Weissella sinus, application of Weissella sinus and method for fermenting salt-reduced soy sauce

Abstract

The invention discloses Weissella cibaria, application of the Weissella cibaria and a method for fermenting salt-reduced soy sauce, and belongs to the technical field of food fermentation. Weissella cibaria Wc-M1 has better antibacterial ability and acid production characteristic under low salt concentration. The Weissella cibaria Wc-M1 is applied to fermentation of the salt-reduced soy sauce; the salt-reduced soy sauce fermentation method comprises the following steps: raw material treatment; making koji; uniformly mixing the finished product koji with saline water according to a certain mass ratio to prepare salt-reduced soy sauce mash, inoculating the bacterium liquid of the Weissella cibaria Wc-M1 into the salt-reduced soy sauce mash, and performing soy sauce salt-reduced fermentation at a certain temperature for 90 days to prepare salt-reduced raw soy sauce; performing squeezing; blending and sterilizing; the salt-reduced soy sauce fermented by the Weissella cibaria Wc-M1 has good physicochemical properties, the contents of acids, esters and guaiacol are particularly prominent, and the salt-reduced soy sauce is endowed with a strong flavor characteristic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of food fermentation technology, in particular to a Weissella cibaria and application thereof and a method for reducing salt content of soy sauce fermentation. BACKGROUND

[0002] Soy sauce originated in China and evolved from sauce and fermented soybeans, and has a long history and culture, and is a typical traditional oriental seasoning. As the first country in the production and consumption of soy sauce, large-scale soy sauce enterprises in China mostly adopt high-salt dilute-state fermentation process to produce soy sauce with rich ester and alcohol flavors. However, most of the high-salt dilute-state soy sauce on the market has a salt content of 16-22%, which conflicts with the current low-salt healthy diet culture, and thus, the reduction of salt content of soy sauce to replace high-salt soy sauce has become an irreversible trend.

[0003] However, the current production of soy sauce with reduced salt content by diluting high-salt dilute-state soy sauce has the following problems: (1) dilution will cause a decrease in the content of amino acid nitrogen, resulting in a decrease in the grade of soy sauce, and the addition of monosodium glutamate is usually used to ensure the grade of soy sauce, but such products are not clean label products, and thus, the market acceptance is decreased; (2) dilution of salt also leads to a decrease in the content of soluble salt-free solids in soy sauce and an increase in water activity, which is not conducive to shelf life stability and requires the use of preservatives, which is not in line with the requirements of clean label; and (3) direct low-salt fermentation, when the salt concentration is lower than 14%, the difficulty of preventing and controlling contamination by miscellaneous bacteria during fermentation increases rapidly, and the risk of deterioration of the flavor of soy sauce also increases. To solve the above problems, the fermentation technology can be changed. Organic acids (lactic acid, acetic acid, etc.) and ethanol produced by microorganisms during soy sauce fermentation can well inhibit the spoilage microorganisms in soy sauce, and are beneficial to the flavor of soy sauce, and thus, the screening of acid-producing bacteria or ethanol-producing yeast and the application thereof in soy sauce fermentation can effectively achieve the preservation and flavor enhancement during the fermentation of soy sauce with reduced salt content, and ensure the quality of soy sauce with reduced salt content.

[0004] Weissella cibaria is a kind of lactic acid bacteria widely used in food fermentation, and is also a dominant bacterium in soy sauce fermentation, which has the characteristics of strong acid production, bacteriostasis and probiotics, and the exploration of the application of Weissella cibaria in the preservation and flavor enhancement of soy sauce fermentation with reduced salt content will help to solve the above technical difficulties in the production of soy sauce with reduced salt content.

