Pedobacter sp. strain 2-79

Abstract

The application discloses a Pediococcus acidilactici for improving color and flavor of low-salt solid-state soy sauce and belongs to the technical field of bioengineering. The application obtains a Pediococcus acidilactici WT1 which can grow well under higher temperature and certain salinity by being separated and screened from soy sauce mash. The P.acidilactici WT1 is strengthened to carry out low-salt solid-state soy sauce fermentation, so that the amino acid nitrogen of the soy sauce is increased by 9.43%, the contents of succinic acid and tartaric acid in the soy sauce are respectively increased by 71.87% and 5.43 times, the contents of umami and sweet amino acids are respectively increased by 35.16% and 13.44%, the total amount of volatile flavor substances is increased by 19.95%, and the color rate is increased by 21.36%. By strengthening the P.acidilactici WT1, the contents of main flavor substances, i.e., guaiacol and phenol in the soy sauce are respectively increased by 35.49% and 50.64%, and the contents of benzyl alcohol and isoamyl alcohol are respectively increased by 16.83% and 21.86%. Therefore, strengthening the P.acidilactici WT1 in the low-salt solid-state soy sauce fermentation process has important significance for improving the quality and flavor of the low-salt solid-state soy sauce.

Description

Technical Field

[0001] This invention relates to a strain of Pediococcus lactis that improves the color and flavor of low-salt solid soy sauce, belonging to the field of bioengineering technology. Background Technology

[0002] Soy sauce is a condiment made from soybeans, wheat, and other raw materials through fermentation by microorganisms such as Aspergillus oryzae, yeast, and bacteria. It has a unique flavor and delicious taste. Currently, there are two main production methods for soy sauce: high-salt liquid-state fermentation and low-salt solid-state fermentation. Soy sauce fermented using the low-salt solid-state method has the advantages of better coloring and a more prominent soy aroma, but it still lags behind soy sauce brewed using the high-salt liquid-state method in terms of umami and flavor.

[0003] The fermentation temperature for producing low-salt solid soy sauce is relatively high (45–55℃), and the fermentation cycle is relatively short (20–30 days). Therefore, low-salt solid soy sauce has issues in terms of color, quality, and flavor, such as a tendency to darken in color, relatively low amino acid nitrogen content, and low content of flavor compounds, which need improvement. Previous studies have shown some effectiveness in improving the flavor of low-salt solid soy sauce by optimizing raw material composition and fermentation processes. For example, using wheat bran enzymatic hydrolysis for fermentation increased the amino acid nitrogen content of soy sauce by 33.68% and 4-ethylguaiacol by 66.18%. Optimizing the fermentation process, such as lowering the fermentation temperature, reduced the adverse effects of high temperatures on enzyme activity and microbial growth and metabolism, resulting in a 20% increase in amino acid nitrogen content and a 10% increase in alcohol content. The formation of flavor compounds in soy sauce is related to the interactions between different types of microorganisms, such as Aspergillus oryzae, yeast, and lactic acid bacteria. By using biotechnological methods such as isolation, selection, and mutagenesis of fermentation strains, *Aspergillus oryzae* strains with strong enzyme production capabilities, high stress resistance, and suitability for different soy sauce brewing processes have been screened, which has also improved raw material utilization and soy sauce flavor to some extent. Fungi are important microorganisms that synthesize volatile flavor compounds during soy sauce fermentation. Because they are not heat-resistant, bacteria with good stress resistance are potential functional microorganisms for improving the quality of low-salt solid soy sauce. For example, *Bacillus* and *Staphylococcus* can promote soy sauce fermentation by secreting proteases and amylases, and can also metabolize and produce pyrazines and acids to make the soy sauce flavor richer. Lactic acid bacteria are common microorganisms involved in fermented foods, and they are widely used to improve the taste and aroma of fermented foods. *Pediococcus* is a type of lactic acid bacteria found in fermented foods such as yogurt, kimchi, and soy sauce. Previous studies have confirmed that using it in the sausage making process can reduce nitrate content and increase flavor substances such as isoamyl acetate, isoamyl hexanoate, and hexyl acetate; adding Pediococcus during donkey milk fermentation can improve the color index and sensory quality of yogurt, and increase acetaldehyde content and enhance aroma.

