Koji making method for improving flavor amino acid content and oxidation resistance of soy sauce

Abstract

The invention discloses a koji-making method for improving the flavor amino acid content and oxidation resistance of soy sauce, and belongs to the technical field of biological engineering. The pediococcus pentosaceus LS1 strain which is resistant to salt and has the best flavor amino acid producing capacity and acid producing capacity is obtained, aspergillus oryzae Huniang 3.042, aspergillus niger Huniang 3.350 and pediococcus pentosaceus LS1 are combined for starter propagation, and compared with single-bacterium starter propagation, composite starter propagation can effectively improve the content of functional components of finished starter, and the content of total polyphenols and total flavonoids is increased. For fermentation, mixed starter propagation does not affect normal fermentation of the soy sauce, the functional components, the antioxidant ability, the flavor amino acid content and the organic acid content are all increased, the total phenol content is respectively increased by 5.13% and 1.71% compared with the contrast and the content of total flavonoids respectively increased by 57.82% and 8.13% compared with the contrast and the content of total flavonoids added with the aspergillus niger 3.350, and the content of total flavonoids added with the aspergillus niger 3.350 is respectively increased by 5.13% and 1.71% compared with the contrast and the content of total flavonoids added with the aspergillus niger. And the total amount of flavor amino acids and the scavenging rate of free radicals are also improved.

Description

Technical Field

[0001] The invention relates to a koji making method for improving the umami amino acid content and antioxidant property of soy sauce, belonging to the technical field of bioengineering. Background Art

[0002] Koji making is a key step in soy sauce brewing. The quality of the koji affects the taste, color and aroma of the soy sauce. Poor quality koji will make the soy sauce taste bland, dull in color, and even produce peculiar smell. The quality of the koji will directly affect the content and activity of the enzyme system in the fermentation, and then affect the decomposition of the raw materials and the formation of the flavor of the soy sauce. At present, most of my country uses Aspergillus oryzae Shanghai Brewing 3.042 for single-bacteria koji making, which has the advantages of simple operation and fast production. However, pure koji making will result in a relatively single koji enzyme system, so that the soy sauce raw materials cannot be fully fermented, which in turn affects the quality and flavor of the soy sauce.

[0003] It has been proven in practice that mixed koji with multiple strains can improve the quality and flavor of soy sauce. Studies have found that mixed koji has higher enzyme activity and richer enzyme system, which can improve the utilization rate of soy sauce raw materials, improve the quality of soy sauce, and is conducive to the release of bioactive ingredients (such as total flavonoids, total phenols, etc.) during the fermentation process of soy sauce. Fermentation with mixed koji with richer enzyme system can not only improve the utilization rate of soy sauce raw materials, improve the quality of soy sauce, but also increase the content and effect of functional ingredients in soy sauce, and has good development prospects.

[0004] Current research mainly focuses on adding microorganisms during the fermentation process of soy sauce, but there are problems such as the limited salt tolerance of the strains resulting in insignificant effects, and the addition of strains during the fermentation process may lead to increased secondary precipitation of soy sauce due to cell residues. In addition, when the strains are added during the fermentation stage, their metabolic activities may have complex effects on the fermentation cycle and the production of flavor substances. If they are added at the wrong time, the fermentation cycle may be prolonged or the flavor formation may be affected. Summary of the invention

[0005] There are differences in the raw materials, temperature, humidity, pH and other conditions required for the growth of different microorganisms. In the process of mixed bacteria koji making, it is necessary to find the optimal conditions for the symbiosis of lactic acid bacteria and Aspergillus oryzae to ensure that both can grow well. The interaction between multiple microorganisms is still unclear. The symbiotic relationship between lactic acid bacteria and Aspergillus oryzae may affect their respective growth and metabolism, and it is necessary to study the interaction mechanism between them. The addition of lactic acid bacteria may affect the activity of the enzyme system in the koji material. It is necessary to study the effect of lactic acid bacteria on the activity of the Aspergillus oryzae enzyme system and the specific effect of this effect on the quality of soy sauce. Lactic acid bacteria have a strong acid production ability, and it is necessary to control the acid production by controlling the amount of strain added to avoid excessive pH reduction leading to koji acid spoilage.

