Bacillus amyloliquefaciens for improving color and luster of soy sauce, inhibiting bacteria and enhancing aroma and application of bacillus amyloliquefaciens

Abstract

The invention provides bacillus amyloliquefaciens capable of improving color and luster of soy sauce, inhibiting bacteria and enhancing aroma and application of the bacillus amyloliquefaciens, and belongs to the field of microorganisms, the bacillus amyloliquefaciens Z-WQ20 is preserved in China General Microbiological Culture Collection Center on June 18, 2024, the preservation number is CGMCC NO.30996, and the preservation number is CGMCC NO.30996. The bacillus amyloliquefaciens Z-WQ20 is preserved in the China General Microbiological Culture Collection Center on June 18, 2024. The preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the strain disclosed by the invention can inhibit bacteria, remove black, improve color and luster and increase ester in the soy sauce fermentation production process, has the efficacy of enhancing aroma, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a bacillus amyloliquefaciens strain capable of improving the color of soy sauce, inhibiting bacteria and enhancing flavor, and application thereof. Background Art

[0002] Soy sauce is a condiment made from soybeans, wheat and other raw materials, which is fermented by microorganisms to enhance the aroma and flavor. However, it is easily contaminated by harmful pathogens during the fermentation process, which affects the quality of soy sauce.

[0003] At the same time, soy sauce can color food. The coloring ability of soy sauce on food and ingredients is mainly related to its color rate index. In addition to adding caramel color, browning is an important reaction in the color generation of soy sauce during the fermentation process. It is the main reaction related to the increase of color rate in the production process of soy sauce. Moderate browning helps soy sauce form an attractive reddish brown, while excessive browning will make the color of soy sauce too dark, affecting the sensory and use value.

[0004] At present, there is no report on strains that can solve the problems of fermented soy sauce being too dark in color, inhibiting bacteria, and enhancing flavor. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] In view of the above problems or problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide Bacillus amyloliquefaciens Z-WQ20.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: a strain of Bacillus amyloliquefaciens that improves the color, antibacterial and flavor of soy sauce: Bacillus amyloliquefaciens Z-WQ20, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on June 18, 2024, with a deposit number of CGMCCNO.30996, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] As a preferred embodiment of the Bacillus amyloliquefaciens of the present invention, the Bacillus amyloliquefaciens grows in an environment of 15% NaCl w / w and pH=4 and has good stress resistance.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide the use of Bacillus amyloliquefaciens Z-WQ20 in the preparation of fermented soy sauce.

[0011] As a preferred solution of the application of the present invention, it includes:

[0012] After Bacillus amyloliquefaciens Z-WQ20 was activated, it was centrifuged at 4°C, 8000r / min for 10min, the supernatant was discarded, and the mixture was washed twice with sterile saline to adjust to 10 6 cfu / ml bacterial suspension;

[0013] Adding it into the mash and fermenting it for 15 to 18 days to obtain fermented soy sauce;

[0014] The amyloliquefaciens bacillus has the effect of removing black and improving the color of soy sauce during the color formation of soy sauce.

[0015] As a preferred embodiment of the application of the present invention, the Bacillus amyloliquefaciens is used to increase ester substances in the preparation of soy sauce.

[0016] As a preferred embodiment of the application of the present invention, the ester substances include ethyl benzoate, ethyl trans-4-decenoate, ethyl linoleate, ethyl palmitate, methyl 9-octadecene-12-ynoate, ethyl 9-hexadecenoate, ethyl linoleate, ethyl pentadecanoate, methyl cis-octadecanoate, isopropyl linoleate, ethyl arachidonic acid, methyl cis-8,11,14-eicosatetrienoate, ethyl acetate, ethyl heptadecanoate, pentamethyl furan bromide, ethyl tetradecanoate, diethyl succinate, ethyl linolenate, methyl palmitate, ethyl oleate, ethyl octadecanoate, 2-methylpropyl hexadecanoate, ethyl laurate and ethyl phenylacetate.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of Bacillus amyloliquefaciens in agricultural biological control, wherein the Bacillus amyloliquefaciens inhibits the activity of Staphylococcus gallinarum and Fusarium oxysporum.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides Bacillus amyloliquefaciens Z-WQ20, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on June 18, 2024, with a deposit number of CGMCCNO.30996, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the strain grows in an environment of 15% NaCl w / w, pH=4, and has good stress resistance;

[0020] Adding Bacillus amyloliquefaciens during the soy sauce fermentation production process can inhibit bacteria, remove black spots, improve color, increase esters, and enhance flavor, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0022] Figure 1 The colony morphology and optical microscope examination images of Bacillus amyloliquefaciens on LB nutrient agar in the examples of the present invention are shown.