[0004] Functional bacteria that can tolerate low-salt solid-state fermentation conditions (higher temperature and salt stress) were isolated and screened from soy sauce mash, and the functional bacteria were enhanced to carry out low-salt solid-state soy sauce fermentation. This reveals the feasibility of using functional microorganisms for low-salt solid-state soy sauce fermentation and is of great significance for improving the quality and flavor of low-salt solid-state soy sauce. Summary of the Invention

[0005] This invention provides a strain of Pediococcus acidilactici WT1, which was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 26263, located at the Institute of Microbiology, Chinese Academy of Sciences.

[0006] The present invention also provides the application of the aforementioned Pyrococcus WT1 in improving the flavor of soy sauce.

[0007] The present invention also provides a microbial agent containing Pediococcus lactis WT1 or its fermentation broth, or containing lysate of Pediococcus lactis WT1.

[0008] In one embodiment of the present invention, the amount of *Pediococcus lactis* WT1 added to the microbial agent is at least 1 × 10⁻⁶. 8 CFU / g.

[0009] The present invention also provides a fermentation agent containing the *Pediococcus lactis* WT1 or containing the microbial agent.

[0010] In one embodiment of the present invention, the amount of *Pediococcus lactis* WT1 added to the fermenting agent is at least 1 × 10⁻⁶. 8 CFU / g.

[0011] The present invention also provides a chemical containing the *Pediococcus lactis* WT1 or the microbial agent or the above-mentioned fermentation agent.

[0012] In one embodiment of the present invention, the amount of *Pediococcus lactis* WT1 added to the fermenting agent is at least 1 × 10⁻⁶. 8 CFU / g.

[0013] The present invention also provides the application of the aforementioned *Pediococcus lactis* WT1 or the aforementioned microbial agent in the preparation of chemicals that improve the color and flavor of low-salt solid soy sauce.

[0014] The present invention also provides a method for improving the color and flavor of low-salt solid soy sauce, wherein the method comprises adding the *Pediococcus lactis* WT1, the microbial agent, or the fermentation agent during the fermentation process of the low-salt solid soy sauce.

[0015] In one embodiment of the present invention, the method includes raw material processing, koji making, fermentation, and soy sauce extraction; on day 0 of the start of fermentation, the above-mentioned Pediococcus lactis WT1 or the above-mentioned microbial agent or the above-mentioned fermentation agent is added.

[0016] The present invention also provides the application of the aforementioned *Pediococcus lactis* WT1, the aforementioned microbial agent, the aforementioned fermenting agent, or the aforementioned method in fermented soy sauce.

[0017] Beneficial effects

[0018] (1) This invention isolates and screens a *Pediococcus acidilactici* WT1 strain that grows well at relatively high temperatures and certain salinity levels from soy sauce mash. Strengthening *P. acidilactici* WT1 for low-salt solid-state soy sauce fermentation, compared to the control without added bacterial suspension, increases the amino acid nitrogen content of soy sauce by 9.43%, succinic acid and tartaric acid content by 71.87% and 5.43 times respectively, umami and sweet amino acid content by 35.16% and 13.44% respectively, color percentage by 21.36%, and red and yellow indices by 0.36 and 0.32 respectively.

[0019] (2) By strengthening P. acidilactici WT1, this invention increases the total amount of volatile flavor compounds by 19.95% compared to the control without added bacterial suspension. Specifically, the contents of the main flavor compounds guaiacol and phenol increased by 35.49% and 50.64%, respectively, while the contents of phenylethanol and isoamyl alcohol increased by 16.83% and 21.86%, respectively. Therefore, strengthening P. acidilactici WT1 during the fermentation of low-salt solid-state soy sauce is of great significance for improving the quality and flavor of low-salt solid-state soy sauce.

[0020] Preservation of biological materials

[0021] A strain of *Pediococcus acidilactici* WT1, classified as *Pediococcus acidilactici*, was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.26263. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0022] Figure 1 A phylogenetic tree of the strains constructed based on the 16S rRNA sequence.

[0023] Figure 2The images show the growth of bacteria in the fermented soybean paste; (a) shows the growth under a culture condition of 45°C; and (b) shows the growth under a culture condition of 8% NaCl.