[0006] Technical solution:

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

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

[0009] (1) The characteristics of lactic acid bacteria isolated from soybean paste 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 bacteria koji making.

[0010] (2) Different mixed bacteria koji making groups were designed, with only Aspergillus oryzae Shanghai Brewing 3.042 as the control group. The total phenolic and flavonoid contents of the koji in different groups were investigated to explore the effect of mixed bacteria koji making on the antioxidant capacity of soy sauce koji.

[0011] (3) To investigate the effects of mixed bacteria koji and control group koji on the quality of soy sauce, and to conduct comparative studies on the physical and chemical indices, functional components, antioxidant capacity, and free amino acids of soy sauce fermented with different koji groups.

[0012] The present invention provides a Pediococcus pentosaceus LS1, which has been deposited in the China Center for Type Culture Collection on December 18, 2024, with a deposit number of CCTCC NO: M 20242843.

[0013] The invention provides a microbial agent, which contains the above-mentioned Pediococcus pentosaceus LS1 or its fermentation liquid, or contains the lysate of the Pediococcus pentosaceus LS1, or contains living bacterial cells of the Pediococcus pentosaceus LS1, dry bacterial bodies of the Pediococcus pentosaceus LS1 obtained by freeze-drying, immobilized cells of the Pediococcus pentosaceus LS1, liquid bacterial agent of the Pediococcus pentosaceus LS1, solid bacterial agent of the Pediococcus pentosaceus LS1, or the 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 CFU / g.

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

[0016] The present invention also provides the use of the Pediococcus pentosaceus LS1 and / or the 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 of (a) to (b):

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

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

[0020] The present invention also provides application of the Pediococcus pentosaceus LS1 and / or the microbial preparation 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) increasing the content of total phenols and total flavonoids in the soy sauce system;

[0023] (b) increasing the reducing sugar, total acid and amino acid nitrogen contents of soy sauce;

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

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

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

[0027] (1) Koji making process:

[0028] The steamed soybeans and flour are uniformly mixed in a mass ratio of (6-7): (4-3), and 1.5-3‰ and 0.2-1‰ of the total weight of the raw materials are sequentially added with Aspergillus oryzae AS3.042 koji essence, Aspergillus niger AS3.350 koji essence, the Pediococcus pentosaceus LS1 and / or the microbial preparation, and the koji making conditions are as follows: an environmental humidity of 92-95%, a temperature of 28-32° C., and a fermentation time of 44-48 hours. When the surface of the koji material is covered with yellow-green spores, the koji making is terminated;

[0029] (2) Fermentation process:

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

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

[0032] The present invention also provides the use of the Pediococcus pentosaceus LS1 and / or the microbial preparation in improving the flavor and antioxidant capacity of soy sauce.

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

[0034] Beneficial Effects

[0035] (1) The present invention takes high-salt dilute soy sauce as the research object, and screens out a strain P. pentosaceus LS1 that can tolerate high-salt dilute soy sauce fermentation conditions and produce high umami amino acids by evaluating the salt tolerance and acid production performance of multiple lactic acid bacteria isolated from soy mash.

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

[0037] Biomaterial Deposit

[0038] A strain of Pediococcus pentosaceus LS1, classified and named Pediococcus pentosaceus LS1, was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO:M20242843, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 : Changes in the activities of proteases (neutral and acidic), cellulases and amylases during mixed-bacteria koji making.

[0041] Figure 3 :Dynamic changes of microbial quantity during mixed-bacteria koji making and soy sauce fermentation.

[0042] Figure 4 : Changes of total phenols and total flavonoids contents in koji materials during mixed fungus koji making.

[0043] Figure 5 :Dynamic changes of microbial quantity during mixed-bacteria koji making and soy sauce fermentation.

[0044] Figure 6 :Effects of mixed bacteria koji making on physicochemical parameters during soy sauce fermentation.

