Bacillus subtilis and application thereof in preparation of fish sauce

Abstract

The invention discloses bacillus subtilis and application thereof in preparation of fish sauce, and belongs to the field of microorganisms, the bacillus subtilis WQ1 is preserved in China Center for Type Culture Collection on February 29, 2024, is classified and named as bacillus subtilis WQ1, has the preservation number of CCTCC NO: M 2024356, is preserved in Wuhan University, Wuhan, China, and is applied to fermentation production of fish sauce, and the bacillus subtilis WQ1 has the advantages that the bacillus subtilis WQ1 can be applied to fermentation production of fish sauce; ketone and alcohol compounds in the fish sauce can be increased, and the fish sauce has a good effect in promoting fat increasing, aroma increasing and quality increasing of the fish sauce and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a bacillus subtilis and application thereof in preparing fish sauce. Background Art

[0002] Fish sauce, also known as fish soy sauce in southern my country, is generally made from cheap fish and shrimp as fermentation raw materials, and the fat and protein in the shrimp are decomposed by the participation of various microorganisms, and finally made after a long fermentation. It contains a large amount of small molecules such as taurine and amino acids, as well as essential trace elements such as iron, calcium, and sodium. The filtered fish sauce is orange-yellow or even brown-red in color, and the taste of the fish sauce is slightly fresh, salty, and slightly sweet. The natural fermentation method of brewing fish sauce is to ferment under sunlight at room temperature, using light and oxygen, the fish's own enzyme system, and salt-tolerant yeast, lactic acid bacteria, acetic acid bacteria and other microorganisms in the air. The processing of fish sauce is mainly based on traditional natural fermentation. This process will have complex and unstable microbial composition, a variety of miscellaneous bacteria, poor controllability of product flavor quality, many safety hazards, and a long fermentation cycle. It brings huge challenges to accurately understand the fermentation process and achieve product quality control, and is not conducive to the industrialization of fish sauce products. Low-salt heat preservation fermentation. The combination of low salt and heat preservation can not only inhibit the reproduction of microorganisms, activate various enzymes such as protease contained in the raw materials, but also remove the odor in the fermentation liquid and inhibit the corruption of microorganisms, thereby improving the quality of fish sauce. According to measurements, fish sauce contains various amino acids required by the human body, especially glutamic acid, which is rich in content and is the main component of monosodium glutamate. In addition, it also contains a variety of inorganic salts and phosphorus, magnesium, iron, calcium and iodized salt required by the human body.

[0003] In the fermentation process of fish sauce, the diversity of microbial flora and the complex metabolic reactions of microorganisms play a vital role. These microorganisms include bacteria, fungi and other microorganisms, which participate in key reactions such as lipid oxidation, carbohydrate fermentation and protein degradation during the fermentation process, thereby promoting the formation of flavor substances. The activities of these microorganisms not only affect the richness of the flavor, but also directly affect the quality and taste of fish sauce. The key to the rapid fermentation of fish sauce by inoculation is to select the right microbial strains.

[0004] Research usually focuses on screening strains that can grow effectively in high-salt environments and produce the desired flavor and aroma. Common strains include Bacillus, lactic acid bacteria, yeast, and halophilic bacteria. Yeast usually works in the middle of fermentation, competing with lactic acid bacteria to become the dominant flora in this stage. Bacillus mainly works in the middle and late stages of fermentation and is the main substrate for protein-rich foods (such as fish).

[0005] Currently, fish sauce fermented by yeast has poor flavor and poor effect in producing flavor substances. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of 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.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] To solve the above technical problems, the present invention provides the following technical solution: Bacillus subtilis WQ1 was deposited in the China Center for Type Culture Collection on February 29, 2024, classified and named Bacillus subtilis WQ1, with a collection number of CCTCC NO: M 2024356, and the collection address is Wuhan University, Wuhan, China.

[0009] As a preferred embodiment of the Bacillus subtilis of the present invention, the Bacillus subtilis grows in an environment of pH=4.5 and 15% NaCl (w / w) and has a strong stress resistance.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a use of Bacillus subtilis WQ1 in the preparation of fish sauce.

[0011] As a preferred embodiment of the application of the present invention, the Bacillus subtilis increases the amino acid nitrogen and total nitrogen in the fish sauce.

[0012] As a preferred embodiment of the application of the present invention, the Bacillus subtilis increases the total amount of organic acid in the fermented fish sauce.

[0013] As a preferred embodiment of the application of the present invention, the ketone compounds with caramel aroma and their aldehyde derivatives include 2,3-butanedione, 3-hydroxy-2-butanone, 3-methylbutyraldehyde, methylthioaldehyde, benzaldehyde, heptadecanal, nonanal, phenylacetaldehyde and decanal.