[0024] Figure 3 This represents the changes in the total number of bacteria and the number of Pediococci during fermentation.

[0025] Figure 4 This represents the change in amino acid nitrogen content during the fermentation process of soy sauce.

[0026] Figure 5 The analysis of volatile flavor compounds in soy sauce includes: A) comparison of the total amount of volatile flavor compounds; and B) comparison of the main volatile flavor compounds.

[0027] Figure 6 A comparison of color indicators for soy sauce. Detailed Implementation

[0028] The culture media involved in the following examples are as follows:

[0029] MRS medium (g / L): peptone 10, beef extract 8, yeast extract 4, glucose 20, dipotassium hydrogen phosphate 2, diammonium hydrogen citrate 2, sodium acetate 5, magnesium sulfate 0.2, manganese sulfate 0.04, Tween-80 1, pH 6.8.

[0030] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.4.

[0031] The Aspergillus oryzae 3.042 used in the following examples was purchased from Shanghai Difa Brewing Biological Products Co., Ltd.

[0032] Example 1: Isolation and Identification of Bacteria in Soybean Paste

[0033] The specific steps are as follows:

[0034] (1) Low-salt solid-state soy sauce fermentation:

[0035] Steamed soybean meal (121℃, 30 min) was mixed with flour (m:m = 5:1), and Aspergillus oryzae 3.042 was inoculated at 1.5‰ of the total raw material mass; the koji was obtained by culturing at 95% humidity and 30℃ for 48 h.

[0036] The koji is mixed with 14% (w / v) brine in a 1:1 ratio to form soy sauce mash, with a final salt concentration of 7% to 8% (w / v) in the soy sauce mash.

[0037] Pack 200g of fermented soybean paste into a 250mL beaker, press it lightly to a thickness of 6cm, and cover it with non-iodized salt (to a thickness of 3cm). The initial fermentation temperature is 40℃, and the temperature is increased to 42℃ at 1℃ / day. On the 4th day, the temperature is increased to 45℃ and fermentation continues for 25 days.

[0038] (2) Bacterial isolation and screening:

[0039] Take 25g of fermented soybean paste sample and add 225mL of sterile physiological saline. Shake at 220r / min for 1h at 37℃. Spread 100μL of the liquid serial dilution onto MRS and LB solid agar plates containing 2g / L sorbic acid and incubate at 37℃ for 24h. Pick single colonies, streak to isolate and purify three times, then incubate on isolation medium to late logarithmic phase, and finally store at -80℃.

[0040] (3) Bacterial species identification:

[0041] Genomic DNA was extracted from the bacterial strain using a bacterial genome extraction kit from Shanghai Sangon Biotech Co., Ltd. Using this DNA as a template, PCR amplification was performed using the universal 16S rRNA primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR products were sent to Tianlin Biotechnology Co., Ltd. (Wuxi) for sequencing. The sequencing results were BLASTed using NCBI, and a phylogenetic tree of the strain was constructed using MEGA-X software.

[0042] The results showed that 18 strains of bacteria belonging to 6 genera (Bacillus, Pediococcus, Weissella, Staphylococcus, Enterococcus, and Enterobacter) were isolated from the fermented soybean mash. Figure 1 ),include:

[0043] 5 strains of Bacillus:

[0044] Bacillus velezensis BLS1, Bacillus pumilus DXYB1, Bacillus subtilisKC1, Bacillus halotolerans NY1, Bacillus amyloliquefaciens JDF01;

[0045] 9 strains of lactic acid bacteria:

[0046] Pediococcus acidilactici WT1 (the aforementioned Pediococcus acidilactici WT1 was deposited on December 26, 2022 at the China General Microbiological Culture Collection Center, with accession number CGMCCNO.26263) and WT4, Pediococcus pentosaceus WT6 and WT7, Weissella paramesenteroides WS1 and WS7, Enterococcus durans DL1, Enterococcus faecium FC9 and FC10;

[0047] Two strains of Staphylococcus:

[0048] Staphylococcus saprophyticus FSPT1 and Staphylococcus pasteuri BSPT1;

[0049] Two strains of Enterobacteriaceae:

[0050] Enterobacter hormaechei HS1 and Enterobacter ludwigii LDWX1.