[0005] Therefore, the present application designs a kind of Weissella cibaria and application thereof and a method for reducing salt content of soy sauce fermentation to solve the above problems. SUMMARY

[0006] The present application aims to provide a kind of Weissella cibaria and application thereof and a method for reducing salt content of soy sauce fermentation to solve the problems raised in the above background.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A Weissella cibaria strain is deposited in China General Microbiological Culture Collection Center (CGMCC), named Weissella cibaria Wc-M1 (Weissella cibaria Wc-M1), with a preservation number of CGMCC NO: 32972. The preservation date is December 9, 2024, and the address of the preservation unit is located at No. 1, Xibaheyi, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences. The DNA sequence of the Weissella cibaria Wc-M1 is SEQ ID NO: 1.

[0009] The above-mentioned Weissella cibaria Wc-M1 provided by the present application is an advantageous strain isolated and screened from traditional high-salt thin-state fermentation soy sauce; the classification and characteristics of the Weissella cibaria Wc-M1 include morphology, 16S rDNA sequence analysis, and bacteriostatic ability, acid-producing ability.

[0010] Through detection and analysis, the above-mentioned Weissella cibaria Wc-M1 is a strain with good acid-producing and bacteriostatic characteristics. The present application adds this strain to the fermentation of salt-reduced soy sauce, determines the quality indicators and flavor substance content of the salt-reduced soy sauce, and determines the application process in the fermentation of salt-reduced soy sauce.

[0011] Based on the characteristics and advantages of the Weissella cibaria Wc-M1 strain, the present application provides an application of the Weissella cibaria Wc-M1 in salt-reduced fermented soy sauce.

[0012] The present application also provides a method for fermenting salt-reduced soy sauce, which is realized based on the above-mentioned Weissella cibaria and includes the following steps:

[0013] S1, raw material processing and koji preparation: the raw material soybean meal is cooked, and the wheat is roasted; after mixing, Aspergillus oryzae Huon 3.042 is inoculated, and the fermented koji is obtained;

[0014] S2, preparation of salt-reduced soy sauce with finished koji: the finished koji is mixed with brine at a mass ratio of 1:2 to prepare salt-reduced soy sauce with a salt content of 10% to 14%;

[0015] S3, Weissella culture: the Weissella cibaria Wc-M1 is cultured in a modified MRS medium to an appropriate bacterial suspension concentration to prepare an inoculum;

[0016] S4, fermentation by adding Weissella: the salt-reduced soy sauce is fermented for 90 days, with temperature control fermentation and moderate stirring; after adding an appropriate amount of Weissella cibaria Wc-M1 within 0 to 30 days, the fermentation is continued for 60 days, and the inoculum amount is 3% to 8% (v / w) of the mass of the mash;

[0017] S5, squeezing and blending: the salt-reduced soy sauce is obtained by clarifying and diatomite filtering, white sugar and yeast extract are added for blending, and the product is obtained by high-temperature instant sterilization, cooling and membrane filtration.

[0018] Preferably, in step S1, the cooking temperature of the soybean meal is 125-145°C, and the cooking time is 15-30 minutes; the frying temperature of the wheat is 220-250°C, the frying time is 2-5 minutes, and the frying moisture is controlled at 2-6%; the soybean meal and the wheat flour are mixed at a mass ratio of (5-7):(5-3) for koji making; the inoculation amount of Aspergillus oryzae is 3-5 parts per million of the total mass of the mixed raw materials; the koji making temperature is 30-34°C, the relative humidity is 85-90%, the koji is turned in time when it is clumped, and the koji making time is 40-44 hours.

[0019] Preferably, in step S2, the mass fraction of the salt water is 15-21 g / 100 mL, the product koji is mixed with the salt water at a ratio of 1:2, and the salt concentration of the prepared salt-reduced soy sauce mash is 10%-14%.

[0020] Preferably, in step S3, the improved MRS medium is prepared as follows: 5-10 g of proteose peptone, 5-10 g of beef infusion powder, 3-5 g of yeast extract powder, 20-50 g of glucose, 1-2 mL of Tween-80, 1-2 g of potassium phosphate dibasic, 3-5 g of sodium acetate, 1-2 g of triammonium citrate, 0.1-0.5 g of magnesium sulfate, 0.05-0.25 g of manganese sulfate, 5-1 g of NaCl, and 1000 mL of distilled water, and the pH is adjusted to 5.5-5.8, and the medium is sterilized at 115-121°C for 20 minutes; 15-20 g of agar powder is added for preparation of a solid medium.

