Strain for efficiently reducing purine content of fermented squid flavor soy sauce and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a strain for efficiently reducing the purine content of fermented squid-flavor soy sauce and application thereof. The present application successfully screens and verifies Leuconostoc mesenteroides strain NS-01. The strain NS-01 is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Yard 3, Chaoyang District, Beijing, on January 9, 2026, and has a preservation number of CGMCC No. 37336. The strain is outstanding in the production of fermented squid-flavor soy sauce and the development of low-purine fermented food, can specifically degrade four types of main purines, namely, adenine, guanine, hypoxanthine and xanthine, effectively solves the problem of excessive purine in traditional squid-flavor soy sauce, and provides a core strain for the industrialization of low-purine seafood soy sauce.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain that efficiently reduces the purine content of fermented squid-flavored soy sauce and its application. Background Technology

[0002] Soy sauce, a traditional fermented condiment in East Asia, is widely used for its unique flavor and nutritional value. In recent years, however, with increasing consumer demand for healthy foods, the high purine content of soy sauce has gradually attracted attention. Excessive accumulation of purine metabolites (such as uric acid) is closely related to metabolic diseases such as gout and hyperuricemia. Traditional soy sauces (especially flavored soy sauces made from fish and seafood, such as squid-flavored soy sauce) often have high levels of purines (adenine, guanine, hypoxanthine, and xanthine) in the finished product due to the decomposition of nucleotide-rich raw materials and microbial metabolism during fermentation, limiting their applicability to certain populations.

[0003] Currently, technologies for reducing purine content in fermented foods mainly include physical adsorption, enzymatic hydrolysis, and microbial transformation. For example, while activated carbon or resin adsorption can partially remove purines, it easily leads to the loss of flavor substances and is costly; exogenous addition of purine oxidase can degrade purines, but it suffers from poor enzyme stability and complex processes. In contrast, utilizing functional microorganisms to simultaneously degrade purines during fermentation is considered a more promising solution due to its green, economical, and compatible characteristics with traditional processes. Existing studies have shown that some lactic acid bacteria (such as Lactobacillus) can transform purines through metabolic pathways, but their efficiency is limited (usually below 20%), and they lack adaptability to complex matrices (such as seafood-flavored soy sauce), making it difficult to simultaneously achieve purine degradation and flavor preservation.

[0004] Furthermore, the production of squid-flavored soy sauce often relies on the co-fermentation of specific raw materials (such as squid by-products) with complex microbial communities. The high-protein, high-salt environment places higher demands on the tolerance and metabolic activity of functional strains. Existing research largely focuses on purine control in soybean-based soy sauce, while the development of dedicated strains for seafood-flavored soy sauce remains a gap. Therefore, screening strains with efficient purine degradation capabilities, salt tolerance, and flavor-regulating properties is crucial to overcoming the technological bottleneck of low-purine seafood soy sauce.

[0005] *Weissella*, an important member of the lactic acid bacteria group, is widely found in fermented foods. Its acid-producing, extracellular polysaccharide-producing, and antibacterial properties have been used to improve food texture and shelf life. However, the potential of this genus in purine metabolism has not been fully explored, especially its application in seafood soy sauce systems, which has not been reported. There is an urgent need to provide a new strain that can fully utilize its potential in purine metabolism. Summary of the Invention

[0006] The purpose of this invention is to provide a strain that can efficiently reduce the purine content of fermented squid-flavored soy sauce and its application. This strain has a strong ability to degrade the total purine content of soy sauce and can effectively degrade the total purine content of fermented squid-flavored soy sauce.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a strain that effectively reduces the purine content of fermented soy sauce. The strain is named NS-01 and classified under the genus *Leuconostoc mesenteroides*. The strain NS-01 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on January 9, 2026, with accession number CGMCC No. 37336.

[0008] The present invention also provides a method for screening the strain, comprising the following steps: (1) Enrichment: Take Shanghai dwarf green samples, cut them into pieces and shake them for culture. Take 1 mL of the supernatant and inoculate it into 50 mL of LMRS liquid medium. Culture continuously for 3-5 days at 25-35℃ and 150-250 rpm. (2) Spread culture: The enriched bacterial solution was serially diluted to 10. -1 -10 -8 Take 100 μL of 10 -6 -10 -8 Spread the diluted solution onto MRS solid medium and incubate upside down at 30°C for 3-5 days. (3) Isolation and purification: The cultured bacterial cells were inverted and cultured on MRS solid medium for 2 days. Single colonies were selected and streaked 6-8 times to isolate and purify them to obtain single colonies. (4) Screening: The purified single colony was inoculated into soy sauce and cultured for 3 days. The purine content of the supernatant was determined by HPLC. The strain with the best effect in reducing the total purine content was selected as NS-01.