[0051] Example 2: Comparison of the temperature and salt tolerance of bacteria in fermented soybean paste

[0052] Since the fermentation temperature for low-salt solid soy sauce is relatively high, strains that can grow well at 45℃ and 8% NaCl will have greater potential for use in the fermentation of low-salt solid soy sauce.

[0053] (1) Temperature resistance characteristics analysis:

[0054] Cultivation of lactic acid bacteria:

[0055] Single colonies from the culture dishes of lactic acid bacteria screened in Example 1 were inoculated into MRS liquid medium and cultured at 37°C for 10-12 h to the logarithmic growth phase. Lactic acid bacteria were then inoculated into fresh MRS liquid medium at an inoculation rate of 2% (v / v) and incubated at 45°C for 12 h.

[0056] Culture of other bacteria:

[0057] Single colonies from the culture dishes of lactic acid bacteria screened in Example 1 were inoculated into LB liquid medium and cultured at 37°C for 10-12 h to the logarithmic growth phase. Then, LB liquid medium was inoculated at 2% (v / v) and cultured at 45°C with shaking at 220 r / min for 12 h.

[0058] Take 1 mL of bacterial culture and measure the absorbance at 600 nm using a UV spectrophotometer. The results are as follows. Figure 2 As shown in 'a'.

[0059] The results showed that strains of the genus *Pediococcus* grew better at 45°C than strains of the genera *Weissella*, *Enterobacter*, *Enterococcus*, *Staphylococcus*, and *Bacillus*.

[0060] Among them, P. acidilactici WT1 showed the best growth at 45℃, with an OD of 600 The OD reached 2.44; strain Pediococcus pentosaceus WT6 showed the best growth among Pediococcus pentosaceus species. 600 It can reach 1.98.

[0061] (2) Salt tolerance analysis:

[0062] Cultivation of lactic acid bacteria:

[0063] Single colonies from the culture dishes of lactic acid bacteria screened in Example 1 were inoculated into MRS liquid medium and cultured at 37°C for 10-12 h until the logarithmic growth phase. Lactic acid bacteria were then inoculated into fresh MRS liquid medium containing 8% NaCl at an inoculation rate of 2% (v / v) and cultured statically at 37°C for 12 h.

[0064] Culture of other bacteria:

[0065] Single colonies from the culture dishes of lactic acid bacteria screened in Example 1 were inoculated into LB liquid medium and cultured at 37°C for 10-12 h until the logarithmic growth phase. They were then inoculated into LB liquid medium containing 8% NaCl at a 2% (v / v) inoculation rate and cultured at 37°C with shaking at 220 r / min for 12 h.

[0066] Take 1 mL of bacterial culture and measure the absorbance at 600 nm. The results are as follows: Figure 2 As shown in b in the figure.

[0067] The results showed that the effect of adding 8% NaCl to the culture medium on the strains was similar to that under 45℃ culture conditions, with strains of the genus *Pediococcus* growing better than those of the genera *Weissella*, *Enterobacter*, *Enterococcus*, *Staphylococcus*, and *Bacillus*. Among them, *P. acidilactici* WT1 showed the best growth, with an OD... 600 Reached 0.73; P. pentosaceus WT6OD 600 It can reach 0.63.

[0068] The above results indicate that *P. acidilactici* WT1 and *P. pentosaceus* WT6 are the strains investigated that can grow well at 45℃ and 8% NaCl, and since they belong to different genera and species, they have the potential to be used in the fermentation of low-salt solid-state soy sauce. Therefore, the effects of enhanced fermentation of *P. acidilactici* WT1 and *P. pentosaceus* WT6 on the physicochemical properties and flavor of low-salt solid-state soy sauce will be investigated subsequently.

[0069] Example 3: Effect of Pediococcus fermentation on bacterial count in low-salt solid soy sauce mash

[0070] The specific steps are as follows:

[0071] 1. Low-salt solid-state soy sauce fermentation:

[0072] (1) Composition:

[0073] Steamed soybean meal (121℃, 30 min) was mixed with flour (m:m = 5:1), and Aspergillus oryzae 3.042 was inoculated at 1.5‰ of the total raw material mass; the koji was obtained by culturing at 95% humidity and 30℃ for 48 h.