[0021] Preferably, in step S3, the Weissella cibaria Wc-M1 is inoculated on an improved MRS slant with a salt concentration of 5-8%, and cultured at 30-35°C for 24-36 hours; the Wc-M1 strain is transferred to an improved MRS liquid medium with a salt concentration of 5-8% at 3-5 rings, and cultured at 30-35°C with 120 rpm oscillation for 18-24 hours until OD 600 ≥1; the Wc-M1 strain is suspended in an improved MRS liquid medium with a salt concentration of 8-12% at an inoculation amount of 5% (v / v), and cultured at 30-35°C with 120 rpm oscillation for 36-42 hours until OD 600 ≥1.2, as the inoculum for salt-reduced soy sauce fermentation.

[0022] Preferably, in step S4, the salt-reduced soy sauce mash is fermented for 90 days, the whole process is constant temperature control at 28-35 DEG C, the initial pH value of fermentation is natural; 0-20d, compressed air stirring once every 2-5 days; 20-50d, compressed air stirring once every 7-14 days; 50-90d, compressed air stirring once every 14-21 days; 0-30d of fermentation, inoculate Weissella cibaria Wc-M1, and the inoculation amount is 1x10 8 ~9x10 10 CFU per L of salt-reduced soy sauce mash.

[0023] Preferably, in step S5, the proportion of the preparation is: the fresh soy sauce is obtained by clarifying and filtering with diatomite; the preparation adopts 0-3% of white granulated sugar and 0-0.3% of yeast extract, and the salt-reduced soy sauce does not need to add MSG; sterilization is carried out by adopting 115-130 DEG C, 5-15s of cooling, and then membrane filtration to obtain the salt-reduced finished product soy sauce.

[0024] Compared with the prior art, the beneficial effects of the present application are: the Weissella cibaria Wc-M1 is isolated from soy sauce mash for the first time, and there is no reported application example of the Weissella cibaria Wc-M1 in salt-reduced soy sauce fermentation at present, the present application applies the Weissella cibaria Wc-M1 to the fermentation of salt-reduced soy sauce, and determines and analyzes the content of total acid, amino acid nitrogen, reducing sugar and volatile flavor components in the salt-reduced fermentation soy sauce with the addition of the Weissella cibaria Wc-M1, and the salt-reduced fermentation soy sauce with the addition of the Weissella cibaria Wc-M1 has good quality and flavor characteristics. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0026] Figure 1 The colony morphology diagram of the present application after inoculating and culturing the Weissella cibaria Wc-M1 for 48h.

[0027] Figure 2 The gram staining diagram of the present application after inoculating and culturing the Weissella cibaria Wc-M1 for 48h.

[0028] Figure 3 The 16S rDNA sequence fragment electropherogram of the present application of the Weissella cibaria Wc-M1.

[0029] Figure 4 The schematic diagram of the change of the total acid content of the salt-reduced soy sauce fermentation of the present application of the Weissella cibaria Wc-M1 under different salt concentrations with time;

[0030] Figure 5 Figure for reducing sugar content of salt-reduced soy sauce fermented by Weissella cibaria Wc-M1 under different salt concentrations changing with time;

[0031] Figure 6 Figure for amino acid nitrogen content of salt-reduced soy sauce fermented by Weissella cibaria Wc-M1 under different salt concentrations changing with time;

[0032] Figure 7 Figure for key flavor substances of salt-reduced soy sauce fermented by Weissella cibaria Wc-M1;

[0033] Figure 8 Figure for sensory evaluation score of salt-reduced soy sauce fermented by Weissella cibaria Wc-M1. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0035] I. Strain screening

[0036] Weissella strains were isolated and screened from soy sauce mash by using modified MRS medium containing vancomycin (0.2 g / L), natamycin (0.2 g / L), NaCl (80 g / L) and agar powder (15 g / L);

