A strain of *Weissella violaceum* and its application in flavor improvement of soy sauce

Abstract

This invention discloses a strain of *Westernella greenis* and its application in flavor improvement in soy sauce, belonging to the field of microbial fermentation agent technology. The *Westernella greenis* F2 provided by this invention can significantly enhance the flavor of fermented seasonings, increase the content of volatile substances in fermented soy sauce, and increase the content of characteristic flavor compounds such as isoamyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, guaiacol, furfural, and pyrazines. Simultaneously, it increases the levels of ethyl benzoate and ethyl acetate, which can mask undesirable flavors, demonstrating very broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation agent technology, specifically to a strain of *Westernella greenis* and its application in improving the flavor of soy sauce. Background Technology

[0002] Soy sauce, a traditional Chinese condiment, boasts a unique flavor and rich nutritional value. High-salt, low-temperature fermentation is one of the most widely used soy sauce fermentation processes. It involves using soybeans or defatted soybeans, wheat or wheat flour as raw materials, which are steamed, fermented with Aspergillus oryzae to form a koji (fermentation starter), and then mixed with brine to create a mash. This mash is then fermented to produce soy sauce. Because this method involves low fermentation temperatures and high brine concentrations, the fermentation period is generally 4-6 months. Lactic acid bacteria and yeast play a major role in the flavor of soy sauce fermentation. These microorganisms fully decompose nutrients such as proteins and starches in the fermentation raw materials, producing various small-molecule compounds that impart a rich aroma and savory flavor to the soy sauce.

[0003] Soy sauce is primarily produced through natural fermentation, and the artificial addition of microbial fermentation agents is an effective way to enhance its flavor. For example, salt-tolerant yeasts (CN114480150B) can increase the content of pyrazine compounds in soy sauce; novel Aspergillus oryzae strains can shorten spore germination time, inhibit the proliferation of miscellaneous bacteria during the koji-making stage, and improve the flavor quality of soy sauce (CN117660196A); and Bacillus amyloliquefaciens (CN116376736A) can enhance the umami flavor of soy sauce obtained through low-salinity fermentation. Current flavor improvement technologies mainly rely on sensory evaluation, which has a certain degree of subjectivity. Volatile compounds such as phenylethanol, isoamyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, ethyl benzoate, ethyl acetate, furfural, benzaldehyde, phenylacetaldehyde, and guaiacol contribute positively to the flavor of soy sauce. Therefore, new technologies should be developed to increase the content of these compounds. Developing new lactic acid bacteria strains suitable for brewing soy sauce is an urgent technical problem to be solved in this field for improving soy sauce flavor. Weissella is a type of lactic acid bacteria found in fermented foods. This bacteria may accelerate protein hydrolysis and increase the content of volatile esters, aldehydes, alcohols and ketones to give the food a rich aroma and fruity flavor. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of green Weissella to enhance the flavor of brewed soy sauce.

[0005] This invention is achieved through the following technical solution:

[0006] A strain of *Weissella viridescens* F2, accession number CGMCC No. 22644, was deposited on May 31, 2021, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 10010, China.

[0007] This invention provides a method for improving the flavor of brewed soy sauce using *Weissella viridescens* F2, comprising the following steps: using defatted soybeans and roasted wheat as raw materials, preparing koji with *Aspergillus oryzae*, and then carrying out high-salt fermentation using a high-salt, thin-state soy sauce brewing method. *Weissella viridescens* is introduced during the early stage of fermentation (0-15 days, preferably 15 days) to achieve a concentration of (1×10⁻⁶) in the mash. 6 )-(1×10 8 The soy sauce concentrate is obtained by pressing, filtering, and collecting the concentrate after fermentation for 120-180 days at a concentration of CFU / g.