[0009] Furthermore, in step (1), the shaking culture conditions are 30℃ and 200rpm, and the culture is carried out continuously for 4 hours before inoculation culture in MRS liquid medium.

[0010] The present invention also provides an application of the strain described above in reducing the purine content of fermented soy sauce.

[0011] Furthermore, the purines include adenine, guanine, hypoxanthine, and xanthine.

[0012] The present invention also provides the application of the strain described herein in the preparation of low-purine fermented soy sauce.

[0013] Furthermore, when applying the strain NS-01, it is cultured into a seed culture and inoculated into the soy sauce fermentation system at a 2% inoculation rate. The culture is then shaken and cultured for 4 days at 30℃ and 200rpm.

[0014] The present invention also provides an application of the strain described above in the preparation of low-purine soy products, including tofu and fermented black beans.

[0015] The present invention also provides the application of the strain described above in the preparation of low-purine aquatic fermented products, including fish sauce and shrimp paste.

[0016] The present invention also provides a compound microbial agent containing the strains, the compound microbial agent further comprising aroma-producing strains or texture-improving strains, the compound microbial agent being used to reduce the purine content of fermented foods, the fermented foods including fermented soy sauce, soy products and fermented aquatic products.

[0017] The beneficial effects of this invention are as follows: This invention successfully screened and verified the *Weissella cibaria* strain NS-01, which demonstrates significant advantages in the production of fermented squid-flavored soy sauce and the development of low-purine fermented foods. Regarding purine degradation, HPLC analysis showed that it reduced the total purine content of squid-flavored soy sauce from 86.67 g / L in the control group to 53.38 g / L, a reduction rate of 38.4%, far exceeding the efficiency of existing lactic acid bacteria (less than 20%). Furthermore, it can specifically degrade four major purines: adenine, guanine, hypoxanthine, and xanthine, effectively solving the problem of excessive purine levels in traditional squid-flavored soy sauce and providing a core strain for the industrialization of low-purine seafood soy sauce.

[0018] In terms of process compatibility and cost-effectiveness, strain NS-01 exhibits excellent salt tolerance and environmental adaptability. It can grow stably in the high-protein, 10% brine fermentation environment of squid-flavored soy sauce without requiring significant adjustments to existing processes such as mash temperature control and stirring cycles, making it perfectly compatible with traditional production processes. Compared to the drawbacks of physical adsorption methods, which are prone to flavor loss and increased costs, and exogenous enzymatic hydrolysis methods, which suffer from poor enzyme stability and complex processes, this strain simultaneously degrades purines during fermentation. This preserves the fresh and savory aroma of squid in the soy sauce while eliminating the need for additional adsorbents or enzyme preparations, significantly reducing production energy consumption and costs, thus meeting the food industry's requirements for "green, economical, and efficient" production.

[0019] Furthermore, strain NS-01 possesses DPPH free radical antioxidant activity. Its fermentation metabolites, such as acetic acid, lactic acid, and butyric acid, significantly improve the DPPH free radical scavenging rate, enhancing antioxidant performance. This adds oxidative stability to the already low-purine squid-flavored soy sauce, extending its shelf life and endowing it with dual health attributes of "low purine + antioxidant," thus broadening its market positioning. Simultaneously, this strain exhibits excellent growth characteristics, rapidly proliferating in both MRS medium and soy sauce fermentation systems. It can be compounded with aroma-producing and texture-improving strains to create complex inoculants that optimize food quality. The technological approach can also be extended to low-purine research in soy products such as tofu and fermented black beans, as well as aquatic fermented products like fish sauce and shrimp paste, filling the gap in dedicated purine-reducing strains for seafood-flavored fermented foods. This provides theoretical and practical support for the health-oriented upgrading of the fermented food industry, offering both economic and social benefits. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The image shows the PCR electrophoresis detection of strain NS-01 in this invention, where the DL2000 bands are distributed at 2000, 1000, 750, 500, 250, and 100 bp. Figure 2 The EJA1402369-1492R spectrum of strain NS-01 in this invention; Figure 3 This is a blast comparison image of strain NS-01 in this invention with the NCBI NT database. Figure 4 This is a diagram illustrating the identification of closely related species of strain NS-01 in this invention; Figure 5 This is a graph showing the DPPH free radical antioxidant activity of strain NS-01 in this invention. Figure 6 This is a graph showing the determination of purine content in fermented soy sauce before and after inoculation with strain NS-01 in this invention. Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0023] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0024] The MRS culture medium used for screening and identification of the strains in this invention was prepared according to the following method: MRS medium formulation: 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 2.0g dipotassium hydrogen phosphate, 2.0g triammonium citrate, 5.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate, 0.05g Tween 80, add ultrapure water to 1000mL, adjust pH to 6.2-6.6, autoclave at 115℃ for 15min (Note: 20.0g agar powder is added to MRS solid medium, but not to liquid medium).

