A koji-making method for improving the umami amino acid content and the antioxidant property of soy sauce

Abstract

The application discloses a koji-making method for improving the content of umami amino acids and the antioxidation of soy sauce and belongs to the technical field of bioengineering. The application obtains a Pediococcus pentosaceus LS1 strain with the best salt tolerance, umami amino acid production capacity and acid production capacity, and combines Aspergillus oryzae Huon 3.042 with Aspergillus niger Huon 3.350 and the Pediococcus pentosaceus LS1 to make koji, compared with single-bacterium koji-making, the compound koji-making can effectively improve the content of functional components of koji, and the content of total polyphenols and total flavones is increased. For fermentation, the mixed-bacterium koji-making does not affect the normal fermentation of soy sauce, and the functional components, the antioxidation capacity, the content of umami amino acids and the content of organic acids are all improved, the total phenol content is increased by 5.13% and 1.71% compared with the control and the addition of Aspergillus niger 3.350 respectively, the total flavone content is increased by 57.82% and 8.13% respectively, and the total content of umami amino acids and the free radical clearance rate are also improved.

Description

Technical Field

[0001] This invention relates to a method for making koji (fermentation starter) to improve the content of umami amino acids and antioxidant properties in soy sauce, belonging to the field of bioengineering technology. Background Technology

[0002] Koji (fermentation starter) making is a crucial step in soy sauce brewing. The quality of the koji affects the soy sauce's taste, color, and aroma. Poor-quality koji will result in a bland taste, dull color, and even off-flavors. The quality of the koji directly impacts the content and activity of enzymes during fermentation, thus affecting the decomposition of raw materials and the formation of soy sauce flavor. Currently, most koji making in my country uses Aspergillus oryzae var. oryzae (Hu Niang 3.042) as a single-strain method, which has the advantages of simple operation and rapid production. However, pure-strain koji making results in a relatively simple enzyme system, preventing the soy sauce raw materials from fully fermenting, thereby affecting the soy sauce's quality and flavor.

[0003] The ability of mixed-strain koji making to improve the quality and flavor of soy sauce has been proven in practice. Studies have found that mixed-strain koji making has higher enzyme activity and a richer enzyme system, which can improve the utilization rate of raw materials for soy sauce, improve soy sauce quality, and facilitate the release of bioactive components (such as total flavonoids and total phenols) during soy sauce fermentation. Using mixed-strain koji making with a richer enzyme system for fermentation can not only improve the utilization rate of raw materials for soy sauce and improve soy sauce quality, but also increase the content and effectiveness of functional components in soy sauce, showing promising development prospects.

[0004] Current research mainly focuses on adding microorganisms during the fermentation process of soy sauce. However, there are problems such as limited salt tolerance of the strains leading to insignificant effects, and the addition of strains during fermentation may increase secondary precipitation in soy sauce due to cell residue. Furthermore, when strains are added during the fermentation stage, their metabolic activities may have a complex impact on the fermentation cycle and the formation of flavor substances. If the timing of addition is inappropriate, it may prolong the fermentation cycle or affect the formation of flavor. Summary of the Invention

[0005] Different microorganisms require varying conditions for growth, including raw materials, temperature, humidity, and pH. In mixed-culture koji preparation, it's crucial to identify the optimal conditions for the symbiotic relationship between lactic acid bacteria and *Aspergillus oryzae* to ensure their healthy growth. The interactions between various microorganisms are not yet fully understood; the symbiotic relationship between lactic acid bacteria and *Aspergillus oryzae* may affect their respective growth and metabolism, necessitating further research into their interaction mechanisms. The addition of lactic acid bacteria may affect the activity of enzymes in the koji material, requiring investigation into the impact of lactic acid bacteria on the enzyme activity of *Aspergillus oryzae* and the specific effects of this impact on soy sauce quality. Lactic acid bacteria possess a strong acid-producing capacity; therefore, controlling the amount of strain added is necessary to manage acid production and prevent excessive pH reduction leading to koji rancidity.

[0006] Technical solution:

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for making koji that can improve the content of umami amino acids and antioxidant properties in fermented soy sauce.

[0008] The complete technical means and methods of this invention are as follows:

[0009] (1) The characteristics of the lactic acid bacteria isolated from the fermented mash were analyzed, and the salt tolerance and metabolic acid production capacity of different lactic acid bacteria were investigated to determine the functional lactic acid bacteria suitable for mixed fermentation.

[0010] (2) Different mixed-culture koji-making groups were designed, with the control group using only Aspergillus oryzae Hu Niang 3.042 for koji-making. The total phenol and total flavonoid content of the koji in different groups was investigated to explore the effect of mixed-culture koji-making on the antioxidant properties of soy sauce koji.

[0011] (3) The effects of mixed-culture koji and control group koji on the quality of soy sauce were investigated, and the physicochemical indicators, functional components, antioxidant capacity, and free amino acids of soy sauce fermentation in different koji groups were compared.

[0012] This invention provides a strain of Pediococcus pentosaceus LS1, which was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCC NO:M 20242843.

[0013] The present invention provides a microbial inoculant containing Pediococcus pentosaceus LS1 or its fermentation broth, or containing Pediococcus pentosaceus LS1 lysate, or containing live cells of Pediococcus pentosaceus LS1, freeze-dried dried cells of Pediococcus pentosaceus LS1, immobilized cells of Pediococcus pentosaceus LS1, liquid inoculant of Pediococcus pentosaceus LS1, solid inoculant of Pediococcus pentosaceus LS1, or Pediococcus pentosaceus LS1 strain in any other form.