[0023] Figure 2 This is a graph showing the growth data of Bacillus amyloliquefaciens under stress in an embodiment of the present invention.

[0024] Figure 3 This is a graph showing the antibacterial activity of Bacillus amyloliquefaciens in an embodiment of the present invention, wherein the left one is the antibacterial zone of Staphylococcus aureus, and the right one is the antibacterial zone of Fusarium oxysporum. Specific implementation methods

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0029] The Bacillus amyloliquefaciens Z-WQ20 in the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on June 18, 2024, with a deposit number of CGMCCNO.30996, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0030] Example 1: Isolation of Bacillus amyloliquefaciens

[0031] The strain was from soybean paste and the samples were taken by five-point method. After being thoroughly mixed, the samples were placed in sterile glycerol EP tubes and stored in a -80℃ refrigerator.

[0032] 5.0 g of soybean paste sample stored at -80 °C was weighed under sterile conditions, mixed with 45 mL of sterile saline, and shaken in a shaker for 2 h to disperse the microbial cells. -1 Dilutions of different concentrations were prepared and serial dilutions were performed.

[0033] Take 0.1mL of each dilution to 10 -3 , 10 -4 and 10 -5 The diluted solution was inoculated into LB nutrient agar medium. After spreading, the plates were placed in a 30°C incubator for 48 hours. The colonies producing pigments were observed and picked, and repeated streaking purification was performed to obtain pure strains.

[0034] Streak the plate on LB solid medium and incubate at 30℃ for 48h to observe the colony morphology: colony size, color, shape, colony edge, protrusion, etc. Figure 1 .

[0035] The picked colonies are milky white in color; they are mainly round in shape, moist and sticky on the surface, easy to pick up, and have a smooth surface.

[0036] At the same time, the screened strains cannot ferment galactose, maltose, sucrose, trehalose, melibiose, lactose, cellobiose, and starch; they can assimilate glucose, galactose, sorbose, ribose, xylose, L-arabinose, D-arabinose, sucrose, maltose, trehalose, and cellobiose.

[0037] The optimum growth temperature is 30±2℃, and the maximum temperature is 45℃.

[0038] Example 2: Molecular Biological Identification of Bacillus amyloliquefaciens

[0039] Pick up millet-sized colonies (clone diameter is 0.1-0.3 cm) and put them into the TE solution. Try to avoid picking up any solid culture medium (mixed culture medium will affect the subsequent genome extraction effect).

[0040] Add 100 μL TE solution to fully resuspend the precipitate, then add 20 μL lysozyme and 10 μL RNaseA, mix well, incubate at 37 °C for 2.5 hours, and shake every 30 minutes;

[0041] Add 100 μL of LYS solution (preheated at 55°C), gently pipette to mix, then add 20 μL of proteinase K solution and mix thoroughly;

[0042] Incubate at 55°C for 30 min, add 220 μL of EXT solution, invert several times until mixed, and centrifuge at 10,000 × g for 5 min;

[0043] Transfer the supernatant to a new 1.5mL EP tube and add 220μL of anhydrous ethanol. Invert several times until mixed. Place the DNA spin column in a 2mL collection tube, transfer all samples to the DNA spin column, and centrifuge at 10,000×g for 1 minute. Discard the waste liquid from the centrifugation and reload the DNA spin column into the 2mL collection tube. Add 500μL of PD solution, centrifuge at 10,000×g for 1 minute, discard the waste liquid, and reload the DNA spin column into the 2mL collection tube. Add 600μL PW solution, centrifuge at 10,000×g for 1 minute, and discard the waste liquid.

[0044] Refill the DNA spin column into a 2 mL collection tube and repeat the process. Centrifuge at 12,000 rpm for 2 minutes.

[0045] Re-install the DNA centrifuge column into a clean 1.5 mL EP tube and let it dry at room temperature for 5 minutes; add 50-100 μL of Solution Buffer or water to the DNA centrifuge column; let it stand at room temperature for 3 to 5 minutes, and centrifuge at 12,000 rpm for 2 minutes to collect the genomic DNA.