[0008] In a preferred embodiment, the method for improving the flavor of brewed soy sauce using *Westernella greenis* includes the following steps: Wheat is washed and stir-fried over low heat until golden brown, then ground and sieved (50-100 mesh); defatted soybeans are soaked in water for 2-3 hours, then sterilized at 121℃ for 15 minutes. When the defatted soybeans cool to approximately 40℃, roasted wheat flour is added to them at a mass ratio of defatted soybeans to roasted wheat flour of 7:3. Then, 0.2% w / w of commercially available soy sauce koji is added, the koji thickness is 2-3 cm, the koji temperature is 30℃, and the koji time is 48 hours. The koji obtained from the raw material koji is added to brine at a mass ratio of 1:1.9 (salt water concentration is 23%), the fermentation temperature is 26℃, and the mixture is stirred once daily for the first 30 days of fermentation, and once weekly thereafter. *Westernella greenis* F2 is added after 15 days of fermentation to achieve a *Westernella greenis* content of 10% in the fermented mash. 7 The concentration was approximately CFU / mL. After fermentation for 180 days, the soy sauce concentrate was collected by pressing and filtration. The preservation number of the *Westernella greenis* F2 strain is CGMCC No. 22644.

[0009] In a preferred embodiment, the preparation method of the *Westernella greenis* F2 bacterial suspension is as follows: *Westernella greenis* F2 is streaked onto an MRS agar plate and incubated at 30°C with oxygen for 24 hours. A single colony is then picked and inoculated into MRS broth medium and incubated at 30°C with oxygen for 12 hours. The bacterial cells are collected by centrifugation and diluted with 0.85% physiological saline to a final concentration of (1×10⁻⁶). 6 )~(1×10 8 CFU / mL;

[0010] The MRS agar medium consists of the following components: peptone 10.0 g / L, beef meal 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, and a pH of 6.5 ± 0.2 at 25°C.

[0011] The MRS broth culture medium consisted of: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80, with a pH of 5.7 ± 0.2 at 25°C.

[0012] The present invention also provides the application of Weissella viridescens F2 in brewing seasonings, including but not limited to soy sauce.

[0013] The present invention also provides the application of Weissella viridescens F2 in improving the flavor of soy sauce.

[0014] In one embodiment, the improvement of soy sauce flavor includes increasing the content of characteristic flavor compounds such as isoamyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, guaiacol, furfural, and pyrazines, while increasing the content of ethyl benzoate and ethyl acetate, which can mask undesirable flavors.

[0015] In one embodiment, 1×10⁻⁶ ppm is inoculated into the soy sauce raw materials. 6 )-(1×10 8 Fermentation was carried out using CFU / mL of *Westernella greenis* F2.

[0016] Beneficial effects:

[0017] The *Westernella greenis* F2 provided by this invention, when added as a starter culture in high-salt, dilute-state fermented soy sauce, significantly increases the content of the main volatile flavor alcohols, phenols, aldehydes, and pyrazines in the soy sauce by 63.32%, 54.05%, 38.35%, and 70.53%, respectively. It also significantly increases the content of characteristic flavor compounds 3-methylthiopropanol, guaiacol, furfural, and ethyl benzoate by 110.34%, 59.87%, 55.27%, and 81.92%, respectively. This significantly enhances the flavor of the fermented seasoning and has very broad application prospects.

[0018] Preservation of biological materials

[0019] A strain of *Weissella viridescens* F2, taxonomically named *Weissella viridescens*, was deposited on May 31, 2021, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 22644. Detailed Implementation

[0020] The invention will be further illustrated below through specific implementation examples.

[0021] The *Weissella viridescens* F2 species involved in the following examples is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 22644.

[0022] The above-mentioned *Westernella greenis* F2 strain was isolated from fermented food and identified as *Westernella greenis* using the universal bacterial primer 27F / 1492R for 16S rDNA sequencing.

[0023] The preservation method of the green Weissella F2 is as follows: Select a well-grown single colony from an MRS agar plate and inoculate it into 5 mL of MRS broth medium. Incubate at 30°C with oxygen for 24 h. Take 1 mL of the culture medium and add it into a cryovial containing 1 mL of 30% glycerol. Store at -80°C.

