7.8 mm; mobile phase: 2.5 mM H2SO4; column temperature: internal 35°C, external 60°C; detection wavelength: 210 nm; flow rate: 0.5 mL / min; injection volume: 20 μL; upper pressure limit: 1000 psi.

[0062] Sample treatment: The sample was diluted ten-fold with sterile water and filtered through a membrane before loading.

[0063] 4.2 Salt-free lactic acid fermentation experiment using soy sauce koji as culture medium:

[0064] According to 300g wheat flour and 700g soybeans (the weight of soybeans here is based on the weight before soaking), mix the soybeans with 2.5 times their weight of cold water and soak them in a refrigerator at 4-10℃ for about 15 hours. After soaking, drain the water with gauze. Then, wrap the soybeans with gauze or non-woven fabric and place them in a sterilizer at 118℃ for 15 minutes. Aspergillus oryzae spores (3.042 Aspergillus oryzae spore powder purchased from Shanghai Brewing) are used as koji fermentation strains and mixed evenly with wheat flour in proportion (0.07g). When the steamed soybeans are cooled to 32±1℃, add the wheat flour mixed with the strains to the soybeans, stir evenly and then carry out koji fermentation ( Figure 3 The fermentation process lasts for about 40 hours until the koji material is mature, at which point it can be used as a culture medium. When preparing the culture medium, the ratio of koji material to water should be 1:6 (i.e. 15 grams of koji material corresponds to 90 grams of water), and the addition ratio of the bacterial liquid is 5% (equivalent to 5 ml). Samples are taken every 24 hours to test the lactic acid content. For detailed results, see Figure 4 Under salt-free fermentation conditions, the lactic acid concentration showed a dynamic trend of first increasing rapidly and then decreasing with the extension of fermentation time.

[0065] 4.3 Optimization of lactic acid fermentation test using soy sauce koji as culture medium:

[0066] Accurately weigh wheat flour (300g) and soybeans (700g) in a dry weight ratio of 3:7 (the weight of the soybeans here is based on the weight before soaking). Mix the soybeans with 2.5 times their weight in cold water and soak in a refrigerator at 4-10°C for approximately 15 hours. After soaking, drain the soybeans with gauze. Wrap the soybeans with gauze or non-woven fabric and steam in an autoclave at 118°C for 15 minutes. Mix Aspergillus oryzae spores with the wheat flour at a 0.1% inoculation ratio. When the steamed soybeans cool to 30-35°C, add the wheat flour mixed with the bacteria to the soybeans, stir thoroughly, and then proceed to koji making and fermentation. The fermentation process should continue for 38 hours until the koji is fully mature. After packaging the mature koji, mix it with water in a ratio of 1:6 and inoculate 5% of the total volume with lactic acid bacteria for lactic acid fermentation. After 24 hours of fermentation, add salt at a mass volume ratio of 18g:100mL. Samples are taken every 24 hours to test the lactic acid content. For detailed results, see Figure 5 After a day of lactic acid fermentation, lactic acid rapidly accumulated in the fermentation liquid. With the addition of high salt, the lactic acid content dropped slightly and then gradually stabilized.

[0067] 5. Salt tolerance screening:

[0068] High salt separation medium (g / 100 mL): Add 0 g, 8 g, 10 g, 12 g, 14 g, and 16 g of NaCl to MRS liquid medium, respectively, and add 18 g of agar powder.

[0069] 100 μL of culture medium was taken from each glycerol tube of Lactococcus cremoris, Lactobacillus fermentans, Lactobacillus paracasei, Weissella fusion, Lactobacillus plantarum, Lactobacillus plantarum 2, Lactobacillus plantarum 3, and Lactobacillus rhamnosus and inoculated into 10 mL of MRS broth. The culture was activated at 30°C and 180 rpm for 16 hours. 2 μL of each lactic acid bacteria strain was spotted onto a high-salt isolation medium plate, with three replicates per sample.