[0046] DNA was stored at -20℃. Universal primers ITS1:TCCGTAGGTGAACCTGCGG; ITS4:TCCTCCGCTTATTGATATGC. The extracted DNA sample was diluted appropriately and used as a PCR template for amplification using 1×TSE101 Gold Mix from Qingke. The sequencing results were spliced ​​using ContigExpress, and the inaccurate parts at both ends were removed. The spliced ​​sequences were compared in the NCBI database (blast.ncbi.nlm.nih.gov), and the identification result was Bacillus amyloliquefaciens.

[0047] The nucleotide sequence (Seq_1) of Bacillus amyloliquefaciens in the present invention is:

[0048]

[0049] Example 3: Determination of stress resistance of Bacillus amyloliquefaciens

[0050] Pour 2 mL of bacterial solution frozen at -80°C into 100 mL of LB liquid culture medium (1% tryptone, 1% sodium chloride, 0.5% yeast powder, adjusted to pH = 7), and culture at 30°C with a shaker at 120 rpm for 48 h. Then, inoculate the cultured bacterial solution into 100 mL of fresh LB liquid at a ratio of 2%, and culture at 30°C with a shaker at 120 rpm until OD 600 = 0.7, which is considered to be completed.

[0051] The activated strain was inoculated in batches with an inoculum size of 2% into LB liquid culture medium containing 0%, 5%, 10%, 15%, and 20% NaCl (w / w); LB liquid culture medium with a pH of 3, 3.5, 4, 4.5, 5, 5.5, and 6, and cultured at 30°C for 120 h. The culture solution was centrifuged to collect the bacteria, and dried in an oven at 55°C for 24 h to constant weight. The dry weight was used to reflect the biomass.

[0052] The results of the test are as follows Figure 2 Considering that Bacillus amyloliquefaciens needs to play a practical role in soy sauce fermentation, the biomass greater than 0.01 g / L was selected as the threshold to evaluate the stress resistance of Bacillus amyloliquefaciens; it was found that the tolerance limit thresholds of NaCl and pH were 15% (w / w) and 4, respectively.

[0053] Example 4: Inhibitory effect of Bacillus amyloliquefaciens Z-WQ20 on pathogenic microorganisms

[0054] (1) Preparation of fermentation supernatant:

[0055] A loop of purified single colony was picked and inoculated into LB liquid culture medium, and cultured with shaking at 220 rpm at 37°C for 24 h to obtain the fermentation broth of Bacillus amyloliquefaciens Z-WQ20;

[0056] The supernatant was collected by centrifugation at 10,000 rpm for 10 min at 4°C, filtered using a 0.22 μm sterile membrane, and stored at -20°C for later use; it was naturally thawed at room temperature before use.

[0057] (2) Preparation of indicator bacteria solution

[0058] Take 25% glycerol solution of pathogenic bacteria Staphylococcus aureus, streak culture on solid culture medium, then pick out revived single colonies and inoculate them into liquid culture medium, and culture them until OD600 is 1.0-1.2.

[0059] Take 25% glycerol solution of pathogenic fungus Fusarium oxysporum, streak culture on solid culture medium, then pick out revived single colonies and inoculate them into liquid culture medium, and culture them until OD600 is 1.0-1.2.

[0060] (3) Determination of antibacterial ability:

[0061] Prepare a 1.5% pure agar plate, pour it, cool and solidify it, place the sterile Oxford cups on the left and right, dilute the bacterial solution of Staphylococcus aureus to be tested in sterile saline by 100 times, take 100 μL of the diluted bacterial solution in LB culture medium containing 0.7% agar cooled to about 50°C, shake it gently to disperse the bacterial solution, and pour it on the pure agar culture medium placed in the Oxford cups, and wait for the agar to cool.

[0062] After the agar has cooled, the Oxford cup was taken out, 100 μL of the fermentation supernatant of Bacillus amyloliquefaciens Z-WQ20 was added to the left well, and sterile LB medium was added to the right well as a blank control; after diffusion in a 4°C refrigerator for 4 hours, it was placed in an incubator at 37°C and cultured upright for 14-18 hours, and the diameter of the Staphylococcus aureus inhibition zone was measured.