[0074] (2) Preparation of fermented soybean paste

[0075] The koji (fermented starter culture) is mixed with 14% (w / v) brine in a 1:1 ratio, and then lactic acid bacteria are added to form the soy sauce mash. The final salt concentration in the soy sauce mash is 7%–8% (w / v), while the final lactic acid bacteria concentration in the soy sauce mash system is 10%. 8 CFU / g.

[0076] The lactic acid bacteria cells were prepared by inoculating single colonies of P. acidilactici WT1 and P. pentosaceus WT6 from culture dishes into liquid MRS medium, culturing at 37°C for 10-12 h to the logarithmic growth phase, and centrifuging at 4°C and 8000 rpm for 2 min to obtain the bacterial cells.

[0077] (3) Fermentation

[0078] Place 200g of fermented soybean paste into a 250mL beaker, press it lightly to a thickness of 6cm, cover it with non-iodized salt (to a thickness of 3cm) to begin fermentation; the initial fermentation temperature is 40℃, and the temperature is increased to 42℃ at 1℃ / day. On the 4th day, the temperature is increased to 45℃ and fermentation continues for 25 days.

[0079] Samples were taken on days 0, 1, 2, 3, 4, 9, 14, and 25 of fermentation and stored at -80℃.

[0080] Control group: The control group is the original process, without the addition of any bacteria; specifically:

[0081] (1) Composition:

[0082] Steamed soybean meal (121℃, 30 min) was mixed with flour (m:m = 5:1), and Aspergillus oryzae 3.042 was inoculated at 1.5‰ of the total raw material mass; the koji was obtained by culturing at 95% humidity and 30℃ for 48 h.

[0083] (2) Preparation of fermented soybean paste

[0084] The koji is mixed with 14% (w / v) brine in a 1:1 ratio to form soy sauce mash, with a final salt concentration of 7% to 8% (w / v) in the soy sauce mash.

[0085] (3) Fermentation

[0086] Place 200g of fermented soybean paste into a 250mL beaker, press it lightly to a thickness of 6cm, cover it with non-iodized salt (to a thickness of 3cm) to begin fermentation; the initial fermentation temperature is 40℃, and the temperature is increased to 42℃ at 1℃ / day. On the 4th day, the temperature is increased to 45℃ and fermentation continues for 25 days.

[0087] Samples were taken on days 0, 1, 2, 3, 4, 9, 14, and 25 of fermentation and stored at -80℃.

[0088] 2. Effect of Pediococcus fermentation on bacterial count in low-salt solid-state soy sauce mash

[0089] (1) Sample preparation: Take 25g of the fermented mash sample obtained after fermentation in step (3) above and add 225mL of sterile physiological saline. Shake at 37℃ and 220r / min for 1h. Spread 100μL of serially diluted solution on MRS and LB solid culture medium containing 2g / L sorbic acid (MRS for lactic acid bacteria and LB for total bacteria). After incubation at 37℃ for 24h, count the viable bacteria.

[0090] (2) Pediococcus count was performed using colony PCR counting.

[0091] Colonies from the plate were transferred to PCR tubes and amplified using Pediococcus-specific primers Pa-F (5′-CGAACTTCCGTTAATTGATTAT-3′) and Pa-R (5′-ACCTTGCGGTCGTACTCC-3′). The PCR system consisted of: 0.5 μL DNA template (50 ng / μL), 10 μL Taq enzyme, 0.5 μL each of forward and reverse primers (10 μmol / L), and 8.5 μL ddH2O. Amplification conditions were: 94℃ for 3 min, 94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, and 12℃ for 10 min. The product was analyzed by gel electrophoresis; the bands showing the target were Pediococcus. The results are as follows: Figure 3 As shown.

[0092] The results showed that on the 4th day of fermentation (sampling was done based on temperature changes; the temperature reached 45℃ on the 4th day, and the difference in viable bacterial count was significant; samples were taken every 5 days thereafter), the total bacterial count in the fermented mash with added P. acidilactici WT1 was 4.8 × 10⁻⁶. 6 The CFU / g was 2.38 orders of magnitude higher than the control group and 1.52 orders of magnitude higher than the total bacterial count in the fermented mash with added P. pentosaceus WT6.