[0014] In one embodiment of the present invention, the amount of Pediococcus pentosaceus LS1 added to the microbial agent is at least 1 × 10⁻⁶. 6 CFU / g.

[0015] The present invention also provides a starter culture, wheat koji, bran koji or other types of fermentation koji containing the above-mentioned Pediococcus pentosaceus LS1 and / or the microbial preparations.

[0016] The present invention also provides the application of the above-mentioned Pediococcus pentosaceus LS1 and / or the above-mentioned microbial preparation in the production of finished koji.

[0017] In one embodiment of the present invention, the application includes, but is not limited to, at least one aspect of (a) to (b):

[0018] (a) Increase the number of microorganisms in the fermentation process of the finished product koji;

[0019] (b) Increase the content of protease activity in the finished product.

[0020] The present invention also provides the application of the above-mentioned Pediococcus pentosaceus LS1 and / or the above-mentioned microbial preparations in soy sauce production.

[0021] In one embodiment of the present invention, the application includes, but is not limited to, at least one of (a) to (d):

[0022] (a) Increase the content of total phenols and total flavonoids in the soy sauce system;

[0023] (b) Increase the content of reducing sugar, total acid, and amino acid nitrogen in soy sauce;

[0024] (c) Enhance the antioxidant capacity of soy sauce;

[0025] (d) Enhance the umami flavor of soy sauce.

[0026] The present invention also provides a method for preparing soy sauce, the method comprising the following steps:

[0027] (1) Koji-making process:

[0028] The cooked soybeans and flour are mixed evenly at a mass ratio of (6-7):(4-3), and Aspergillus oryzae AS3.042 koji, Aspergillus niger AS3.350 koji, Pediococcus pentosus LS1 and / or the microbial preparation are added sequentially at 1.5-3‰ and 0.2-1‰ of the total weight of the raw materials. The koji-making conditions are an ambient humidity of 92-95%, a temperature of 28-32℃, and a fermentation time of 44-48 hours. The koji-making process is ended when the surface of the koji material is covered with yellow-green spores.

[0029] (2) Fermentation process:

[0030] The prepared koji from step (1) is mixed with NaCl-containing brine at a volume ratio of 1:1.8 to 2, so that the salt concentration of the system is 18 to 20%, and fermented at 28℃-30℃ for 40 to 60 days.

[0031] In one embodiment of the present invention, the inoculum amount of the Pediococcus pentosaceus LS1 agent is at least 6 × 10⁻⁶. 6 CFU / g.

[0032] The present invention also provides the application of the above-mentioned Pediococcus pentosaceus LS1 and / or the above-mentioned microbial preparations in improving the flavor and antioxidant capacity of soy sauce.

[0033] The present invention also provides soy sauce obtained by the above preparation method.

[0034] Beneficial effects

[0035] (1) This invention takes high-salt dilute soy sauce as the research object. By evaluating the salt tolerance and acid production performance of multiple strains of lactic acid bacteria isolated from soy sauce mash, a strain P. pentosaceus LS1 that can tolerate high-salt dilute soy sauce fermentation conditions and produces high levels of umami amino acids was screened.

[0036] (2) This invention uses Aspergillus oryzae AS 3.042 as the main koji-making strain, and adds P. pentosaceus LS1 and Aspergillus niger AS 3.350 for mixed koji-making. Under the premise of keeping the physicochemical indicators of soy sauce fermentation unchanged and ensuring normal fermentation of soy sauce, the total phenol and total flavonoid contents are increased by 5.13% and 57.82%, respectively; the DPPH free radical scavenging rate and reducing power are increased by 41.79% and 10.95%, respectively; the sweet amino acid content is increased by 37.30%; and the contents of threonine, aspartic acid and glutamic acid, which are umami amino acids, are increased by 85.08%, 39.77% and 39.35%, respectively.

[0037] Preservation of biological materials

[0038] A strain of Pediococcus pentosaceus LS1, classified as Pediococcus pentosaceus LS1, was deposited on December 18, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20242843, located at Wuhan University, Wuhan, China. Attached Figure Description

[0039] Figure 1 Survival rate (A), acid production capacity (B), antibacterial capacity (C), and ability of culture medium system to produce free amino acids (D) of lactic acid bacteria under different salt concentrations.

[0040] Figure 2 Changes in the activities of protease (neutral and acidic), cellulase, and amylase during mixed culture koji preparation.

[0041] Figure 3 Dynamic changes in the number of microorganisms during the mixed-culture fermentation of soy sauce.

[0042] Figure 4 Changes in the total phenol and total flavonoid content of the starter culture during mixed culture starter culture production.

[0043] Figure 5 Dynamic changes in the number of microorganisms during the mixed-culture fermentation of soy sauce.

[0044] Figure 6 The effect of mixed-culture koji preparation on the physicochemical indicators of soy sauce fermentation.

[0045] Figure 7 The effects of mixed-culture koji preparation on the total phenolic, total flavonoid, and antioxidant capacity of soy sauce.

[0046] Figure 8 The effect of mixed-culture koji preparation on the content of free amino acids (A), especially the content of umami amino acids (B).

[0047] Among them, A: only A.oryzae Hu Niang 3.042 is added for koji making; AA: both A.oryzae Hu Niang 3.042 and A.niger 3.350 are added for koji making; AAP: A.oryzae Hu Niang 3.042, A.niger 3.350 and P.pentosaceusLS1 are added for koji making. *, p<0.05; **, p<0.01; ***, p<0.001. Detailed Implementation

[0048] The indicator bacteria B. halodurans, S. saprophyticus, and B. subtilis mentioned in the following examples are all from the strains preserved in this laboratory.