[0063] Prepare 1.5% pure agar plates, pour them, cool and solidify them, evenly place five sterile Oxford cups, dilute the bacterial solution of the pathogen Fusarium oxysporum to be tested in sterile physiological saline by 100 times, take 100 μL of the diluted bacterial solution in 0.7% agar PDA culture medium cooled to about 50°C, shake it gently to disperse the bacterial solution, and pour it on the pure agar culture medium with the Oxford cups placed, and wait for the agar to cool.

[0064] After the agar has cooled, take out the Oxford cup, add 100 μL of fermentation supernatant of Bacillus amyloliquefaciens Z-WQ20 to three wells in a straight line, and add sterile PDA medium to the wells on the left and right sides as a blank control; place in a 4°C refrigerator for diffusion for 4 hours, place in an incubator at 37°C for 14-18 hours, and measure the diameter of the inhibition zone of Fusarium oxysporum. Figure 3 .

[0065] Bacillus amyloliquefaciens Z-WQ20 has a good inhibitory effect on Staphylococcus aureus, a common pathogen in soy sauce, and Fusarium oxysporum, a common pathogen in agricultural soil, with the diameters of the inhibition zones being approximately 22.5±1.0mm and 27.4±1.0mm.

[0066] Figure 3 The antibacterial effect of Bacillus amyloliquefaciens Z-WQ20 on Staphylococcus aureus and Fusarium oxysporum.

[0067]

[0068] Example 5: Comparison of the ability of Bacillus amyloliquefaciens to remove black spots and improve color

[0069] Pour 2 mL of bacterial solution frozen at -80°C into 10 mL of LB medium and culture at 30°C with a shaker at 120 rpm for 48 h. Then add 2% of the cultured bacterial solution to 100 mL of fresh LB medium and culture at 30°C with a shaker at 120 rpm until OD600 = 0.7, which is considered to be activated.

[0070] Preparation of mash: Mix the cooked soybeans and flour in a ratio of 100:40 and cool to about 30°C, then add 0.3% koji and mix again, pile them on a bamboo plate, and then place the bamboo plate in an incubator at about 30°C and a relative humidity of about 90% for incubation;

[0071] When the koji material appears white and lumps, and the product temperature reaches 35℃, turn the koji for the first time to loosen it. After turning the koji, level the koji material and lower the product temperature to 30-32℃. When the product temperature rises and the koji material lumps again, turn the koji for the second time. After turning the koji for the second time, wait for the koji block to turn green, mix it with 18°Be` / 20℃ salt solution and ferment it. When making mash, the amount of salt solution used is 2-2.5 times the amount of raw materials.

[0072] The activated strain fermentation liquid was centrifuged at 4°C, 8000r / min for 10 min, the supernatant was discarded, and the mixture was washed twice with sterile saline (mass fraction of 0.85%) and adjusted to 10 6 cfu / ml of bacterial suspension was added to the mash and fermented for 15 days. At the same time, a control group was set up, which was the mash fermented without adding bacteria.

[0073] The coloring effect of soy sauce is mainly characterized by the color rate (EBC), and the red index and yellow index are used to characterize the color rendering of the main colors.

[0074] The experimental group and the control group were diluted 20 times and then filtered. 3 ml was taken into a cuvette to measure the OD 610 OD 510 OD 460 Substitute the value at into the formula to calculate the color rate (EBC), red index and yellow index.

[0075] Color ratio (EBC) = (OD 610 ×20000) / 0.076

[0076] Red index = 10 × lg OD 510 / OD 610

[0077] Yellow index = 10 × lg OD 460 / OD 610

[0078] The test showed that the color rate of the experimental group's soy sauce was 13.0% lower than that of the control group, the red index of the experimental group was 22.0% higher than that of the control group, and the yellow index was 19.67% higher. This showed that the color of the soy sauce became lighter, and there was a positive correlation between the red index and the yellow index, which increased. The soy sauce was bright and not dark in proportion, and showed a distinct reddish brown, while the brown color of the control group was more obvious.

[0079] Table 1 Soy sauce color index

[0080]

[0081] Example 6: Application and effect of Bacillus amyloliquefaciens in brewing soy sauce

[0082] Pour 2 mL of bacterial solution frozen at -80°C into 10 mL of LB medium and culture at 30°C with a shaker at 120 rpm for 48 h. Then add 2% of the cultured bacterial solution to 100 mL of fresh LB medium and culture at 30°C with a shaker at 120 rpm until OD600 = 0.7, which is considered to be activated.