[0037] A 10 g sample of soy sauce mash fermented for 45 days was placed in a beaker, 90 mL of sterile normal saline and glass beads were added, and the mixture was shaken at 100 rpm for 5 min. After standing at room temperature for 5 min, 1 mL of bacterial suspension was taken, gradient diluted and plated on MRS separation plates containing vancomycin (0.2 g / L) and natamycin (0.2 g / L), and incubated at 37℃ in an anaerobic tank for 1-3 days. Short rod-shaped bacterial single colonies with smooth and opaque surface, central elevation, adhesion, neat edge, milky white color and good growth were selected, and the strains were subcultured on modified MRS slant for preservation after multiple purification.

[0038] II. Strain identification

[0039] 1. Morphological characteristics

[0040] (1) The fermentation broth of the pure culture strain was gradient diluted and plated on modified MRS solid medium, and incubated at 30℃ for 48 h. The colony morphology was observed, as shown in Figure 1The colony surface is smooth and opaque, the center is raised, has viscosity, the edge is neat and milky white round, which is consistent with the colony morphology characteristics of Weissella.

[0041] (2) Gram staining was performed on Weissella cibaria Wc-M1, and the bacterial morphology was observed under an oil immersion lens. See Figure 2 It was found that the strain was a short rod-shaped bacteria that was gram-positive, non-spore-forming, non-flagellated, and non-motile, which was consistent with the microscopic morphology characteristics of Weissella.

[0042] 2. Molecular biological characteristics

[0043] Genomic DNA of the above strains was extracted using a bacterial kit from Shengong Bioengineering (Wuhan) Co., Ltd., and 16S rDNA was amplified by PCR using universal bacterial primers 27F and 1492R. The PCR product was sent to Shengong Bioengineering Co., Ltd. for sequencing, and the 16S rRNA gene sequence obtained by sequencing was submitted to GenBank for BLAST comparison.

[0044] Primer design:

[0045] Forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 2;

[0046] Reverse primer: 5'-AAGGAGGTGATCCAGCCGCA-3', SEQ ID NO: 3.

[0047] PCR reaction system (50 μL): 2 μL DNA, 2 μL upstream primer (10 μmol / mL), 2 μL downstream primer 10 μmol / mL, 5 μL 10×PCR Buffer, 3 μL Mg 2+ (25 mol / mL), 4 μL dNTP (2.5 mol / mL), 1 μL Taq DNA polymerase (5 U / μL), 34 μL double distilled water.

[0048] Reaction conditions: 95°C template DNA pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min, and the amplified product was stored at 4°C.

[0049] The PCR product was sent to Shenguo Bioengineering Company for sequencing, and the sequencing results were compared in the Genbank database of NCBI (National Center for Biotechnology Information). The results were identified as Weissella cibaria, named Wc-M1, and the DNA sequence of Weissella cibaria Wc-M1 was SEQ ID NO: 1.

[0050] III. Antibacterial characteristics of Weissella cibaria Wc-M1

[0051] 1. Indicator bacteria and culture: The main contaminant bacteria in soy sauce, Bacillus licheniformis, Bacillus megaterium, Bacillus cereus and the conditional pathogenic bacteria Escherichia coli were selected as the antibacterial indicator bacteria, and the LB culture medium was cultured at 37°C with shaking until the logarithmic phase (bacterial concentration was 10 7 CFU / mL) for standby.

[0052] 2. Preparation of cell-free supernatant of Weissella cibaria Wc-M1: The seed liquid of Weissella cibaria Wc-M1 was adjusted to OD 600 1, inoculated into modified MRS liquid medium at 1% (v / v) inoculation amount, cultured at 30°C, 120 rpm on a shaking table for 48 h, and the fermentation liquid was centrifuged at 4000 rpm for 10 min. The supernatant was collected, and the bacteria were removed by 0.22 μm filter membrane to obtain cell-free supernatant.