[0049] The Aspergillus oryzae AS 3.042 koji used in the following examples was purchased from Shanghai Difa Brewing Biological Products Co., Ltd., and the Aspergillus niger Hu Niang 3.350 koji (Aspergillus niger AS 3.350) was purchased from Shanghai Jiamin Brewing Food Co., Ltd.

[0050] The culture media involved in the following examples are as follows:

[0051] LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL distilled water, 15g agar powder added to solid medium, autoclave at 121℃ for 20min.

[0052] MRS broth medium: Oxoid, UK, solid medium with 15g agar powder, autoclaved at 118℃ for 20min.

[0053] Bengal Red Medium: 5g peptone, 10g glucose, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.1g chloramphenicol, 1000mL distilled water, 15g agar powder added to solid medium, autoclave at 121℃ for 20min.

[0054] The detection methods involved in the following embodiments are as follows:

[0055] 1. Analysis of the characteristics of lactic acid bacteria strains

[0056] (1) Analysis of the salt tolerance of the strain:

[0057] Take the lactic acid bacteria culture, centrifuge at 8000 r / min for 5 min, discard the supernatant, wash the bacterial precipitate twice with sterile physiological saline, and then... 9 Inoculation amounts of CFU / mL were used to inoculate MRS liquid medium containing 180 g / L (equivalent to a salt concentration of 18%) and 160 g / L (equivalent to a salt concentration of 16%), respectively, and incubated at 37°C for 24 h. 100 μL of the culture obtained after incubation was diluted to appropriate ratios (0 h, 24 h) and plated onto MRS solid plates to calculate the survival rate.

[0058] (2) Analysis of organic acid production capacity:

[0059] The content of organic acids was determined by high performance liquid chromatography.

[0060] The primary seed culture was transferred to MRS liquid medium at an inoculation rate of 2% (v / v) and incubated at 37°C for 24 h to obtain the secondary seed culture. The secondary seed culture was centrifuged at 12000 r / min for 8 min, and the supernatant was filtered through a 0.22 μm sterile aqueous filter membrane before being loaded for analysis.

[0061] Samples were derivatized pre-column with o-xylene (OPA). Liquid chromatography system: Agilent 1260, ODSHYPERSIL column (250 mm × 4.6 mm, 2.5 μm); Mobile phase A: 5 g anhydrous sodium acetate, 5 mL tetrahydrofuran, 200 μL triethylamine, pH 7.2; Mobile phase B: 5 g anhydrous sodium acetate, 200 mL ultrapure water, 400 mL methanol, 400 mL acetonitrile, pH 7.2. Injection volume: 10 μL; Elution rate: 1 mL·min⁻¹; Elution time: 40 min; Column temperature: 40 °C; Detector: UV 338 nm.

[0062] The liquid phase conditions are shown in Table 1.

[0063] Table 1: Conditions for Determination of Organic Acid Content by High Performance Liquid Chromatography

[0064]

[0065] 2. Microbial quantity detection

[0066] The number of Aspergillus, lactic acid bacteria, yeast, and bacteria were all determined by viable cell counting. Specifically, 1g of koji or fermented mash was weighed into a sterile tube using an electronic balance in a clean bench, 9mL of physiological saline was added, the mixture was vortexed and diluted serially to the corresponding multiples, and 100μL of the diluted solution was spread onto a counting plate.

[0067] 3. Detection of enzyme activity in curing process

[0068] (1) Neutral protease activity and acidic protease activity: According to SB / T 10317-1999 standard, weigh 2g of ground koji material, add 40mL of deionized water, and extract at 40℃ for 2h. Experimental group: 1mL each of diluted and preheated enzyme solution and preheated casein (reaction substrate), react in a water bath at 40℃ for 10min, and add 2mL of TCA solution. Control group: 1mL of enzyme solution and 2mL of TCA solution are mixed, incubated for 10min, and 1mL of substrate is added. After the reaction is terminated, both experimental and control groups are incubated for 20min, centrifuged at 8000r / min for 5min, 0.5mL of supernatant is taken, 2.5mL of 0.4mol / L Na2CO3 solution and 0.5mL of Folin-Ciocalteu reagent are added, mixed well, and developed in a water bath for 20min. The absorbance value is measured at a wavelength of 660nm. The standard curve for tyrosine is y = 0.0099x - 0.0208 (R² = 0.9994).

[0069] (2) Amylase activity: Experimental group: 1 mL each of enzyme solution and starch solution (reaction substrate), reacted at 37℃ for 1 h, and then 1 mL of 1 mol / L NaOH was added; Blank group: 1 mL each of enzyme solution and NaOH solution, incubated in a water bath for 1 h, and then 1 mL of reaction substrate was added. After the reaction, 0.5 mL of the reaction solution was taken, 0.5 mL of DNS reagent was added, boiled for 10 min, cooled to room temperature, and then 4 mL of deionized water was added and mixed well. The absorbance was measured at a wavelength of 540 nm. The maltose standard curve was y = 0.0005x - 0.0427 (R² = 0.999).

[0070] (3) Cellulase activity: In the blank group, 25 μL of enzyme solution was mixed with 475 μL of buffer solution; in the experimental group, 25 μL of enzyme solution was mixed with 475 μL of CMC solution, reacted at 50℃ for 20 min, 0.5 mL of DNS reagent was added, boiled for 5 min, cooled to room temperature, and diluted to 5 mL with deionized water. After mixing thoroughly, the absorbance was measured at a wavelength of 540 nm. The standard curve for glucose content was y = 0.0009x - 0.0383 (R² = 0.9998).