[0045] Figure 7 :Effects of mixed bacteria koji making on the contents of total phenols, total flavonoids and antioxidant capacity of soy sauce.

[0046] Figure 8 :Effect of mixed bacteria koji making on free amino acid content (A), especially umami amino acid content (B).

[0047] Among them, A: only A.oryzae Shanghai Niang 3.042 was added to make koji; AA: A.oryzae Shanghai Niang 3.042 and A.niger 3.350 were added to make koji; AAP: A.oryzae Shanghai Niang 3.042, A.niger 3.350 and P.pentosaceus LS1 were added to make koji. *, p<0.05; **, p<0.01; ***, p<0.001. DETAILED DESCRIPTION

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

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

[0050] The culture medium involved in the following examples is as follows:

[0051] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of distilled water, 15 g of agar powder added to the solid medium, and sterilized by high-pressure steam at 121°C for 20 min.

[0052] MRS broth medium: Oxoid Company, UK, solid medium plus 15g agar powder, sterilized by high pressure steam at 118℃ for 20min.

[0053] Rose Bengal culture medium: 5 g peptone, 10 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.1 g chloramphenicol, 1000 mL distilled water, solid culture medium plus 15 g agar powder, sterilize with high pressure steam at 121°C for 20 min.

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

[0055] 1. Analysis of lactic acid bacteria strain characteristics

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

[0057] Take the lactic acid bacteria liquid, centrifuge at 8000r / min for 5min, remove the supernatant, wash the bacterial precipitate twice with sterile saline, and use 1×10 9 CFU / mL inoculation amount, inoculate into MRS liquid culture medium containing salt (NaCl) of 180g / L (equivalent to salt concentration of 18%) and 160g / L (equivalent to salt concentration of 16%), respectively, and culture at 37℃ for 24h. Take 100μL of the culture solution obtained after culture and dilute it to the appropriate multiple of bacterial solution (0h, 24h) and spread it on MRS solid plate to calculate the survival rate.

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

[0059] The organic acid content was determined by HPLC.

[0060] The primary seed solution was transferred to MRS liquid culture medium at a 2% (v / v) inoculation rate, and cultured at 37°C for 24 hours to obtain the secondary seed solution. The secondary seed solution was centrifuged at 12000r / min for 8 minutes, and the supernatant was filtered through a 0.22μm sterile water filter membrane and then sampled for determination.

[0061] The sample was pre-column derivatized with o-xylene (OPA). Liquid phase system: Agilent 1260, chromatographic column ODSHYPERSIL (250mm×4.6mm, 2.5μm); mobile phase A: 5g anhydrous sodium acetate, 5mL tetrahydrofuran, 200μL triethylamine, pH 7.2; mobile phase B: 5g anhydrous sodium acetate, 200mL ultrapure water, 400mL methanol, 400mL acetonitrile, pH 7.2. Injection volume: 10μL elution rate: 1mL·min-1; elution time 40min; column temperature: 40℃; detector: UV338 nm.

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

[0063] Table 1: HPLC determination conditions for organic acid content

[0064]

[0065] 2. Microbial quantity detection

[0066] The number of Aspergillus, lactic acid bacteria, yeast, and bacteria was determined by the live bacteria counting method, that is, 1 g of koji or mash was weighed into a sterile tube using an electronic balance in a clean bench, 9 mL of normal saline was added, vortexed to mix, gradient diluted to the corresponding multiples, and 100 μL of the dilution was spread on a counting plate.

[0067] 3. Detection of sclerotinase activity

[0068] (1) Neutral protease activity and acidic protease activity: According to SB / T 10317-1999 standard, weigh 2g of ground koji, add 40mL of deionized water, and extract at 40℃ for 2h. Experimental group: 1mL of diluted and preheated enzyme solution and 1mL of 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 is mixed with 2mL of TCA solution, incubated for 10min, and 1mL of substrate is added. After the reaction is terminated, both the experimental group and the control group 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 phenol reagent are added, mixed and colorized in a water bath for 20min, and the absorbance is measured at a wavelength of 660nm. The standard curve of tyrosine was y=0.0099x-0.0208 (R2=0.9994).