[0053] 3. Antibacterial performance of Weissella cibaria Wc-M1: 50 mL of the lower medium was poured into a plate after melting, and after it solidified, the Oxford cups were placed on the surface of the medium in a 3x3 manner. After adding 200 μL of indicator bacteria solution to the upper plate medium cooled to about 56°C, pour into the plate, after it solidifies, pull out the Oxford cup and add 100 μL of Wc-M1 cell-free supernatant in the hole. After placing the plate in a refrigerator at 4°C for 4 h, culture at 37°C for 8 h, observe and measure the diameter of the inhibition zone.

[0054] The results are shown in Table 1, and the four indicator bacteria have good inhibition effect on the four indicator bacteria, and the inhibition zone diameter from large to small is Bacillus cereus, Escherichia coli, Bacillus megaterium and Bacillus licheniformis, and the inhibition zone diameter is 16.51, 15.39, 13.34 and 9.42 mm, respectively. It shows that Weissella cibaria Wc-M1 can effectively inhibit three kinds of soy sauce contaminated Bacillus, and can also inhibit the conditional pathogenic bacteria Escherichia coli.

[0055] Table 1 results of the inhibition test of weissella cibaria wc-m1 (mm)

[0056]

[0057] IV. Acid production performance of weissella cibaria wc-m1

[0058] The activated weissella cibaria wc-m1 bacteria concentration was adjusted to OD 600 =1, inoculated in 10-14g / 100ml salt concentration MRS liquid medium at 1% (v / v) inoculation amount, cultured at 30℃, 120rpm in a shaking flask for 48h, and the total acid content was determined according to national standard GB / T12456-2021.

[0059] The results are shown in table 2, weissella cibaria wc-m1 has good acid production capacity at low salt concentration, compared with the total acid content of 14g / 100ml (0.65g / 100ml), the total acid content is increased by 36.92% and 76.92% respectively at salt concentration of 10-12g / 100ml, and the total acid content does not exceed the national standard requirement (≤2.5g / 100ml).

[0060] Table 2 acid production capacity of weissella cibaria wc-m1

[0061]

[0062] V. Expansion culture of weissella cibaria

[0063] Weissella cibaria wc-m1 was inoculated in modified MRS slant with salt concentration of 5%, cultured at 30℃ for 24h, and then 5 circles of weissella cibaria wc-m1 slant strain were transferred to modified MRS liquid medium with salt concentration of 8%, cultured at 30℃, 120rpm for 22h, and the OD 600 was 1.08 (≥1); the weissella cibaria wc-m1 bacteria suspension was transferred to modified MRS liquid medium with salt concentration of 10%, the inoculation amount was 5% (v / v), and cultured at 30℃, 120rpm for 42h, and the OD 600 was 1.23 (≥1.2), the bacteria were collected by centrifugation, and used as inoculum for salt-reduced soy sauce fermentation.

[0064] VI. Application of weissella cibaria wc-m1 in salt-reduced soy sauce fermentation

[0065] Through detection and analysis, the above-mentioned weissella cibaria wc-m1 of the application is a strain with good acid production and inhibition characteristics, the application creatively adds this strain in salt-reduced soy sauce fermentation, determines the quality indicators and flavor substance content of salt-reduced soy sauce, and determines the application process in salt-reduced soy sauce fermentation.

[0066] A method for fermenting reduced-sodium soy sauce, using the aforementioned *Westernella vulgaris* Wc-M1, specifically includes the following steps:

[0067] (1) The temperature range for steaming soybean meal is 135℃ and the steaming time is 20 minutes; the temperature for roasting wheat is 250℃ and the roasting time is 2 minutes. The two raw materials are mixed in a 5:5 ratio.

[0068] (2) Inoculate with 0.05% Aspergillus oryzae spore powder; control the koji-making temperature at 32℃, the relative humidity at 90%, and ventilate the koji-making process for 40 hours to obtain the finished koji, with the moisture content controlled at around 32%.

[0069] (3) Mix the finished koji with 15, 18, and 21 g / 100 mL of salt water at a mass-volume ratio of 1:2 to make low-sodium soy sauce mash with salt contents of 10, 12, and 14% respectively, and the pH value is natural.