[0024] The cultivation method of the green Weissella F2 is as follows: streak the preserved green Weissella on MRS agar plates, incubate at 30°C with oxygen for 24 hours, pick a single colony and inoculate it into MRS broth medium, incubate at 30°C with oxygen for 12 hours.

[0025] The MRS agar medium consisted of: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 14.0 g / L agar, and 1.0 g / L Tween 80, with a pH of 6.5 ± 0.2 at 25°C.

[0026] The MRS broth culture medium consisted of: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween 80, with a pH of 5.7 ± 0.2 at 25°C.

[0027] The commercially available soy sauce koji used in the following examples was purchased from Yishui Jinrun Biotechnology Co., Ltd., and its main component is Aspergillus oryzae; defatted soybeans were purchased from China National Cereals, Oils and Foodstuffs Corporation (Rizhao) Co., Ltd. Other experimental materials were conventional experimental raw materials or reagents.

[0028] Example 1

[0029] 1. Composition:

[0030] After washing the wheat, stir-fry it at 100℃ over low heat for 15 minutes until the surface is golden brown, then grind and sieve it (50-100 mesh). Soak defatted soybeans in water for 2-3 hours, then sterilize them at 121℃ for 15 minutes. When the defatted soybeans cool to about 40℃, add roasted wheat flour with a final mass concentration of 30% to the defatted soybeans, and then add 0.2% w / w of commercially available soy sauce koji. The koji thickness is 2-3cm, the koji temperature is 30℃, and the koji time is 48 hours to obtain the koji.

[0031] 2. Fermentation:

[0032] Add salt water (23% concentration) to the koji obtained in step 1 at a mass ratio of 1:1.9. After addition, the resulting solution contains 53% koji and 23% sodium chloride. The fermentation temperature is 26℃. Stir once daily for the first 30 days of fermentation, and once weekly thereafter. Inoculate with *Westernella greenis* F2 (CGMCC No. 22644) on day 15 of fermentation to achieve a *Westernella greenis* F2 content of 10% in the fermented mash. 7 The soy sauce is fermented for 180 days at around CFU / g, then pressed, filtered, and the original liquid is collected.

[0033] The preparation method of the *Westernella greenis* F2 bacterial suspension is as follows: *Westernella greenis* F2 is streaked on MRS agar plates and incubated at 30°C with oxygen for 24 hours. Single colonies are picked and inoculated into 5 ml of MRS broth medium and incubated at 30°C with oxygen for 12 hours. After centrifugation at 10000 r / min for 10 min, the bacterial cells are collected and the cells are prepared into 10... 8 CFU / mL bacterial suspension.

[0034] The original soy sauce solution obtained in this embodiment was used to detect the types and contents of volatile flavor compounds in the fermented soy sauce by SPME-GC-MS. The peak area of ​​the internal standard 2-octanol was used for semi-quantitative analysis of the compounds. The detection results are shown in Table 1.

[0035] Example 2

[0036] 1. Composition:

[0037] After washing the wheat, stir-fry it at 100℃ over low heat for 15 minutes until the surface is golden brown, then grind and sieve it (50-100 mesh). Soak defatted soybeans in water for 2-3 hours, then sterilize them at 121℃ for 15 minutes. When the defatted soybeans cool to about 40℃, add roasted wheat flour with a final mass concentration of 30% to the defatted soybeans, and then add 0.2% w / w of commercially available soy sauce koji. The koji thickness is 2-3cm, the koji temperature is 30℃, and the koji time is 48 hours to obtain the koji.

[0038] 2. Fermentation:

[0039] Add the prepared koji (fermented starter culture) and brine at a mass ratio of 1:1.9 (23% brine concentration). Ferment at 26°C. Stir once daily for the first 30 days of fermentation, and once weekly thereafter. Inoculate with *Westernella greenis* F2 (CGMCC No. 22644) on day 15 of fermentation to achieve a *Westernella greenis* content of 10% in the fermented mash. 7 The soy sauce concentrate is collected after fermentation for 120 days at a concentration of approximately CFU / mL and then pressed and filtered.