[0069] (2) Amylase activity: Experimental group: 1 mL of enzyme solution and starch solution (reaction substrate), react at 37°C for 1 h, add 1 mL of 1 mol / L NaOH; Blank group: 1 mL of enzyme solution and 1 mL of NaOH solution, add 1 mL of reaction substrate after 1 h in water bath. After the reaction, take 0.5 mL of reaction solution, add 0.5 mL of DNS reagent, boil for 10 min, cool to room temperature, add 4 mL of deionized water and mix well. The wavelength of absorbance measurement is 540 nm. The maltose standard curve is y=0.0005x-0.0427 (R2=0.999).

[0070] (3) Cellulase activity: The blank group was a mixture of 25 μL enzyme solution and 475 μL buffer solution; the experimental group was a mixture of 25 μL enzyme solution and 475 μL CMC solution, reacted at 50°C for 20 min, added with 0.5 mL DNS reagent, boiled for 5 min, cooled to room temperature, fixed to 5 mL with deionized water, mixed evenly, and measured absorbance 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: Refer to the direct drying method in GB 5009.3-2016. First, weigh the weighing bottle with an electronic balance and record the weight of the bottle. Weigh 2g of koji, dry it in an oven at 105℃ for 2h, then measure the total weight and calculate the moisture content.

[0072] 4. Determination of physical and chemical indicators of soy sauce fermentation samples

[0073] Take 10g of mash in a 50mL centrifuge tube, centrifuge at 12000r / min for 30min, take the supernatant and filter it with a filter membrane with a pore size of 0.22μm, and then measure the physical and chemical indicators. Use a precision pH meter to measure the pH of the mash; the total acid and amino acid nitrogen content are determined by titration; the reducing sugar content is determined by 3,5-dinitrosalicylic acid method. For specific methods, refer to GB 18186-2000 National Standard for Brewing Soy Sauce.

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

[0075] (1) Total phenol content:

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

[0077] (2) Total flavonoid content:

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

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

[0080] Experimental group: 1 mL of sample diluent was added with 1 mL of 200 μM / L DPPH ethanol solution, mixed and reacted for 30 min in the dark;

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

[0082] Blank group: 1 mL of deionized water and 1 mL of anhydrous ethanol were mixed. The absorbance measurement wavelength was 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: 1 mL of sample diluent was mixed with 2.5 mL of PBS solution (pH = 6.6) and 2.5 mL of 1% potassium ferricyanide solution, reacted at 50°C for 20 min, 2.5 mL of 10% TCA solution was added, centrifuged at 5000 r / min for 10 min, 2.5 mL of supernatant was taken, 2.5 mL of deionized water and 0.5 mL of 1% ferric chloride solution were added in sequence, mixed and placed at room temperature for 10 min, and the absorbance was measured at a wavelength of 700 nm. The reducing power of the sample was expressed by the Trolox content, and the Trolox standard curve was 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, place it at room temperature away from light for 30 minutes, filter it with a water filter membrane with a pore size of 0.22 μm, and take the filtrate for determination. The umami amino acid content is determined by high performance liquid chromatography. The specific conditions are shown in Table 2.

[0087] Table 2: HPLC conditions for determination of free amino acid content

[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 the mash sample (25 g) and add 225 mL of sterile saline at 37 °C and 220 r·min -1 Oscillate for 1 hour. Take 100 μL of the gradient dilution liquid and apply it to MRS medium containing 2 g / L sorbic acid, and culture at 37°C for 24 hours. Pick a single colony, streak it, isolate and purify it 3 times, and then store it at -80°C.

[0093] (2) Screening of strains:

[0094] Pediococcus strain screening: Pick the colonies on the plate into PCR tubes and amplify using Pediococcus specific primers Pa-F (5'-CGAACTTCCGTTAATTGATTAT-3') and Pa-R (5'-ACCTTGCGGTCGTACTCC-3'). PCR system: DNA template 0.5 μL (50 ng·μL -1), Taq enzyme 10 μL, upstream and downstream primers 0.5 μL each (10 μmol·L-1), ddH2O8.5 μL; amplification conditions: 94℃ 3min, 94℃ 30s, 58℃ 30s, 72℃ 1min, 12℃ 10min. The product was analyzed by gel electrophoresis. The target band was Pediococcus.