[0070] (4) The *Westernella esculenta* Wc-M1 bacterial solution was inoculated into the above-mentioned low-sodium soy sauce mash. The fermented soy sauce mash with a salt content of 12% and without the addition of *Westernella esculenta* Wc-M1 was used as the control group (CK). Fermentation was carried out at 30°C for 90 days. From 0 to 20 days, the mixture was stirred with compressed air once every 2 days. From 20 to 50 days, the mixture was stirred with compressed air once every 7 days. From 50 to 90 days, the mixture was stirred with compressed air once every 14 days.

[0071] (5) Following step (4) above, on the 20th day of fermentation of the reduced-sodium soy sauce, add 1×10 mg of the reduced-sodium soy sauce mash per L. 8 The CFU inoculation amount was used to inoculate Weissella esculenta Wc-M1 into the reduced-salt soy sauce mash, and the mash was cultured for 70 days to obtain reduced-salt fermented raw soy sauce.

[0072] VII. Physicochemical properties and volatile flavor compound analysis of reduced-salt fermented soy sauce:

[0073] The physicochemical properties and volatile flavor compounds of the reduced-sodium soy sauce prepared in Example 6 were analyzed. The results are shown in [link to relevant documentation]. Figures 4-7 See Table 3. The determination method for total acid content followed the People's Republic of China National Standard GB / T12456-2021; the determination method for reducing sugar content followed the People's Republic of China National Standard GB / T5009.7-2016; the determination method for amino acid nitrogen followed the People's Republic of China National Standard GB / T5009.39-2003; and volatile flavor compounds were analyzed using solid-phase microextraction-gas chromatography-mass spectrometry.

[0074] Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) analysis method:

[0075] Two mL of a soy sauce sample fermented by Weissella taurids Wc-M1 under low-salt conditions for 90 days was placed in a 20 mL gas chromatography-mass spectrometry (GC-MS) vial, and 2 μL of a 0.816 μg / μL 2-methyl-3-heptanone solution was added as an internal standard.

[0076] SPME conditions: Shaker heating temperature 50℃, heating and shaking time 20min, extraction 20min followed by sample injection;

[0077] GC conditions: Inlet temperature 250℃, resolution time 5 min, temperature program conditions: 40℃ held for 1 min, temperature increased to 120℃ at 5℃ / min, held for 2 min, temperature increased to 250℃ at 10℃ / min, held for 5 min, carrier gas was high-purity helium, carrier gas flow rate was 1.0 mL / min, and the injection was splitless.

[0078] MS conditions: EI ion source, electron energy 70 eV, emission current 200 μA, transfer line and ion source temperature 250 °C, ion fragment mass scan range 40–500 m / z.

[0079] Depend on Figures 4-6 It can be seen that, overall, the total acid content of *Westernella tamariscina* Wc-M1 fermentation under reduced salt conditions (10%, 12%, and 14%) for 90 days was 1.39, 1.74, and 1.32 g / 100 mL, respectively. This indicates that *Westernella tamariscina* Wc-M1 has strong acid-producing properties during the fermentation of reduced-salt soy sauce mash, which is beneficial to the conversion and accumulation of organic acids in the mash, thereby improving the flavor. At 90 days, compared with the control group, the reducing sugar content decreased by 12.57% at 12% salt content, and the amino acid nitrogen content reached as high as 1.21 g / 100 mL, exceeding that of premium soy sauce by 51.25% (premium soy sauce amino acid nitrogen ≥ 0.8 g / 100 mL), thus improving the quality of the reduced-salt soy sauce.

[0080] like Figure 7 As shown in Table 3, compared with the control group (CK) without the addition of *Wesleyanus scutellariae* Wc-M1, 13 volatile flavor compounds with a VIP value >1 were found in the reduced-sodium soy sauces with different salt contents (10%, 12%, and 14%), including 6 alcohols, 3 acids, 3 aldehydes and ketones, and 1 ester. The analysis of the types and contents of volatile flavor compounds in reduced-sodium soy sauces fermented with *Wesleyanus scutellariae* Wc-M1 at different salt contents revealed that the 12% salt content reduced-sodium soy sauce had the most types of volatile flavor compounds, with acids, esters, and guaiacol being particularly prominent. Compared with the CK group, the volatile flavor compound content was increased by 2193.71 μg / L, indicating that the reduced-sodium soy sauce fermented with *Wesleyanus scutellariae* Wc-M1 at a salt content of 12% had the best quality and flavor.