[0083] The activated strain fermentation liquid was centrifuged at 4°C, 8000r / min for 10 min, the supernatant was discarded, and the mixture was washed twice with sterile saline (mass fraction of 0.85%) and adjusted to 10 6 cfu / ml of bacterial suspension was added to the mash and fermented for 15 days. At the same time, a control group was set up, in which the mash was fermented without adding Bacillus amyloliquefaciens.

[0084] The volatile components in the fermentation broth were determined by HS-SPME-GC-MS. 2 mL of sample was taken into a 15 mL headspace bottle, 30 μL of 2-octanol (20 ppm) was added as an internal standard, and the sample was balanced at 60 °C and 600 r / min for 20 min. At the same temperature and speed, the aged 50 / 30 μm DVB / CAR / PMDS extraction head was inserted for headspace adsorption for 30 min. After the adsorption, the extraction head was inserted into the injection port of the gas chromatography (GC) and desorbed at 250 °C for 15 min. GC conditions: the starting temperature was 40 °C for 30 min, then increased to 150 °C at 4 °C / min for 1 min, and then increased to 250 °C at 8 °C / min for 6 min.

[0085] Helium was used as the carrier gas, the injection port temperature was 250 °C, and the injection time was 1 min.

[0086] MS conditions: ion source temperature 200 °C, interface temperature 220 °C, solvent delay time 1.5 min, ionization mode EI, 70 eV scanning mass range 33-500 m / z, scanning rate 3.00 scans / s.

[0087] Compounds with similarity ≥ 80% were selected according to the NIST11 database search, and qualitative analysis was performed by combining ion fragments and retention index. 2-octanol was used as the internal standard substance and the internal standard method was used for quantification.

[0088] After testing, the experimental group detected more ester substances, especially ethyl linoleate, ethyl benzoate, ethyl palmitate, ethyl myristate, ethyl octadecanoate, etc. These substances increase the fruity and waxy aromas, making the overall flavor of the soy sauce richer and softer.

[0089] Table 2 Volatile flavor substances of soy sauce esters

[0090]

[0091]

[0092] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. Bacillus amyloliquefaciens Z-WQ20 was deposited in the General Microbiology Center of China Culture Collection Administration on June 18, 2024, with the deposit number CGMCCNO.30996, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Bacillus amyloliquefaciens according to claim 1, wherein: The amyloliquefaciens bacillus grows in an environment of 15% NaCl w / w and pH=4.0 and has a strong stress resistance ability.

3. Use of the Bacillus amyloliquefaciens according to claim 1 or 2 in the preparation of fermented soy sauce.

4. The use according to claim 3, characterized in that: include, After Bacillus amyloliquefaciens Z-WQ20 was activated, it was centrifuged at 4°C, 8000r / min for 10min, the supernatant was discarded, and the mixture was washed twice with sterile saline to adjust to 10 6 cfu / ml bacterial suspension; Adding it into the mash and fermenting it for 15 to 18 days to obtain fermented soy sauce; The amyloliquefaciens bacillus has the effect of removing black and improving the color of soy sauce during the color formation of soy sauce.

5. The use according to claim 3, characterized in that: The Bacillus amyloliquefaciens increases ester substances in the preparation of soy sauce.

6. The use according to claim 5, characterized in that: The ester substances include ethyl benzoate, ethyl trans-4-decenoate, ethyl linoleate, ethyl palmitate, methyl 9-octadecene-12-ynoate, ethyl 9-hexadecenoate, ethyl linoleate, ethyl pentadecanoate, methyl cis-octadecanoate, isopropyl linoleate, ethyl arachidonic acid, methyl cis-8,11,14-eicosatetrienoate, ethyl acetate, ethyl heptadecanoate, pentamethyl furan bromide, ethyl tetradecanoate, diethyl succinate, ethyl linolenate, methyl palmitate, ethyl oleate, ethyl octadecanoate, 2-methylpropyl hexadecanoate, ethyl laurate and ethyl phenylacetate.

7. Use of the Bacillus amyloliquefaciens according to claim 1 or 2 in agricultural biological control.

8. The use according to claim 7, characterized in that: The Bacillus amyloliquefaciens inhibits the activity of Staphylococcus aureus.

9. The use according to claim 7, characterized in that: The Bacillus amyloliquefaciens has the activity of inhibiting Fusarium oxysporum.

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