[0071] (4) Moisture content of koji material: Refer to the direct drying method in GB 5009.3-2016. First, weigh the weighing bottle with an electronic balance and record the weight. Weigh 2g of koji material, dry it in an oven at 105℃ for 2 hours, and then measure the total weight to calculate the moisture content.

[0072] 4. Determination of physicochemical properties of fermented soy sauce samples

[0073] Take 10g of fermented soy sauce mash into a 50mL centrifuge tube, centrifuge at 12000r / min for 30min, collect the supernatant and filter it through a 0.22μm pore size filter membrane for physicochemical index determination. The pH of the fermented soy sauce mash was measured using a precision pH meter; the total acid and amino acid nitrogen content were determined by titration; the reducing sugar content was determined by the 3,5-dinitrosalicylic acid method. Specific methods refer to GB 18186-2000, the national standard for brewed soy sauce.

[0074] 5. Determination of total phenols, total flavonoids, and antioxidant capacity.

[0075] (1) Total phenol content:

[0076] After mixing 1 mL of sample diluent with 4.5 mL of deionized water, 500 μL of Folin-Ciocalteu reagent was added, and the mixture was allowed to stand at room temperature for 10 min. Then, 4 mL of 7.5% (w / v) Na₂CO₃ solution was added, mixed well, and allowed to stand in the dark for 2 h. The absorbance was measured at 765 nm. The gallic acid standard curve was y = 0.009x + 0.0127(R²). 2 =0.9979).

[0077] (2) Total flavonoid content:

[0078] Dilute the sample with methanol to a suitable concentration, centrifuge at 8000 rpm for 15 min, take 1 mL of the supernatant and mix with 4 mL of 70% ethanol and 0.3 mL of 5% NaNO2 solution, let stand for 5 min. Then add 0.3 mL of 10% Al(NO3)3 solution, mix well and let stand for 6 min. Then add 4 mL of 1 mol / L NaOH solution and 0.4 mL of 30% ethanol solution, mix well and let stand for 10 min. The absorbance is measured at 510 nm. The rutin standard curve is y = 1.1657x + 0.0025 (R² = 0.9999).

[0079] (3) DPPH free radical scavenging rate:

[0080] Experimental group: Add 1 mL of 200 μM / L DPPH ethanol solution to 1 mL of sample dilution solution, mix well and react in the dark for 30 min.

[0081] Control group: 1 mL each of sample diluent and anhydrous ethanol were mixed;

[0082] Blank group: 1 mL each of deionized water and anhydrous ethanol were mixed. The absorbance was measured at a wavelength of 517 nm.

[0083] DPPH free radical scavenging rate = [1 - (absorbance of experimental group - absorbance of control group) / absorbance of blank group] × 100%.

[0084] (4) Reducing power: Take 1 mL of sample dilution, mix it with 2.5 mL of PBS solution (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide solution, react at 50℃ for 20 min, add 2.5 mL of 10% TCA solution, centrifuge at 5000 r / min for 10 min, take 2.5 mL of supernatant, add 2.5 mL of deionized water and 0.5 mL of 1% ferric chloride solution in sequence, mix well, and let stand at room temperature for 10 min. The absorbance is measured at a wavelength of 700 nm. The reducing power of the sample is expressed as the Trolox content. The Trolox standard curve is y = 3.946x + 0.034(R 2 =0.995).

[0085] 6. Determination of umami amino acid content

[0086] Take 1 mL of soy sauce sample, dilute it 20 times with 5% (w / v) trichloroacetic acid solution, and let it stand at room temperature in the dark for 30 min. Filter the solution through an aqueous filter membrane with a pore size of 0.22 μm and collect the filtrate for analysis. The content of umami amino acids was determined by high performance liquid chromatography. Specific conditions are shown in Table 2.

[0087] Table 2: Conditions for Determination of Free Amino Acid Content by High Performance Liquid Chromatography

[0088]

[0089] Example 1: Isolation and identification of Pediococcus pentosaceus LS1

[0090] The specific steps are as follows:

[0091] (1) Bacterial isolation and screening:

[0092] Take a sample of fermented soybean paste (25g) and add it to 225mL of sterile physiological saline. Incubate at 37℃ for 220r·min. -1 Shake for 1 hour. Spread 100 μL of the serially diluted liquid onto MRS medium containing 2 g / L sorbic acid and incubate at 37°C for 24 hours. Pile single colonies for isolation and purification three times, and then store at -80°C.

[0093] (2) Screening of strains:

[0094] Pediococcus strain screening: Colonies from the plate were picked and transferred to PCR tubes, and amplified using Pediococcus-specific primers Pa-F (5'-CGAACTTCCGTTAATTGATTAT-3') and Pa-R (5'-ACCTTGCGGTCGTACTCC-3'). The PCR system was: 0.5 μL DNA template (50 ng / μL) -1The reagents included 10 μL of Taq polymerase, 0.5 μL each of forward and reverse primers (10 μmol·L⁻¹), and 8.5 μL of 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 band appearing was identified as Pediococcus.

[0095] (3) Bacterial species identification

[0096] 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'-AGAGTTTGATCCTGGCTCAG3') and 1492R (5'-GGTTACCTTGTTACGACTT3'). 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 MEGAX software.

[0097] The results showed that it was identified as Pediococcus pentosaceus and named Pediococcus pentosaceus LS1.