[0093] The amount of Pediococcus in the fermented mash with added P. acidilactici WT1 was 4.5 × 10⁻⁶. 6 The CFU / g level was 4.59 orders of magnitude higher than the control group and 1.42 orders of magnitude higher than the Pediococcus count in the fermented mash with added P. pentosaceus WT6. This indicates that the addition of P. acidilactici WT1 can enhance the activity of this bacterium in low-salt solid-state fermentation.

[0094] Example 4: Effects of enhanced Pediococcus fermentation on the color of low-salt solid soy sauce and its influence on amino acid nitrogen content.

[0095] Coloring effect is one of the important sensory characteristics of soy sauce produced by the low-salt solid-state process. The coloring effect of soy sauce is mainly characterized by the color rate to represent the depth of the soy sauce color, and by the red index and yellow index to represent the coloring of the main colors.

[0096] 1. Low-salt solid-state soy sauce fermentation:

[0097] The specific fermentation method is the same as in Example 3, with koji making, fermentation of soy sauce mash and fermentation carried out separately; samples were taken on days 0, 1, 2, 3, 4, 9, 14 and 25 of fermentation and stored at -80℃.

[0098] Set up a control group:

[0099] The specific method is the same as in Example 3, with koji making, fermentation of soy sauce mash and fermentation carried out respectively; samples were taken on fermentation days 0, 1, 2, 3, 4, 9, 14 and 25 and stored at -80℃.

[0100] 2. Effects of Pediococcus fermentation on color index and amino acid nitrogen content of low-salt solid-state fermented soybean paste.

[0101] (1) Color index determination:

[0102] Take 1 mL of the final soy sauce product diluted 100 times and measure the absorbance at wavelengths of 460 nm, 510 nm and 610 nm respectively. Calculate the color rate, red index and yellow index using formulas (1), (2) and (3) respectively.

[0103] Color index (EBC) = ((OD) 610 ×20000)) / 0.076(1);

[0104] Red Index = 10 × lg(OD) 510 / OD 610 (2);

[0105] Yellow Index = 10 × lg(OD) 460 / OD 610 (3).

[0106] The results are shown in Table 1. Figure 6 As shown.

[0107] Table 1 Color index of soy sauce samples

[0108]

[0109]

[0110] The results showed that enhanced fermentation with *P. acidilactici* WT1 significantly deepened the color of soy sauce, increasing the color percentage by 21.36% compared to the control, and increasing the red and yellow indices by 0.36 and 0.32 respectively. This indicates that enhanced fermentation with *P. acidilactici* WT1 makes the low-salt solid soy sauce more reddish and translucent, significantly improving its coloring effect.

[0111] (2) Determination of amino acid nitrogen

[0112] Sample processing: 5g of fermented mash was collected at the end of fermentation days 0, 1, 2, 3, 4, 9, 14, and 25, respectively. 45mL of sterile water was added, and the mixture was ultrasonically agitated for 30min. The supernatant was then centrifuged at 6000r / min for 5min, and the amino acid nitrogen content was determined.

[0113] Titration was used, referring to "SB / T 10311-1999 Low-Salt Solid-State Fermentation Soy Sauce Brewing Process Specification". The results are as follows: Figure 4 As shown.

[0114] Depend on Figure 4 It can be seen that strengthening the fermentation of P. acidilactici WT1 is beneficial to increasing the amino acid nitrogen content in soy sauce, reaching 1.74 g / 100 g at the end of fermentation, which is 9.43% higher than the control group and 6.10% higher than P. pentosaceus WT6.

[0115] Example 5: Improvement of organic acids, flavor amino acids, and volatile flavor compounds in low-salt solid soy sauce by enhanced Pediococcus fermentation

[0116] 1. Low-salt solid-state soy sauce fermentation:

[0117] The specific fermentation method is the same as in Example 3, with koji making, fermentation of soy sauce mash and fermentation carried out separately; samples were taken on days 0, 1, 2, 3, 4, 9, 14 and 25 of fermentation and stored at -80℃.