[0025] Example 1 The specific purification and screening method for the strains in this invention includes the following steps: (1) Enrichment: Take the Shanghai Dwarf Green Seed sample sold on the market, cut it into pieces, and place it in an Erlenmeyer flask for shaking culture. Take 1 mL of the supernatant and inoculate it into 50 mL of MRS liquid culture medium. Culture continuously at 30℃ and 200 rpm for 4 days to enrich the bacterial cells. The shaking culture conditions are 30℃ and 200 rpm for 4 hours.

[0026] (2) Spread culture: The enriched bacterial culture was serially diluted, and 100 μL was spread on MRS solid medium and incubated at 30°C for 3 days. The gradient dilution for plating culture was performed as follows: the enriched bacterial culture sample was serially diluted to 10-1. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 and 10 -8 The liquid, 100 uL of 10 -6 10 -7 and 10 -8 The diluted solution was evenly spread on MRS solid culture medium and incubated upside down for 3 days.

[0027] (3) Isolation and purification: The cultured bacteria are isolated and purified in MRS solid medium. After inverting the culture for 2 days, single colonies are selected and streaked in MRS solid medium for isolation and purification 6-8 times until single colonies are obtained.

[0028] (4) Screening: The purified single colony was inoculated into squid-flavored soy sauce and cultured for 3 days. The purine content in the supernatant of the fermented squid-flavored soy sauce was determined by HPLC. The strain with the lowest total purine content was the strain NS-01 of this invention.

[0029] Example 2: Molecular biological identification of strain NS-01 After culturing strain NS-01 in MRS liquid medium with shaking for 3 days, centrifugation was performed. The precipitate was washed with sterile PBS buffer, centrifuged again, and the bacterial cells were collected. This process was repeated twice. The bacterial cells were then identified using Sanger sequencing. Specifically, DNA was extracted from the strain using an Ezup column-based genome extraction kit, amplified by PCR, and sequenced. The resulting PCR electrophoresis results are shown in the figure. Figure 1 As shown, the EJA1402369-1492R spectrum of this strain is as follows: Figure 2 As shown, the contig sequence of this strain is shown in SEQ ID NO.1. The sequencing results of this strain were compared with the NCBI NT database BLAST (the results are shown in...). Figure 3 As shown), strains with high sequence similarity were selected for close-related species identification (the results are shown in the figure). Figure 4 As shown). The results indicate that this strain belongs to the genus Leuconostoc (…). Weissella cibaria The strain was named NS-01 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 9, 2026, and the accession number is CGMCC No. 37336.

[0030] Example 3: Determination of DPPH free radical antioxidant capacity of strain NS-01 The NS-01 strain was cultured in a shaker to prepare a seed culture, which was then inoculated into MRS liquid fermentation medium at a 2% inoculum. The culture was incubated at 30°C and 200 rpm for 4 days. 10 mL of the fermentation broth was centrifuged at 4°C and 10,000 rpm for 10 min. The DPPH content of the supernatant was determined using a DPPH kit. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the DPPH content of the NS-01 sample was significantly higher than that of the control group, which indicates that the strain has the antioxidant capacity of DPPH free radicals. Its antioxidant capacity comes from the metabolic products of lactobacillus growth, such as acetic acid, lactic acid, butyric acid and other substances.