[0095] (3) Identification of bacterial species

[0096] The genomic DNA of the strain was extracted using the bacterial genome extraction kit of Shanghai Shenggong Biological Company, and PCR amplification was performed using the 16S rRNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG3') and 1492R (5'-GGTTACCTTGTTACGACTT3') as a template. The PCR product was sent to Tianlin Biotechnology Wuxi Co., Ltd. for sequencing, and the sequencing results were compared by BLAST at NCBI, and the strain phylogenetic tree was constructed using MEGAX software.

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

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

[0099] Example 2: Testing of the performance of Pediococcus pentosaceus LS1 strain

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

[0101] 1. Analysis of salt tolerance of strains:

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

[0103] Use an inoculation loop to pick up a single colony of lactic acid bacteria obtained by streaking on the plate in the early stage, inoculate it into 10 mL of MRS medium, culture it at 37°C, and culture it statically until the absorbance measured at a wavelength of 600nm is about 2. Take 200 μL and transfer it to 10 mL of MRS medium, and culture it statically at 37°C for 10 to 12 hours until the logarithmic growth phase.

[0104] (2) Take the above lactic acid bacteria solution, centrifuge at 8000r / min for 5min, remove the supernatant, wash the bacterial precipitate twice with sterile saline, discard the supernatant, and use 1×10 8The inoculum size was 2.577 CFU / mL, and the culture medium was inoculated with MRS liquid culture medium containing 180 g / L (equivalent to 18% salt concentration) and 160 g / L (equivalent to 16% salt concentration) of NaCl, respectively. The culture was resuspended and cultured at 37°C for 24 h.

[0105] 100 μL of the culture solution obtained after cultivation was taken respectively, diluted to an appropriate multiple of bacterial solution (0h, 24h), and spread on an MRS solid plate to 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 16% salt concentration and 84.02% after 24 hours of culture at 18% salt concentration.

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

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

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

[0110] (1) Preparation of primary seed solution

[0111] The single colony of lactic acid bacteria obtained by streaking on the plate in the early stage was picked up with an inoculation loop and inoculated into 10 mL of MRS medium. The culture temperature was 37°C and cultured at 37°C for 10 to 12 hours until the logarithmic growth phase.

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

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

[0114] Table 3: Organic acids and their contents 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 in the table are strains screened in the laboratory at the same time.

[0117] In the MRS culture medium system, Pediococcus can secrete 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, of which 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 solution

[0120] The single colony of lactic acid bacteria obtained by streaking on the plate in the early stage was picked up with an inoculation loop and inoculated into 10 mL of MRS medium. The culture temperature was 37°C and cultured at 37°C for 10 to 12 hours until the logarithmic growth phase.

[0121] (2) The seed solution obtained in step (1) was inoculated into MRS liquid culture medium at an inoculum rate of 2% (v / v), and the culture medium was cultured at 37°C for 24 h. -1 , centrifuge for 10 minutes to obtain the fermentation supernatant.

[0122] Take 1 ml of fermentation supernatant and dilute it 5 times with 5% (w / v) trichloroacetic acid, put it on the ultrasonic instrument for 10 minutes, let it stand for 5 minutes to prevent overheating, then ultrasonicate for 10 minutes, let it stand for 30 minutes. After centrifugation, filter the supernatant with a 0.22 μm sterile water filter membrane for later use.

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

[0124] 4. Analysis of antibacterial properties of strains:

[0125] Agar diffusion method, inhibition indicator bacteria are B.halodurans, S.saprophyticus, B.subtilis.

[0126] (1) Culture of strains

[0127] The indicator bacteria B.halodurans, S.saprophyticus and B.subtilis were inoculated into LB liquid medium respectively and heated at 37℃ and 220r·min -1 Culture for 12h.