[0118] Set up a control group:

[0119] The specific method is the same as in Example 3, with koji making, fermentation of soy sauce mash and fermentation carried out respectively; samples were taken on fermentation days 0, 1, 2, 3, 4, 9, 14 and 25 and stored at -80℃.

[0120] 2. Enhance the effects of Pediococcus fermentation on organic acids, flavor amino acids, and volatile flavor compounds in low-salt solid soy sauce.

[0121] Sample preparation: Remove the top salt seal of the fermented mash obtained on day 25 of fermentation, add the same mass of 100℃ hot water as the fermented mash, soak at 45℃ for 12 hours, centrifuge at 6000r / min for 30 minutes, take the supernatant and add 5% (w / v) trichloroacetic acid at a ratio of 1:1 (v / v), treat at room temperature in the dark for 30 minutes, filter with a 0.22μm aqueous filter membrane and take the filtrate for testing.

[0122] (1) Determination of organic acid content

[0123] High-performance liquid chromatography (HPLC) was used. The HPLC system was a Shimadzu 20A; the column was an Aminex-HPX-87H (300 mm × 7.8 mm, 9 μm); the mobile phase was 5 mmol / L dilute sulfuric acid; the injection volume was 10 μL; the elution rate was 0.5 mL / min; the elution time was 30 min; the column temperature was 40℃; and the detector was UV 210 nm. The results are shown in Table 2.

[0124] Table 2 Comparison of organic acid content in soy sauce samples

[0125]

[0126] Organic acids are important components of soy sauce flavor. For example, succinic acid and its sodium salt can regulate saltiness and enhance umami. Tartaric acid is an important flavor-enhancing organic acid in low-salt solid soy sauce, which can improve the richness of soy sauce.

[0127] The results showed that the organic acid content in soy sauce fermented with enhanced P. acidilactici WT1 was increased compared with the control and P. pentosaceus WT6. The total organic acid content in soy sauce fermented with enhanced P. acidilactici WT1 was 30.04 g / L, which was 48.56% higher than the control and 12.59% higher than P. pentosaceus WT6 (Table 2).

[0128] Among them, the succinic acid content in soy sauce fermented with enhanced P. acidilactici WT1 was 71.87% higher than that in the control, and the tartaric acid content was 5.43 times higher than that in the control.

[0129] (2) Determination of free amino acids

[0130] High-performance liquid chromatography (HPLC) was used. Samples were derivatized pre-column with o-xylene (OPA). The HPLC system was an Agilent 1260, with an ODS HYPERSIL column (250 mm × 4.6 mm, 2.5 μm). Mobile phase A consisted of 5 g anhydrous sodium acetate, 5 mL tetrahydrofuran, and 200 μL triethylamine, pH 7.2. Mobile phase B consisted of 5 g anhydrous sodium acetate, 200 mL ultrapure water, 400 mL methanol, and 400 mL acetonitrile, pH 7.2. Injection volume was 10 μL; elution rate was 1 mL / min; elution time was 40 min; column temperature was 40 °C; and the detector was UV 338 nm. The results are shown in Table 3.

[0131] Table 3 Amino acid content in soy sauce samples

[0132]

[0133]

[0134] The results showed that the total amino acid content in the soy sauce fermented with P. acidilactici WT1 was 21.59 g / L, which was 18.43% higher than the control and 16.33% higher than P. pentosaceus WT6.

[0135] Aspartic acid and glutamic acid are the main umami substances in soy sauce. The total amount of these two umami amino acids in soy sauce fortified with P. acidilactici WT1 fermentation increased by 35.16% compared with the control, the sweet amino acids (serine and alanine) increased by 13.44%, and the proportion of bitter amino acids decreased by 3.28%.

[0136] The above results indicate that enhancing the fermentation of P. acidilactici WT1 can significantly increase the content of umami and sweet amino acids in soy sauce and reduce the proportion of bitter amino acids, thereby helping to improve the flavor and quality of soy sauce.

[0137] (3) Volatile flavor compounds were determined by solid phase microextraction combined with gas chromatography-mass spectrometry (SPME-GC-MS).