[0040] The preparation method of the *Westernella greenis* F2 bacterial suspension is as follows: *Westernella greenis* F2 is streaked on MRS agar plates and incubated at 30°C with oxygen for 24 hours. Single colonies are picked and inoculated into 5 ml of MRS broth medium and incubated at 30°C with oxygen for 12 hours. After centrifugation at 10000 r / min for 10 min, the bacterial cells are collected and the cells are prepared into 10... 8 CFU / mL bacterial suspension.

[0041] The original soy sauce solution obtained in this embodiment was used to detect the types and contents of volatile flavor compounds in the fermented soy sauce by SPME-GC-MS. The peak area of ​​the internal standard 2-octanol was used for semi-quantitative analysis of the compounds. The detection results are shown in Table 1.

[0042] Example 3

[0043] 1. Composition:

[0044] After washing the wheat, stir-fry it at 100℃ over low heat for 15 minutes until the surface is golden brown, then grind and sieve it (50-100 mesh). Soak defatted soybeans in water for 2-3 hours, then sterilize them at 121℃ for 15 minutes. When the defatted soybeans cool to about 40℃, add roasted wheat flour with a final mass concentration of 30% to the defatted soybeans, and then add 0.2% w / w of commercially available soy sauce koji. The koji thickness is 2-3cm, the koji temperature is 30℃, and the koji time is 48 hours to obtain the koji.

[0045] 2. Fermentation:

[0046] Add the koji obtained in step 1 to the brine at a mass ratio of 1:1 (the brine concentration is 12%). Fermentation temperature is 40℃ for the first 10 days and 35℃ for days 10-30. Stir once daily for days 0-10, every two days for days 10-20, and twice weekly for days 20-30. Inoculate with *Westernella greenis* F2 (CGMCC No. 22644) on day 10 of fermentation to achieve a *Westernella greenis* content of 10% in the fermented mash. 7 The soy sauce is fermented for 30 days at around CFU / g, then pressed, filtered, and the original liquid is collected.

[0047] The preparation method of the *Westernella greenis* F2 bacterial suspension is as follows: *Westernella greenis* F2 is streaked on MRS agar plates and incubated at 30°C with oxygen for 24 hours. Single colonies are picked and inoculated into 5 ml of MRS broth medium and incubated at 30°C with oxygen for 12 hours. After centrifugation at 10000 r / min for 10 min, the bacterial cells are collected and the cells are prepared into 10... 8 CFU / mL bacterial suspension.

[0048] The original soy sauce solution obtained in this embodiment was used to detect the types and contents of volatile flavor compounds in the fermented soy sauce by SPME-GC-MS. The peak area of ​​the internal standard 2-octanol was used for semi-quantitative analysis of the compounds. The detection results are shown in Table 1.

[0049] Comparative Example 1: Natural Fermentation Preparation of Fermented Soy Sauce

[0050] For the specific implementation method, please refer to Example 1. The difference is that in the fermentation step, Weissella greenis F2 is not added, and natural fermentation is carried out.

[0051] Comparative Example 2: Soy sauce fermented with single-strain halophilic tetracoccus ATCC 33315

[0052] For the specific implementation method, refer to Example 1, except that in the fermentation step, halophilic tetracoccus ATCC 33315 is used to replace green Weissella F2.

[0053] The preparation method of the halophilic tetracoccus ATCC 33315 bacterial suspension is as follows: Halophilic tetracoccus ATCC 33315 is streaked on MRS agar plates and incubated at 30°C for 24 hours. A single colony is picked and inoculated into 5 ml of MRS broth medium and incubated at 30°C for 12 hours. After centrifugation at 10000 r / min for 10 min, the bacterial cells are collected. The bacterial cells are then diluted with sterile physiological saline (0.85% sodium chloride aqueous solution, w / v, g / mL) to prepare 10... 8 CFU / mL bacterial suspension.

[0054] The original soy sauce solution obtained in this comparative example was used to detect the types and contents of volatile flavor compounds in the fermented soy sauce by SPME-GC-MS. The peak area of ​​the internal standard 2-octanol was used for semi-quantitative analysis of the compounds. The detection results are shown in Table 1.