[0128] The selected Pediococcus pentosaceus LS1 strain was inoculated into MRS medium and cultured at 37°C for 24 hours. -1 , centrifuge for 10 min to obtain the fermentation supernatant. Filter the fermentation supernatant with a 0.22 μm sterile water filter membrane for later use.

[0129] (2) Antibacterial experiment

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

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

[0132] The results show:

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

[0134] In summary, the survival rate of the finally screened Pediococcus pentosaceus LS1 was 84.02% in the 18% salt concentration culture medium system, and the total acid production was 27.92 g / L. After research, the antibacterial effect of the Pediococcus pentosaceus LS1 strain was also very superior, and the ability of the strain to produce umami amino acids was also excellent. In the culture medium system, the glutamate content produced by the bacteria was 0.30 g / L, which was better than that of Weissella mesenteroides.

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

[0136] The specific steps are as follows:

[0137] 1. Preparation of finished koji

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

[0139] The single colony of lactic acid bacteria Pediococcus pentosaceus LS1 obtained by streaking on the plate in the previous stage was picked up with an inoculation loop, inoculated into 10 mL of LRS medium, and cultured at 37°C until the absorbance measured at a wavelength of 600 nm was about 2.0, thereby obtaining a seed solution;

[0140] Take 200 μL of the prepared seed solution and transfer it to 10 mL of MRS medium. Incubate at 37°C for 10 to 12 hours until the logarithmic growth phase. At this time, the bacterial concentration is 10 8 CFU / g, dilute with sterile water to the required concentration when using.

[0141] (2) Preparation of finished koji

[0142] Mix soybeans with 4 times the mass of water and soak for 10 hours. Drain the water after fully moistening, and cook at 121℃ for 15 minutes (0.1MPa). Cool to room temperature, and mix the cooked soybeans with flour in a mass ratio of 6:4. And add microorganisms The conditions for koji making are an environmental humidity of 95% and a temperature of 30°C; the koji is turned over every 24 hours and water is added at irregular intervals. The koji making time is 44-48 hours. When the surface of the koji is covered with yellow-green spores, the koji making is finished.

[0143] Among them, the finished koji is divided into three groups by adding different microorganisms, as follows:

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

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

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

[0147] 2. Detect the effect of adding compound bacterial agents on the quality of finished koji

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

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

[0150] Table 4: Effect of compound bacterial agent on the activity of koji enzyme system

[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 added with Aspergillus niger and P. pentosaceus LS1 (AAP group) increased by 34.41% and 27.73% respectively; the acid protease activity increased by 18.68% and 27.53% respectively. In addition, mixed bacteria koji making also has a certain improvement effect on the cellulase activity and amylase activity of the koji material. At 48h, the cellulase activity of the AA and AAP groups was 97.00U / g and 95.89U / g respectively. Compared with pure Aspergillus oryzae koji making, it increased by 8.72% and 7.47% respectively.

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

[0154] Weigh 1g of finished koji and put it in a 15ml sterile centrifuge tube to test the number of viable bacteria. The results are shown in the following table (the values ​​presented in the table are data from 48 hours of koji making, and the values ​​in the table are retained to two decimal places):

[0155] Table 5: Effects of compound microbial agents on koji-forming microorganisms

[0156]

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

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

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

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

[0161]

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

[0167] 1) Preparation of Pediococcus pentosaceus LS1 bacterial agent:

[0168] The inoculation loop was used to pick up a single colony of lactic acid bacteria Pediococcus pentosaceus LS1 obtained by streaking the plate in the early stage, and inoculated into 10 mL of LRS medium, cultured at 37°C, and cultured statically until the absorbance measured at a wavelength of 600 nm was about 2, to obtain a seed solution;

[0169] Take 200 μL of the prepared seed solution and transfer it to 10 mL of MRS medium. Incubate at 37°C for 10 to 12 hours until the logarithmic growth phase. At this time, the bacterial concentration is 10 8 CFU / g, dilute with sterile water to the required concentration when using.

[0170] 2) Preparation of finished koji

[0171] Mix soybeans with 4 times the mass of water and soak for 10 hours. Drain the water after fully moistening and cook at 121℃ for 15 minutes (0.1MPa).