[0138] Take 5 mL of soy sauce sample after fermentation and add 2-octanol (final concentration 100 μg / L) as an internal standard. Extraction conditions: Soy sauce sample (5 mL), incubate at 50℃ for 30 min, then insert the extraction head 1 cm into the liquid surface for 30 min of adsorption, insert into the gas chromatograph injection port, and desorb at 240℃ for 15 min. Gas chromatographic conditions: injection port 250℃, chromatographic column TG-WAMS (60 m × 250 μm × 0.25 μm, Thermo Fisher Scientific), carrier gas high-purity He, splitless injection; gradient temperature program: hold at 45℃ for 1 min, increase to 130℃ at 3℃ / min, increase to 200℃ at 6℃ / min, and then increase to 230℃ at 8℃ / min and hold for 10 min. Mass spectrometry conditions: ionization source (EI), ion source temperature 220℃, interface temperature 250℃, ion source energy 70 eV, full scan range. Flavor compounds were identified by searching the NIST standard library based on retention index (RI) and similarity index (SI) greater than 800. Semi-quantitative analysis of compounds was performed using the peak area of ​​the internal standard 2-octanol. Results are as follows: Figure 5 As shown in Table 4.

[0139] Table 4: Flavor Compounds

[0140]

[0141]

[0142]

[0143]

[0144] The results showed that volatile substances in soy sauce constitute the main flavor of soy sauce, and their types and contents are closely related to the flavor of soy sauce.

[0145] The results are as follows Figure 5 As shown in Figure A, the total volatile flavor compounds in the fortified *P. acidilactici* WT1 fermented soy sauce was 702.94 μg / L, an increase of 19.95% compared to the control group and 4.18% compared to *P. pentosaceus* WT6. The levels of alcohols, ketones, and other compounds in the fortified *P. acidilactici* WT1 fermented soy sauce increased by 16.79%, 17.52%, and 31.48% respectively compared to the control, while the levels of esters and phenols increased by 90.53% and 50.02% respectively. Phenylethanol and isoamyl alcohol are important floral flavor compounds in soy sauce, contributing significantly to its aroma and enhancing its flavor and texture.

[0146] like Figure 5 As shown in Figure B, the levels of phenylethyl alcohol and isoamyl alcohol in the fortified P. acidilactici WT1 fermented soy sauce increased by 16.83% and 21.86% respectively compared to the control. The main phenolic compounds, guaiacol and phenol (accounting for 89.13% of total phenols), in the fortified P. acidilactici WT1 fermented soy sauce increased by 35.49% and 50.64% respectively compared to the control.

[0147] The above results indicate that strengthening P. acidilactici WT1 can significantly increase the content of volatile flavor compounds in low-salt solid soy sauce, especially the main volatile flavor compounds in soy sauce.

[0148] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person 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 strain of Pediococcus lactis ( Pediococcus acidilactici WT1 was deposited on December 26, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.26263.

2. A microbial inoculant, characterized in that, The microbial agent contains Pediococcus lactis WT1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The amount of *Pediococcus lactis* WT1 added to the microbial agent is at least 1 × 10⁻⁶. 8 CFU / g.

4. A fermenting agent, characterized in that, The fermentation agent contains Pediococcus lactis WT1 as described in claim 1 or contains the microbial agent as described in claim 2 or 3.

5. The fermenting agent according to claim 4, characterized in that, The amount of *Pediococcus lactis* WT1 added to the fermenting agent is at least 1 × 10⁻⁶. 8 CFU / g.

6. The use of the *Pediococcus lactis* WT1 of claim 1 or the microbial agent of claim 2 or 3 in the preparation of chemicals for improving the color and flavor of low-salt solid soy sauce.

7. The application according to claim 6, characterized in that, The amount of *Pediococcus lactis* WT1 added to the chemical is at least 1 × 10⁻⁶. 8 CFU / g.

8. A method for improving the color and flavor of low-salt solid soy sauce, characterized in that, The method includes raw material processing, koji making, fermentation, and soy sauce extraction; before fermentation begins, the *Pediococcus lactis* WT1 of claim 1, the microbial agent of claim 2, or the fermenting agent of claim 4 are added.

9. The application of the *Pediococcus lactis* WT1 according to claim 1, or the microbial agent according to claim 2 or 3, or the fermenting agent according to claim 4 or 5, or the method according to claim 8, in the fermentation of low-salt solid soy sauce.

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