[0055] The effects of *Westernella greenis* F2 (CGMCC No. 22644) on flavor compounds in brewed soy sauce are shown in Table 1.

[0056] Table 1. Effects of *Westernella viride* F2 (CGMCC No. 22644) on flavor compounds in brewed soy sauce.

[0057]

[0058]

[0059] Experimental results showed that inoculation with *Westernella viride* F2 (CGMCC No. 22644) significantly improved the flavor of brewed soy sauce, and the volatile flavor compounds in the soy sauce were significantly increased. In Examples 1, 2, and 3, the total amount of volatile flavor compounds increased by 58.82%, 17.40%, and 14.90% compared to the naturally fermented soy sauce in Example 1, respectively. In Table 1, the levels of aldehydes, esters, phenols, and alcohols in Example 1 increased by 38.35%, 22.65%, 54.05%, and 63.32% compared to Example 1, respectively.

[0060] Compared to Comparative Example 2, under the same fermentation conditions, inoculation with *Westernella viride* F2 (CGMCC No. 22644) in Example 1 significantly increased the content of volatile flavor compounds in soy sauce by 40.02%. Among these, the contents of the main characteristic alcohol flavor compounds, isoamyl alcohol, 1-octen-3-ol, and 3-methylthiopropanol, significantly increased by 77.98%, 29.66%, and 58.24%, respectively. 3-Methylthiopropanol has an extremely strong aroma, producing a strong broth-like aroma even at low concentrations, making a significant contribution to the aroma of soy sauce. Esters provide floral and sweet notes to soy sauce. Although the number of ester types in Example 1 was less than in Comparative Example 2, the total ester content increased by 16.91%. The content of ethyl benzoate, which can mask undesirable flavors such as bitterness from fatty acids, significantly increased by 67.11%. Ethyl acetate was only detected in Example 1. The content of pyrazine compounds (2,5-dimethylpyrazine and 2,6-dimethylpyrazine) was significantly increased by 86.17%. Pyrazine compounds have a nutty, roasted aroma and good aroma diffusion, making a unique and significant contribution to the aroma of soy sauce. Coconut aldehyde has a coconut aroma and plays a certain harmonizing role in the aroma of soy sauce; coconut aldehyde was only detected in Example 1. In addition, compared with Comparative Example 2, the key characteristic flavor compounds guaiacol and furfural in soy sauce in Example 1 were increased by 0.43 times and 1.29 times, respectively.

[0061] Therefore, *Westernella greenis* F2 (CGMCC No. 22644) can be used as a fermenting agent for soy sauce products to enhance the flavor of soy sauce.

[0062] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for improving the flavor of soy sauce, characterized by, Adding *Westernella greenii* to the soy sauce fermentation system Weissella viridescens F2, the green Weissella F2, has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 22644; The method comprises the following steps: (1) koji making: adding fried wheat flour with a final mass concentration of 30%-40% to defatted soybeans, then adding soy sauce koji extract with a total raw material mass of 0.2%-0.4% w / w, and then performing koji making to obtain koji; (2) fermentation: preparing the koji obtained in step 1 into a solution with a koji mass fraction of 50%-60% and a sodium chloride mass fraction of 20%-24%, and then performing fermentation; inoculating green Weissella F2 at the 15th day of fermentation; In step (2), Weissella viridescens F2 was inoculated at a final concentration of 1 x 10 7 CFU / g; the fermentation time was 180 days; The improvement of the soy sauce flavor comprises increasing the content of isoamyl alcohol, 1-octen-3-ol, 3-methylthiopropanol, guaiacol, furfural, pyrazine substances, ethyl benzoate and ethyl acetate.

2. The method of claim 1, wherein, In step (1), the koji making is performed at a temperature of 30-35 DEG C for 48-72 h.

3. The method of claim 1, wherein, In step (2), the fermentation temperature is 25-30 DEG C.

4. Use of Weissella viridescens F2 in brewing soy sauce, characterized in that, The green Weissella F2 has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC N0. 22644.

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