[0172] Cool to room temperature, mix the steamed soybeans and flour in a mass ratio of 6:4, and add microorganisms. The koji-making conditions are 90% humidity and 30°C. The koji-turning time is 24 hours, with irregular water replenishment. The fermentation time is 44-48 hours. When the surface of the koji is covered with yellow-green spores, the koji-making is finished.

[0173] Among them, the finished koji is divided into three groups by adding different microorganisms, as follows:

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

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

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

[0177] (2) Soy sauce fermentation:

[0178] Mix 20% (w / v) brine with the koji obtained in step (1) at a mass ratio of 1.8:1, and stir evenly in a fermentation container to make the salt concentration of the system 18g / 100mL. After sealing, place it in a 30°C constant temperature incubator for fermentation for 35 days. Stir once a day in the early stage of fermentation and once every two or three days in the later stage. Take the same thing every 5 days.

[0179] Three groups of soy sauce were obtained:

[0180] A: Soy sauce prepared by adding only Daqu of Aspergillus oryzae AS 3.042;

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

[0182] AAP: Soy sauce made by adding Aspergillus oryzae AS 3.042, Aspergillus niger AS 3.350, and Pediococcus pentosaceus LS1 bacteria to Daqu.

[0183] 2. Experimental results

[0184] The properties of the prepared soy sauce were tested respectively ( Figures 5 to 8 ), the result is as follows:

[0185] (1) Determination of physical and chemical indicators of soy sauce fermentation samples

[0186] 10 g of soy sauce samples were taken respectively and placed in 50 mL centrifuge tubes for determination of physical and chemical indicators. The results are shown in Table 7 below (the values ​​presented in this table are data on the 35th day of fermentation, and the values ​​in the table are retained to two decimal places):

[0187] Table 7: Effects of compound bacterial agents on physical and chemical parameters during soy sauce fermentation

[0188]

[0189] The results showed that at the end of fermentation, the total acid content of the soy sauce in the double-strain fermentation with Aspergillus niger AS 3.350 (AA group) and the triple-strain fermentation with Aspergillus niger AS 3.350 and LS1 (AAP group) was 6.14 g / L and 6.48 g / L, respectively, which was 6.64% and 12.50% higher than that of the soy sauce made with Aspergillus oryzae. The amino acid nitrogen content was 0.41 g / 100 mL and 0.43 g / 100 mL, respectively, which was 14.09% and 19.61% higher than that of the control group. The highest values ​​of reducing sugar content in the soy sauce mash of the double-strain fermentation group (AA group) and the triple-strain fermentation group (AAP group) were 98.29 g / L and 101.94 g / L, respectively. Figure 6 As shown in the figure, the highest reducing sugar content was on the 20th day of fermentation, and it showed a downward trend after 20 days), which was 48.61% and 54.13% higher than that of the control group, respectively, which was related to its higher koji amylase activity ( Figure 2 ), which is more conducive to the degradation of starch raw materials.

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

[0191] Samples were taken for total phenol, total flavonoids and antioxidant capacity, and the results are shown in Table 8 below (the values ​​presented in this table are data on the 35th day of fermentation, and the values ​​in the table are retained to two decimal places):

[0192] Table 8: Effect of compound bacterial agent on the antioxidant capacity of soy sauce

[0193]

[0194] The results showed that existing studies have shown that in the process of analyzing the functional components and antioxidant activity of different raw soy sauces, it was found that the total polyphenols, total flavonoids and melanoidins contents were significantly positively correlated with the antioxidant activity of soy sauce. Mixed fermentation improved the antioxidant capacity of soy sauce to a certain extent. At the end of fermentation, the total phenol content of soy sauce with the addition of Aspergillus niger (AA group) and the addition of Aspergillus niger and P. pentosaceus LS1 mixed fermentation (AAP group) was 3.67g / L and 3.71g / L, which was 3.97% and 5.13% higher than that of the control group, respectively; the total flavonoids content was 0.87g / L and 0.94g / L, respectively, which was 45.78% and 57.82% higher than that of the Aspergillus oryzae control group, respectively. After the fermentation process, the DPPH free radical scavenging rates 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 phenol and flavonoid contents in the soy sauce of the AA and AAP groups were higher than those in the control group.