[0081] Table 3. Volatile flavor compounds in low-sodium fermented soy sauce produced by *Westernella esculenta* Wc-M1.

[0082]

[0083] VIII. Pressing, blending, and sterilization processes for preparing reduced-sodium soy sauce and its sensory evaluation:

[0084] In Example 7, the fermented low-sodium soy sauce was clarified and filtered with diatomaceous earth to obtain raw soy sauce; the blending method used 3% white sugar and 0.3% yeast extract, and this low-sodium soy sauce did not require the addition of monosodium glutamate; sterilization was carried out at 120°C for 15 seconds, followed by cooling, and then membrane filtration to obtain the finished low-sodium soy sauce.

[0085] The sensory evaluation of reduced-sodium soy sauce was conducted based on four indicators: color, taste, aroma, and appearance. The sensory evaluation panel consisted of 10 healthy and professionally trained evaluators (5 men and 5 women, aged 20-45). The scoring criteria are shown in Table 4 below. The final score for each indicator was the average of all scores given.

[0086] like Figure 8 As shown, sensory evaluations were performed on reduced-sodium soy sauce fermented with *Westernella tamariscina* Wc-M1 at different salt contents (10%, 12%, and 14%) and the control group (CK). The total sensory scores for the reduced-sodium group with added Wc-M1 were 56.78, 82.45, and 72.56, respectively, while the total sensory score for the CK group was 67.33. Furthermore, the reduced-sodium group with 12% salt content showed the best appearance, color, aroma, and flavor. In conclusion, *Westernella tamariscina* Wc-M1 achieved the best sensory score and the best enhancement effect at a 12% salt content. The reduced-sodium soy sauce fermented with *Westernella tamariscina* Wc-M1 met the standards for premium-grade soy sauce in terms of quality and flavor, achieving a 25% reduction in salt compared to high-salt, dilute-state fermented soy sauce (salt content ≥16g / mL).

[0087] Table 4 Sensory Evaluation Criteria for Reduced-Salt Fermented Soy Sauce with Weissella esculenta Wc-M1

[0088]

[0089]

[0090] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0091] The preferred embodiments of the application disclosed above are only used to illustrate the present application. The preferred embodiments are not meant to limit the present application to any particular detailed embodiments. Obviously, many modifications and variations are possible in light of the above teachings. It is intended that the scope of the application be limited only by the widest range of equivalency to the claims below and their full scope of equivalents.

Claims

1. A Weisella cibaria, characterized in that, The strain is preserved in the China General Microbiological Culture Collection Center, and is named Weissella cibaria Wc-M1, with a preservation number of CGMCC NO: 32972. The DNA sequence of the Weissella cibaria Wc-M1 is SEQ ID NO:

1.

2. The use of the Weissella cibaria in claim 1 in the fermentation of reduced-salt soy sauce.

3. A method for reducing salted soy sauce fermentation, characterized by, The method is realized based on the Weissella cibaria in claim 1, and comprises the following steps: S1, raw material processing and koji making: the raw material soybean meal is cooked, and the wheat is roasted; after the two are mixed, Aspergillus oryzae Huonqian 3.042 is inoculated, and fermentation is performed to obtain finished koji; S2, preparation of reduced-salt soy sauce: the finished koji is mixed with brine at a mass ratio of 1:2 to prepare reduced-salt soy sauce with a salt content of 10% to 14%; S3, cultivation of Weissella: the Weissella cibaria Wc-M1 is cultured in a modified MRS medium to a proper concentration of bacterial suspension to prepare inoculum; S4, fermentation after adding Weissella: the reduced-salt soy sauce is fermented for 90 days, and the whole process is controlled in temperature and fermented with moderate stirring; after the appropriate amount of Weissella cibaria Wc-M1 is added within 0 to 30 days, the fermentation is continued for 60 days; the amount of added bacteria is 3% to 8% (v / w) of the mass of the soy sauce; S5, pressing and blending: the reduced-salt raw soy sauce is obtained by clarifying and filtering with diatomite; white granulated sugar and yeast extract are added for blending; after high-temperature instant sterilization, the temperature is lowered, and membrane filtration is performed to obtain reduced-salt finished soy sauce.

4. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S1, the cooking temperature of the soybean meal is 125 to 145°C, and the cooking time is 15 to 30 minutes; the cooked soybean meal is yellow-brown in appearance, has no stickiness and no bad smell; the roasting temperature of the wheat is 220 to 250°C, and the roasting time is 2 to 5 minutes; the roasted wheat is yellow-brown in color, uniform in state and free of raw wheat, and the moisture content is 2% to 6%; The soybean meal and the wheat flour are mixed at a mass ratio of (5 to 7):(5 to 3) for koji making; the inoculation amount of Aspergillus oryzae is 3 to 5 parts per million of the total mass of the mixed raw materials; the koji making temperature is 30 to 34°C, and the relative humidity is 85% to 90%; the koji is turned in time when it is clumped, and the koji making is performed for 40 to 44 hours to obtain finished koji with a moisture content of 30% to 34%.

5. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S2, the mass fraction of the brine is 15 to 21 g / 100 mL, and the finished koji is mixed with the brine at a ratio of 1:2 to prepare reduced-salt soy sauce with a salt concentration of 10% to 14%.

6. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S3, the modified MRS medium is prepared: 5 to 10 g of peptone, 5 to 10 g of beef infusion powder, 3 to 5 g of yeast extract powder, 20 to 50 g of glucose, 1 to 2 mL of Tween-80, 1 to 2 g of potassium phosphate dibasic, 3 to 5 g of sodium acetate, 1 to 2 g of triammonium citrate, 0.1 to 0.5 g of magnesium sulfate, 0.05 to 0.25 g of manganese sulfate, 5 to 1 g of NaCl, and 1000 mL of distilled water are added, the pH is adjusted to 5.5 to 5.8, high-temperature sterilization is performed at 115 to 121°C for 20 minutes, and 15 to 20 g of agar powder is added for preparation of a solid culture medium.

7. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S3, Weissella cibaria Wc-M1 is inoculated on modified MRS slant with salt concentration of 5-8%, and incubated at 30-35℃ for 24-36h. Then, 3-5 loops of Weissella cibaria Wc-M1 slant strain are transferred to modified MRS liquid medium with salt concentration of 5-8%, and incubated at 30-35℃ with 120rpm shaking for 18-24h until OD 600 ≥1.

2. The fermentation inoculum can be used for salt-reduced soy sauce fermentation. 600 ≥1.

2. The fermentation inoculum can be used for salt-reduced soy sauce fermentation.

8. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S4, the salt-reduced soy sauce mash is fermented for 90 days, with constant temperature control at 28-35℃, and the initial pH value is natural; 0-20 days, compressed air stirring once every 2-5 days; 20-50 days, compressed air stirring once every 7-14 days; 50-90 days, compressed air stirring once every 14-21 days; 0-30 days of fermentation, inoculating with Weisella cibaria Wc-M1 at an inoculation amount of 1×108-9×1010 CFU per L of salt-reduced soy sauce mash.

9. The method of fermenting reduced-salt soy sauce according to claim 3, characterized by, In step S5, the proportion is adjusted as follows: clear soy sauce is obtained by adding diatomite to the salt-reduced soy sauce mash; white granulated sugar 0-3% and yeast extract 0-0.3% are used for adjustment, and the salt-reduced soy sauce does not need to add MSG; sterilization is carried out at 115-130℃ for 5-15 s, followed by cooling, and membrane filtration to obtain the final product of salt-reduced soy sauce.

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