[0031] Example 4: Determination of purine content in soy sauce after fermentation using strain NS-01 The soy sauce used in this embodiment is squid-flavored soy sauce. Soybean meal and potato flour are mixed in a 6:4 ratio. A 10% brine solution is added, along with 1.1 times the dry weight of the raw materials. After sealing, the fermentation tank is placed in an incubator with an initial temperature of 37°C for 7 days of fermentation. The temperature is then lowered to 35°C and increased by 1°C every two days until reaching 41°C for continued fermentation, which lasts for 42 days. During fermentation, the fermentation mash is typically stirred every 2-3 days to ensure thorough mixing. For the first 3 days of fermentation, the mash is stirred every morning. After fermentation, the mash is centrifuged and filtered to obtain the finished soy sauce.

[0032] The NS-01 strain was cultured in a shaker to obtain a seed culture, which was then inoculated into the above-mentioned soy sauce at an inoculation rate of 2%. The culture was carried out at 30°C and 200 rpm for 4 days. 10 mL of the fermentation broth was centrifuged in a low-temperature centrifuge at 4°C and 10,000 rpm for 10 min. The purine content of the supernatant was determined by HPLC.

[0033] Using 0.02 mol·L -1 Potassium dihydrogen phosphate buffer was used as the mobile phase. Based on the separation properties of the C18 (4.6 mm × 250 mm, 5.0 μm) column, the pH of the mobile phase was adjusted to 3.0 with H3PO4. The mobile phase was then filtered through a 0.22 μm filter membrane. The column temperature was set at 30℃ and the flow rate at 1.0 mL / min. －1 The UV detector wavelength was 254 nm, and the injection volume was 10 μL. The prepared purine stock solution was diluted with ultrapure water to a gradient concentration of 0.05-25 μg·mL. －1 The samples were filtered through a 0.22 μm microporous membrane and analyzed sequentially to create a standard curve. Using the above HPLC conditions and standard curve, the purine content of the samples was determined and calculated. The final purine content results of strain NS-01 before and after fermentation are shown below. Figure 6 As shown, by Figure 6 It can be seen that strain NS-01 can reduce the total purine content of squid-flavored soy sauce from 86.67 g / L in the control group to 53.38 g / L, with a reduction rate of 38.4%.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain that efficiently reduces the purine content of fermented soy sauce, characterized in that, The strain was named NS-01 and classified as Leuconostoc mesenteroides. The strain NS-01 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on January 9, 2026, with accession number CGMCC No. 37336.

2. The method for screening strains as described in claim 1, characterized in that, Includes the following steps: (1) Enrichment: Take Shanghai dwarf green samples, cut them into pieces and shake them for culture. Take 1 mL of the supernatant and inoculate it into 50 mL of LMRS liquid medium. Culture continuously for 3-5 days at 25-35℃ and 150-250 rpm. (2) Spread culture: The enriched bacterial solution was serially diluted to 10. -1 -10 -8 Take 100 μL of 10 -6 -10 -8 Spread the diluted solution onto MRS solid medium and incubate upside down at 30°C for 3-5 days. (3) Isolation and purification: The cultured bacterial cells were inverted and cultured on MRS solid medium for 2 days. Single colonies were selected and streaked 6-8 times to isolate and purify them to obtain single colonies. (4) Screening: The purified single colony was inoculated into soy sauce and cultured for 3 days. The purine content of the supernatant was determined by HPLC. The strain with the best effect in reducing the total purine content was selected as NS-01.

3. The screening method as described in claim 2, characterized in that, In step (1), the shaking culture conditions are 30℃ and 200rpm, and the culture is carried out continuously for 4 hours before inoculation culture in MRS liquid medium.

4. The application of the strain described in claim 1 in reducing the purine content of fermented soy sauce.

5. The application as described in claim 4, characterized in that, The purines include adenine, guanine, hypoxanthine, and xanthine.

6. The application of the strain described in claim 1 in the preparation of low-purine fermented soy sauce.

7. The application as described in claim 6, characterized in that, When applying, strain NS-01 is cultured into a seed culture and inoculated into the soy sauce fermentation system at an inoculation rate of 2%. The culture is then shaken and cultured at 30℃ and 200rpm for 4 days.

8. The application of the strain described in claim 1 in the preparation of low-purine soy products, characterized in that, The low-purine soy products include tofu and fermented black beans.

9. The application of the strain described in claim 1 in the preparation of low-purine aquatic fermented products, characterized in that, The low-purine fermented aquatic products include fish sauce and shrimp paste.

10. A compound bacterial agent comprising the strain of claim 1, characterized in that, The compound microbial agent also includes aroma-producing strains or texture-improving strains. The compound microbial agent is used to reduce the purine content of fermented foods, including fermented soy sauce, soy products, and fermented aquatic products.

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