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

[0196] 1 mL of soy sauce samples were taken for determination of umami amino acid content, and the results are shown in Table 9 below (the values ​​presented in this table are data of soy sauce on the 35th day of fermentation, and the values ​​in the table are retained to two decimal places):

[0197] Table 9: Effect of compound bacterial agent 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 bacteria Aspergillus niger AS 3.350 and P. pentosaceus LS1 (group AAP) increased significantly, with aspartic acid and glutamic acid increasing by 39.77% and 54.24% respectively. Compared with the addition of Aspergillus niger (group AA), the aspartic acid and glutamic acid of soy sauce fermented with mixed bacteria Aspergillus niger AS 3.350 and P. pentosaceus LS1 (group AAP) also increased by 11.15% and 20.49% respectively.

[0200] The above results show that fermenting soy sauce with composite bacteria can effectively improve the flavor of soy sauce.

[0201] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Pediococcus pentosaceus LS1 was deposited in the China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO:M 20242843.

2. A microbial agent, characterized in that: The microbial agent contains the Pediococcus pentosaceus LS1 or its fermentation liquid according to claim 1, or contains the lysate of the Pediococcus pentosaceus LS1, or contains living cells of Pediococcus pentosaceus LS1, dry cells of Pediococcus pentosaceus LS1 obtained by freeze-drying, immobilized cells of Pediococcus pentosaceus LS1, liquid agent of Pediococcus pentosaceus LS1, solid agent of Pediococcus pentosaceus LS1, or Pediococcus pentosaceus LS1 strain in any other form; Preferably, in the microbial agent, the amount of Pediococcus pentosaceus LS1 added is at least: 1×10 6 CFU / g.

3. A starter, wheat koji, bran koji or other types of fermentation koji containing the Pediococcus pentosaceus LS1 according to claim 1 and / or the microbial preparation according to claim 2.

4. Use of the Pediococcus pentosaceus LS1 according to claim 1 and / or the microbial preparation according to claim 2 in the production of finished koji.

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

6. Use of the Pediococcus pentosaceus LS1 according to claim 1 and / or the microbial preparation according to claim 2 in soy sauce production.

7. The use according to claim 6, characterized in that: The application includes but is not limited to at least one of (a) to (d): (a) increasing the content of total phenols and total flavonoids in the soy sauce system; (b) increasing the contents of reducing sugar, total acid and amino acid nitrogen in the soy sauce system; (c) improving the antioxidant capacity in the soy sauce system; (d) Improve the umami flavor of soy sauce.

8. A method for preparing soy sauce, characterized in that: The method comprises the following steps: (1) Koji making process: The steamed soybeans and flour are uniformly mixed in a mass ratio of (6-7): (4-3), and 1.5-3‰ and 0.2-1‰ of the total weight of the raw materials are added to the Aspergillus oryzae AS3.042 and Aspergillus niger AS3.350, respectively. At the same time, the Pediococcus pentosaceus LS1 described in claim 1 and / or the microbial preparation described in claim 2 are added. The koji making conditions are as follows: an environmental humidity of 92-95%, a temperature of 28-32° C., and a fermentation time of 44-48 hours. When the surface of the koji material is covered with yellow-green spores, the koji making is terminated. (2) Fermentation process: The koji prepared in step (1) is mixed with saline containing NaCl in a volume ratio of 1:1.8-2, so that the salt concentration of the system is 18-20%, and fermented at 28°C-30°C for 40-60 days; Preferably, the inoculation amount of Pediococcus pentosaceus LS1 is at least 6×10 6 CFU / g.

9. Use of the Pediococcus pentosaceus LS1 according to claim 1 and / or the microbial preparation according to claim 2 in improving the flavor and antioxidant capacity of soy sauce.

10. Soy sauce obtained by the preparation method according to claim 8.

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