[0098] In the same period, three strains of Pediococcus pentosaceus were screened out and named: P. pentosaceus ZQ3 and P. pentosaceus ZQ4, respectively.

[0099] Example 2: Performance testing of Pediococcus pentosaceus LS1 strain

[0100] Since soy sauce fermentation is a high-salt environment, the salt tolerance and other characteristics of the strains were investigated. Figure 1 The specific steps are as follows:

[0101] 1. Analysis of the salt tolerance of the strain:

[0102] (1) Preparation of lactic acid bacteria culture

[0103] A single colony of lactic acid bacteria obtained from previous streak isolation was picked up with an inoculation loop and inoculated into 10 mL of MRS medium. The culture temperature was 37℃, and the culture was allowed to stand until the absorbance measured at 600 nm was approximately 2. 200 μL of the colony was then transferred to 10 mL of MRS medium and incubated at 37℃ for 10–12 h until the logarithmic growth phase.

[0104] (2) Take the above lactic acid bacteria culture, centrifuge at 8000 r / min for 5 min, discard the supernatant, wash the bacterial precipitate twice with sterile physiological saline, discard the supernatant, and then... 8Inoculations of CFU / mL were inoculated into MRS liquid medium containing 180 g / L (equivalent to a salt concentration of 18%) and 160 g / L (equivalent to a salt concentration of 16%), respectively, resuspended, and incubated at 37°C for 24 h.

[0105] Take 100 μL of the culture medium obtained after cultivation, dilute it to an appropriate multiple (0 h, 24 h), spread it on MRS solid plates, and calculate the survival rate.

[0106] The results showed that the survival rate of Pediococcus pentosaceus LS1 was 94.68% after 24 hours of culture at a salt concentration of 16%; and 84.02% after 24 hours of culture at a salt concentration of 18%.

[0107] The survival rates of Pediococcus pentosaceus ZQ3 and ZQ4 after 24 hours of culture at a salt concentration of 16% were 69.87% and 64.76%, respectively.

[0108] The survival rates of Pediococcus pentosaceus ZQ3 and ZQ4 after 24 hours of culture at 18% salt concentration were 64.96% and 59.21%, respectively.

[0109] 2. Analysis of organic acid production capacity:

[0110] (1) Preparation of primary seed liquid

[0111] Pick a single colony of lactic acid bacteria obtained from the previous streak isolation with an inoculation loop, inoculate it into 10 mL of MRS medium, and incubate at 37℃ for 10–12 h until the logarithmic growth phase.

[0112] (2) The prepared primary seed culture was transferred to MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h to obtain secondary seed culture. The prepared secondary seed culture was centrifuged at 12000 r / min for 8 min, and the supernatant was filtered through a 0.22 μm sterile aqueous filter membrane before being sampled and measured.

[0113] The results are shown in the table below (values ​​in the table are rounded to two decimal places).

[0114] Table 3: Organic acids and their content in fermentation supernatant

[0115]

[0116] The results showed that different species of lactic acid bacteria have different abilities to secrete organic acids. The *Weissella paramesenteroides* WP20 and WS6 listed in the table are strains screened in the laboratory during the same period.

[0117] In the MRS medium system, Pediococcus secretes four main organic acids: malic acid, acetic acid, lactic acid, and citric acid. P. pentosaceus LS1 has the highest acid production, with a total acid production of 27.93 g / L. Acetic acid and lactic acid account for more than 85% of the total organic acid production.

[0118] 3. Determination of free amino acid production capacity:

[0119] (1) Preparation of seed liquid

[0120] Pick a single colony of lactic acid bacteria obtained from the previous streak isolation with an inoculation loop, inoculate it into 10 mL of MRS medium, and incubate at 37℃ for 10–12 h until the logarithmic growth phase.

[0121] (2) The seed culture obtained in step (1) was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v), and after static incubation at 37°C for 24 h, the resulting culture solution was refluxed at 8000 r·min. -1 Centrifuge for 10 minutes to obtain fermentation supernatant.

[0122] Take 1 ml of fermentation supernatant, dilute it 5 times with 5% (w / v) trichloroacetic acid, sonicate it for 10 min, let it stand for 5 min to prevent overheating, sonicate it for another 10 min, and let it stand for 30 min. After centrifugation, filter the supernatant through a 0.22 μm sterile aqueous filter membrane for later use.

[0123] The results showed that the P. pentosaceus LS1 strain had the strongest ability to produce umami amino acids aspartic acid and glutamic acid in the culture medium system, at 0.14 g / L and 0.30 g / L, respectively.

[0124] 4. Analysis of the antibacterial properties of the strain:

[0125] The agar diffusion method was used to inhibit the growth of B. halodurans, S. saprophyticus, and B. subtilis.

[0126] (1) Culture of strains

[0127] Indicator bacteria B. halodurans, S. saprophyticus, and B. subtilis were inoculated into LB liquid medium and incubated at 37°C and 220 rpm. -1 Incubate for 12 hours.

[0128] The selected Pediococcus pentosaceus LS1 strain was inoculated into MRS medium and incubated statically at 37°C for 24 h. The resulting culture was then heated to 8000 rpm. -1 Centrifuge for 10 min to obtain fermentation supernatant. Filter the fermentation supernatant through a 0.22 μm sterile aqueous filter membrane for later use.