[0070] The results of the point plate are shown in Figures 6-10 Among them, 1: Lactococcus creamer; 2: Lactobacillus fermentans; 3: Lactobacillus paracasei; 4: Weissella fusion; 5: Lactobacillus plantarum; 6: Lactobacillus plantarum 2; 7: Lactobacillus plantarum 3; 8: Lactobacillus rhamnosus.

[0071] Based on the above experimental results, the present invention screened out Lactobacillus plantarum 2 and Lactobacillus plantarum 3 with better salt tolerance. These two strains were then acclimated to salt tolerance, ultimately obtaining a strain with excellent salt tolerance, named gxas-G (the original strain before salt tolerance acclimation and screening was Lactobacillus plantarum 2).

[0072] 6. Salt-tolerant domestication and screening of lactic acid bacteria:

[0073] First, Lactobacillus plantarum 2 and Lactobacillus plantarum 3 were inoculated into MRS culture medium containing 8% sodium chloride for preliminary salt tolerance adaptation culture. During the culture process, the growth of the strains was observed every 24 hours. When the strains could grow stably and be passaged at this sodium chloride concentration, the sodium chloride concentration in the culture medium was gradually increased by 1% each time. After the strains could grow stably in a culture medium containing 9% sodium chloride, the sodium chloride concentration was gradually increased to 10% using the same method. The strains were stored in glycerol tubes during each passage. The entire acclimation process lasted about five months, during which the growth rate and lactic acid production of the strains were regularly tested to ensure the stability and functionality of the strains. Through this series of salt tolerance acclimation steps, a strain with stable growth performance and excellent salt tolerance was selected and named gxas-G. This strain not only grows well in a high-salt environment, but also maintains a high lactic acid production.

[0074] 7. Analysis of salt tolerance and acid production performance of the strain after salt tolerance acclimation:

[0075] After completing salt tolerance acclimation and screening, the salt tolerance and acid production performance of the gxas-G strain were analyzed in detail. First, the strain was cultured in different concentrations of sodium chloride medium and the OD values ​​were measured. 600 , pH and lactic acid content ( Figure 11 ) ; The strain was cultured in 10% sodium chloride medium and the OD 600 , pH, lactic acid content and viable bacteria count ( Figure 12 ) to observe their growth and acid production. 100 μL of each strain of Lactobacillus plantarum 2 (using the original strain, which had been acclimated and screened for salt tolerance, as a control) and gxas-G glycerol storage tube were inoculated into 10 mL of MRS broth and activated at 30°C and 180 rpm for 16 hours. A 1% inoculum was then transferred to MRS broth containing 0%, 8%, and 10% NaCl, respectively, and incubated at 30°C and 180 rpm. Samples were taken every 12 hours until the strain showed complete and stable decline. The experimental results showed that the gxas-G strain could grow stably in a medium containing 10% sodium chloride, indicating its good salt tolerance. Furthermore, measurements of the culture pH, viable cell count, and lactic acid concentration revealed that the strain maintained a high viable cell count and lactic acid production in a high-salt environment, indicating that salt tolerance had not affected its acid resistance and production.

[0076] Ultimately, the gxas-G lactic acid strain demonstrated excellent growth and fermentation capabilities at a 10% sodium chloride concentration. Not only did it meet the national standard for "List of Strains Applicable for Food Use, 2022, No. 14," it also exhibited high lactic acid production and demonstrated feasibility for application in soy sauce production.

[0077] The above-mentioned Lactiplantibacillus plantarum gxas-G has been deposited, its deposit number is CGMCC No: 32924, the deposit date is December 5, 2024, the depository is the General Microbiology Center of China Culture Collection Administration (CGMCC), and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0078] Example 2: Application of Lactiplantibacillus plantarum gxas-G in a practical brewing process

[0079] In this example, a soy sauce fermentation experiment was conducted, in which the Lactobacillus plantarum gxas-G strain was applied to a soy sauce system to increase the lactic acid content of the soy sauce.