[0129] (2) Antibacterial test

[0130] Using the double-layer Oxford cup method, pour 10 mL of basal agar (15% w / v) into a petri dish and allow it to solidify; place a sterile Oxford cup on the culture medium. Cool LB and MRS media to approximately 50°C, then add 1 mL of indicator bacteria (final concentration 10). 6 CFU·mL -1 Mix well.

[0131] Measure 15 mL of mixed culture medium from a centrifuge tube and add it to a plate. After it solidifies, remove the Oxford cup. Take 100 μL of lactic acid bacteria (concentration: 1 × 10⁻⁶). 8 The fermentation supernatant (CFU / mL) was collected in the wells, incubated at 4°C for 2 hours, and then cultured at 37°C for 8 hours. The size of the inhibition zone was measured using vernier calipers.

[0132] The results show:

[0133] The inhibition zone of Pediococcus pentosaceus LS1 against Staphylococcus saprophyticus was 21.78±0.5 cm, against Bacillus subtilis it was 12.10±0.5 cm, and against halophilic Bacillus was 19.70±0.5 cm.

[0134] In summary, the selected Pediococcus pentosaceus LS1 strain had a survival rate of 84.02% in an 18% salt concentration culture medium system, with a total acid production of 27.92 g / L. The study also showed that the Pediococcus pentosaceus LS1 strain had excellent antibacterial effect and excellent ability to produce umami amino acids. In the culture medium system, the glutamic acid content produced by this strain was 0.30 g / L, which was superior to that of Enterobacter sieboldii.

[0135] Example 3: Application of Pediococcus pentosaceus LS1 strain in koji production

[0136] The specific steps are as follows:

[0137] 1. Preparation of finished product

[0138] (1) Preparation of Pediococcus pentosaceus LS1 inoculum:

[0139] A single colony of *Pediococcus pentosaceus* LS1 lactic acid bacteria, obtained by streak isolation in the previous stage, was picked up with an inoculation loop and inoculated into 10 mL LMRS medium. The culture was incubated at 37°C until the absorbance measured at 600 nm was about 2.0, thus obtaining the seed culture.

[0140] Transfer 200 μL of the prepared seed culture to 10 mL of MRS medium and incubate at 37°C for 10–12 h until the logarithmic growth phase, at which point the bacterial concentration is 10. 8 CFU / g, dilute with sterile water to the required concentration before use.

[0141] (2) Preparation of finished product

[0142] Soak soybeans in 4 times their weight of water for 10 hours until fully moistened, then drain. Steam at 121℃ for 15 minutes (0.1MPa). Cool to room temperature, then mix the steamed soybeans with flour at a weight ratio of 6:4. And add microorganisms The conditions for making koji are 95% humidity and 30℃. The koji is turned over every 24 hours and water is added intermittently. The koji-making time is 44-48 hours. The koji-making process ends when the surface of the koji material is covered with yellow-green spores.

[0143] The finished koji was divided into three groups by adding different microorganisms, as detailed below:

[0144] A: Add Aspergillus oryzae AS 3.042 at 1.6‰ of the total weight of raw materials;

[0145] AA: Aspergillus oryzae AS 3.042 koji and Aspergillus niger AS 3.350 were added sequentially at 1.6‰ and 0.4‰ of the total weight of the raw materials;

[0146] AAP: Aspergillus oryzae AS 3.042 koji and Aspergillus niger AS 3.350 were inoculated sequentially at 1.6‰ and 0.4‰ of the total weight of the raw materials, respectively, while Pediococcus pentosaceus LS1 inoculum prepared in step (1) was added. The inoculum amount of the inoculum was 6 × 10⁻⁶. 6 CFU / g.

[0147] 2. The effects of adding compound microbial agents on the quality of the finished product were tested separately.

[0148] (1) Determination of enzyme activity in soy sauce koji ( Figure 2 )

[0149] Weigh 2g of the ground koji material into a 50mL centrifuge tube and determine the enzyme activity. The results are shown in the table below (the values ​​presented in the table are data after 48 hours of koji preparation, and the values ​​in the table are retained to two decimal places):

[0150] Table 4: Effects of compound microbial agents on the activity of enzyme systems in koji production

[0151]

[0152] The results are as follows: Compared with the control group, the neutral protease activity of the experimental group inoculated with Aspergillus niger (AA group) and the experimental group supplemented with Aspergillus niger and P. pentosaceus LS1 (AAP group) increased by 34.41% and 27.73%, respectively; the acidic protease activity increased by 18.68% and 27.53%, respectively. Furthermore, mixed-culture koji preparation also showed a certain improvement effect on the cellulase and amylase activities of the koji material. At 48 h, the cellulase activities of the AA and AAP groups were 97.00 U / g and 95.89 U / g, respectively. Compared with koji preparation using pure Aspergillus oryzae, these were increases of 8.72% and 7.47%, respectively.

[0153] (2) Determination of microbial indicators in soy sauce koji ( Figure 3 )

[0154] Weigh 1g of the prepared koji into a 15ml sterile centrifuge tube and test the viable bacteria count. The results are shown in the table below (the values ​​presented in the table are data after 48 hours of koji preparation, and the values ​​in the table are rounded to two decimal places):

[0155] Table 5: Effects of compound microbial agents on microorganisms in koji production

[0156]

[0157] The results showed that at the end of the koji-making process, the number of Aspergillus oryzae in the experimental group with added Aspergillus niger (AA group) and the experimental group with added Aspergillus niger and P. pentosaceus LS1 (AAP group) was slightly lower than that in the control group. This may be related to the competition of Aspergillus niger and lactic acid bacteria for nutrients with Aspergillus oryzae, inhibiting its growth. However, the impact was not significant; the decrease in the number of Aspergillus oryzae was no more than one order of magnitude and did not affect the fermentation of soy sauce. During the koji-making process, all three groups of lactic acid bacteria showed a phenomenon of rapid growth rate in the early stage of koji-making, followed by a slowdown.

[0158] (3) Determination of the content of functional components in soy sauce koji ( Figure 4 )

[0159] Weigh 2g of the ground koji material into a 50mL centrifuge tube and determine the content of functional components. The results are shown in the table below (the values ​​presented in the table are data from 48 hours of koji preparation, and the values ​​in the table are retained to two decimal places):

[0160] Table 6: Effects of compound microbial agents on functional components of fermented koji.

[0161]

[0162] The results are as follows: The total phenol and total flavonoid contents of the koji-making groups (AAP group) made from Aspergillus niger AS 3.350 and P. pentosaceus LS1 were significantly higher than those of the Aspergillus oryzae control group. At the end of koji-making, their contents were 1.25 g / L and 0.70 g / L, respectively, representing increases of 9.65% and 7.00%. This may be related to the higher cellulase activity of the koji materials in this group compared to the control group.

[0163] Example 4: Application of Pediococcus pentosaceus LS1 strain in soy sauce fermentation

[0164] The specific steps are as follows:

[0165] 1. Soy sauce fermentation

[0166] (1) Preparation of finished product

[0167] 1) Preparation of Pediococcus pentosaceus LS1 inoculum:

[0168] A single colony of *Pediococcus pentosaceus* LS1 lactic acid bacteria obtained from previous streak isolation was picked up with an inoculation loop, inoculated into 10 mL LMR S medium, and cultured at 37°C. The culture was then allowed to stand until the absorbance measured at 600 nm was approximately 2, thus obtaining the seed culture.

[0169] Transfer 200 μL of the prepared seed culture to 10 mL of MRS medium and incubate at 37°C for 10–12 h until the logarithmic growth phase, at which point the bacterial concentration is 10. 8 CFU / g, dilute with sterile water to the required concentration before use.

[0170] 2) Preparation of finished product

[0171] Mix soybeans with 4 times their weight of water and soak for 10 hours. After fully moistening, drain the water and steam at 121℃ for 15 minutes (0.1MPa).

[0172] Cool the mixture at room temperature, then mix the cooked soybeans and flour at a mass ratio of 6:4, and add microorganisms. The koji-making conditions are 90% humidity and 30℃. The koji-making time is 24 hours, with water added intermittently. The fermentation time is 44-48 hours. The koji-making process is completed when the surface of the koji material is covered with yellow-green spores.

[0173] The finished koji was divided into three groups by adding different microorganisms, as detailed below:

[0174] A: Add Aspergillus oryzae AS 3.042 at 1.6‰ of the total weight of raw materials;

[0175] AA: Aspergillus oryzae AS 3.042 koji and Aspergillus niger AS 3.350 were added sequentially at 1.6‰ and 0.4‰ of the total weight of the raw materials;

[0176] AAP: Aspergillus oryzae AS 3.042 koji and Aspergillus niger AS 3.350 were inoculated sequentially at 1.6‰ and 0.4‰ of the total weight of the raw materials, respectively, while Pediococcus pentosaceus LS1 inoculum prepared in step (1) was added. The inoculum amount of the inoculum was 6 × 10⁻⁶. 6 CFU / g.

[0177] (2) Soy sauce fermentation:

[0178] A 20% (w / v) brine solution and the koji material obtained in step (1) were mixed at a mass ratio of 1.8:1 and stirred evenly in a fermentation container to achieve a salt concentration of 18 g / 100 mL. The mixture was then sealed and placed in a 30°C constant temperature incubator for 35 days. Stirring was performed once daily during the early stages of fermentation and once every two to three days during the later stages. Samples were taken every five days.

[0179] The three groups of soy sauce were obtained respectively:

[0180] A: Soy sauce prepared by adding only Aspergillus oryzae AS 3.042 to the starter culture;

[0181] AA: Soy sauce prepared by adding Aspergillus oryzae AS 3.042 koji and Aspergillus niger AS 3.350 koji.

[0182] AAP: Soy sauce prepared by adding Aspergillus oryzae AS 3.042 koji, Aspergillus niger AS 3.350, and Pediococcus pentosaceus LS1 inoculants simultaneously;

[0183] 2. Experimental Results

[0184] The properties of the prepared soy sauce were tested separately. Figures 5-8 The results are as follows:

[0185] (1) Determination of physicochemical properties of fermented soy sauce samples

[0186] 10g of soy sauce samples were taken into 50mL centrifuge tubes for physicochemical index determination. The results are shown in Table 7 below (the values ​​presented in this table are from day 35 of fermentation, and the values ​​in the table are rounded to two decimal places):

[0187] Table 7: Effects of compound microbial agents on physicochemical properties during soy sauce fermentation

[0188]

[0189] The results showed that at the end of fermentation, the total acid content in soy sauces fermented with Aspergillus niger AS 3.350 (AA group) and those fermented with Aspergillus niger AS 3.350 and LS1 (AAP group) was 6.14 g / L and 6.48 g / L, respectively, which were 6.64% and 12.50% higher than those produced by Aspergillus oryzae alone. The amino acid nitrogen content was 0.41 g / 100 mL and 0.43 g / 100 mL, respectively, which were 14.09% and 19.61% higher than the control group. In the fermented mash of the AA group and the AAP group, the highest reducing sugar content was 98.29 g / L and 101.94 g / L, respectively (e.g., ...). Figure 6 As shown, the highest reducing sugar content was observed on day 20 of fermentation, after which it showed a decreasing trend. Compared to the control group, the content was 48.61% and 54.13% higher, respectively, which is attributed to the higher amylase activity in the fermented koji (as indicated). Figure 2 This is related to the fact that it is more conducive to the degradation of starchy raw materials.