[0080] The Lactobacillus plantarum gxas-G strain was inoculated into a fermentation system using soy sauce koji as the matrix, and its growth and fermentation under actual brewing conditions were observed.

[0081] Specifically:

[0082] On December 2, 2024, 210g of soybeans were soaked and sterilized at 121℃ for 15min. At 5 pm, 90g of wheat flour was used to prepare the koji.

[0083] On December 4, 2024, a total of 264 g of koji was prepared, divided into 6 equal parts, and used in each group to brew soy sauce. Detailed information for each group is shown in Table 1.

[0084] Table 1 Information of each group

[0085]

[0086]

[0087] During the fermentation process of 0 hours, 24 hours, 48 ​​hours, 72 hours, 5 days, 10 days, and 15 days, the mash was mixed and a 1 mL disposable syringe was inserted from the center of the mash to the bottom to take samples, and then centrifuged. The sampled liquid was filtered through a 0.22 μm water filter membrane, and its lactic acid content was determined. On the 20th day of the fermentation process, the middle and upper layers of the mash were taken and filtered through a neutral filter paper, and then its total acid, amino acid nitrogen and salt content were determined (Table 2), and the lactic acid content of each group at different times was measured ( Figure 14 Although the fermentation process lasted only until the 20th day, the indicators of amino acid nitrogen and total acid had reached a relatively ideal level according to the national standard for soy sauce. Figure 14 The results showed that compared with the control group and the common salt-tolerant plant lactobacillus 2 screened in the early stage, the introduction of plant lactobacillus (Lactiplantibacillus plantarum) gxas-G significantly increased the lactic acid content in soy sauce.

[0088] Table 2 Total acid, amino acid nitrogen and salt content (g / 100mL)

[0089] December 04 sample Amino acid nitrogen (titration method) Total acid Salt Control 1 0.64 0.99 18.3 Control 2 0.47 0.71 19.2 Lactobacillus plantarum 2-1 0.54 1.18 21.4 Lactobacillus plantarum 2-2 0.57 1.21 21.3 gxas-G-1 0.52 1.62 20.6 gxas-G-2 0.54 1.66 21

[0090] The experimental results showed that the lactic acid content of soy sauce samples inoculated with Lactiplantibacillus plantarum gxas-G was significantly increased. This shows that the Lactiplantibacillus plantarum gxas-G strain not only performs well under laboratory conditions but also has high potential application value in actual production. Therefore, the Lactiplantibacillus plantarum gxas-G strain is considered an ideal strain for increasing the lactic acid content of soy sauce and is expected to be widely used in the soy sauce brewing industry.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A salt-tolerant lactic acid bacterium, characterized in that The salt-tolerant lactic acid bacteria is Lactiplantibacillus plantarum gxas-G, which has been deposited in the General Microbiology Center of the China Culture Collection Administration Committee with the deposit number CGMCC No: 32924 and the deposit date of December 5, 2024.

2. Use of the salt-tolerant lactic acid bacteria as claimed in claim 1 in condiment fermentation.

3. The use according to claim 2, characterized in that The condiment includes soy sauce.

4. Use of the salt-tolerant lactic acid bacteria according to claim 1 in increasing the lactic acid content in soy sauce.

5. The use according to any one of claims 2 to 4, characterized in that: The viable count of the salt-tolerant lactic acid bacteria is >2×10 8 CFU / mL.

6. Use of the salt-tolerant lactic acid bacteria according to claim 1 in the preparation of a soy sauce fermentation regulator.

7. A soy sauce fermentation regulator, characterized in that The soy sauce fermentation regulator includes the salt-tolerant lactic acid bacteria according to claim 1.

8. A method for increasing the lactic acid content in soy sauce, characterized in that: The method comprises inoculating the salt-tolerant lactic acid bacteria as claimed in claim 1 into soy sauce for fermentation.

9. The method for increasing the lactic acid content in soy sauce according to claim 8, wherein: The viable count of the salt-tolerant lactic acid bacteria is >2×10 8 CFU / mL.

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