[0190] (2) Determination of total phenols, total flavonoids and antioxidant capacity of fermented soy sauce samples

[0191] Samples were taken and analyzed for total phenols, total flavonoids, and antioxidant capacity. The results are shown in Table 8 below (the values ​​presented in this table are from day 35 of fermentation, and the values ​​are rounded to two decimal places):

[0192] Table 8: Effect of compound microbial agents on the antioxidant capacity of soy sauce

[0193]

[0194] The results showed that previous studies have indicated that the content of total polyphenols, total flavonoids, and melanoidins in soy sauces made from different raw materials were significantly positively correlated with the antioxidant activity of soy sauces. Mixed-culture fermentation improved the antioxidant capacity of the fermented soy sauce mash to some extent. At the end of fermentation, the total phenol content of the mash fermented with Aspergillus niger (AA group) and with mixed-culture fermentation of Aspergillus niger and P. pentosaceus LS1 (AAP group) was 3.67 g / L and 3.71 g / L, respectively, which were 3.97% and 5.13% higher than the control group; the total flavonoid content was 0.87 g / L and 0.94 g / L, respectively, which were 45.78% and 57.82% higher than the Aspergillus oryzae control group. After the fermentation process, the DPPH free radical scavenging rate of the AA and AAP groups increased by 37.71% and 41.79% respectively compared with the control group; the reducing power increased by 8.90% and 10.95% respectively. This may be related to the fact that the total phenols and total flavonoids content in the AA and AAP group soy sauces was better than that in the control group.

[0195] (3) Determination of umami amino acid content

[0196] 1 mL of soy sauce samples were taken for the determination of umami amino acid content. The results are shown in Table 9 below (the values ​​presented in this table are data from soy sauce fermented for 35 days, and the values ​​in the table are rounded to two decimal places):

[0197] Table 9: Effects of Compound Microbial Agents on Umami Amino Acids

[0198]

[0199] The results showed that compared with the Aspergillus oryzae control group (Group A), the umami amino acid content of soy sauce fermented with mixed-culture Aspergillus niger AS 3.350 and P. pentosaceus LS1 (Group AAP) was significantly increased, with aspartic acid and glutamic acid increasing by 39.77% and 54.24%, respectively. Compared with Aspergillus niger (Group AA), the aspartic acid and glutamic acid content of soy sauce fermented with mixed-culture Aspergillus niger AS 3.350 and P. pentosaceus LS1 (Group AAP) also increased by 11.15% and 20.49%, respectively.

[0200] The above results indicate that fermentation of soy sauce with compound microbial agents can effectively improve the flavor of soy sauce.

[0201] 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 microbial inoculant, characterized in that, The microbial inoculants are a strain of Pediococcus pentosaceus LS1, which was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCC NO:M 20242843, as well as Aspergillus oryzae AS3 .042 and Aspergillus niger AS3 .

350.

2. A fermentation agent containing the microbial agent of claim 1.

3. Wheat koji or bran koji containing the microbial agent as described in claim 1.

4. The application of the microbial agent according to claim 1 in the preparation of finished koji.

5. The application according to claim 4, characterized in that, The application includes, but is not limited to, at least one of (a) to (c): (a) Increase the number of microorganisms in the fermentation process of the finished product koji; (b) Increase the content of protease activity in the finished product; (c) Increase the content of total phenols and total flavonoids in the finished product.

6. The application of the microbial agent according to claim 1 in the preparation of soy sauce.

7. The application according to claim 6, characterized in that, The applications include enhancing the flavor and antioxidant capacity of soy sauce.

8. The application according to claim 6, characterized in that, The application includes, but is not limited to, at least one of (a) to (d): (a) Increase the content of total phenols and total flavonoids in the soy sauce system; (b) Increase the content of reducing sugars, total acids, and amino acid nitrogen in the soy sauce system; (c) Enhance the antioxidant capacity of the soy sauce system; (d) Enhance the umami flavor of soy sauce.

9. A method for preparing soy sauce, characterized in that, The method includes the following steps: (1) Koji-making process: Steamed soybeans and flour were mixed evenly at a mass ratio of (6-7):(4-3). Aspergillus oryzae AS3.042 and Aspergillus niger AS3.350 were inoculated sequentially at 1.5-3‰ and 0.2-1‰ of the total weight of raw materials, respectively. At the same time, Pediococcus pentosaceus LS1 was added. It was deposited at the China Center for Type Culture Collection on December 18, 2024, with accession number CCTCC NO:M 20242843. The koji-making conditions were 92-95% humidity, 28-32℃ temperature, and 44-48h fermentation time. The koji-making process was stopped when the surface of the koji material was covered with yellow-green spores, and the koji was obtained. (2) Fermentation process: The prepared koji from step (1) is mixed with NaCl-containing brine at a volume ratio of 1:1.8 to 2, so that the salt concentration of the system is 18 to 20%, and fermented at 28℃-30℃ for 40 to 60 days.

10. The method according to claim 9, characterized in that, The inoculum size of *Pediococcus pentosaceus* LS1 is at least 6 × 10⁻⁶. 6 CFU / g.

11. Soy sauce prepared by any one of claims 9-10.

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