[0014] As a preferred embodiment of the application of the present invention, the content of 2,3-butanedione reaches 28.112 μg / L, and the content of 3-hydroxy-2-butanone reaches 107.142 μg / L.

[0015] As a preferred embodiment of the application of the present invention, the Bacillus subtilis weakens the influence of miscellaneous bacteria on the flavor of fish sauce and inhibits the growth of spoilage microorganisms during the fermentation process.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of Bacillus subtilis in agricultural control.

[0017] Beneficial effects of the present invention:

[0018] (1) The present invention provides a Bacillus subtilis WQ1, which was deposited in the China Center for Type Culture Collection on February 29, 2024, and was classified and named Bacillus subtilis WQ1, with a deposit number of CCTCC NO: M2024356. The deposit address is Wuhan University, Wuhan, China. The Bacillus subtilis WQ1 of the present invention can grow in an environment of pH = 4.5 and 15% NaCl (w / w), and is applied to the fermentation production of fish sauce. It can increase the content of amino acid nitrogen and total nitrogen in fish sauce, and produce a large amount of various organic acids, ketone compounds and aldehyde derivatives in fish sauce. It has good effects in promoting the fat increase, fragrance increase and quality increase of fish sauce, and has good effects in improving the flavor quality of fish sauce.

[0019] (2) The Bacillus subtilis WQ1 of the present invention can be used in industrial production, making full use of the antibacterial properties of Bacillus subtilis and being more widely promoted in the field of agricultural control. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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:

[0021] Figure 1 The colony morphology of Bacillus subtilis on LB nutrient agar in the embodiment of the present invention is shown;

[0022] Figure 2 This is a microscope morphology of Bacillus subtilis in an embodiment of the present invention;

[0023] Figure 3 This is a graph showing the growth data of Bacillus subtilis under stress in an embodiment of the present invention;

[0024] Figure 4 This is a growth curve of Bacillus subtilis in an embodiment of the present invention;

[0025] Figure 5 The results of the protease capacity determination of Bacillus subtilis in the examples of the present invention are as follows;

[0026] Figure 6 The pH value changes after the fish sauce fermentation inoculated with different strains in the embodiment of the present invention;

[0027] Figure 7 The change of amino acid nitrogen content in fish sauce after fermentation with Bacillus subtilis in the embodiment of the present invention;

[0028] Figure 8 The total nitrogen content in the fish sauce after fermentation with Bacillus subtilis in the embodiment of the present invention is changed;

[0029] Fig. 9 The change of organic acid content in fish sauce after fermentation with Bacillus subtilis in the embodiment of the present invention;

[0030] Fig.10 These are the results of the antibacterial test of Bacillus subtilis in the examples of the present invention; wherein, the indicator bacteria in the left figure is Candida albicans, the middle figure is Staphylococcus aureus, the right figure is Salmonella enterica serovar Typhimurium, the upper left corner of the plate is the negative control, the upper right corner is the positive control, and the lower left and lower right are two parallel experimental groups, respectively. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The Bacillus subtilis WQ1 of the present invention was deposited in the China Center for Type Culture Collection on February 29, 2024, classified and named Bacillus subtilis WQ1, with a collection number of CCTCCNO: M 2024356, and the collection address is Wuhan University, Wuhan, China.

[0035] The method for preparing the fish sauce used in the embodiment of the present invention is as follows: the anchovies are cleaned, steamed for 30 minutes, distilled water is added at a mass ratio of 1:1, stirred with a stirrer until homogenized, and weighed;

[0036] The mixture was sterilized under high pressure at 0.1 MPa and 121° C. for 20 min. After cooling to room temperature, 17% salt by weight of the homogenate was added, and 18% Aspergillus oryzae by weight of the anchovy was added. The mixture was fermented at 37° C. for 5 days. After the fermentation was completed, the fermentation liquid was filtered.

[0037] The raw materials in the embodiments of the present invention are all common commercially available products.

[0038] Example 1

[0039] Isolation and screening of Bacillus subtilis:

[0040] Under sterile conditions, 10 g of the traditional fermented soybean paste was weighed and mixed with 90 ml of sterile saline. The mixture was shaken and eluted at 120 r / min for 30 min to disperse the microbial cells. The eluted stock solution was gradient diluted to 10 -1 ~10 -6 .

[0041] Take 0.1mL of each dilution to 10 -3 , 10 -4 and 10 -5 The diluted solution was inoculated into LB nutrient agar medium, with 3 parallels for each gradient. After spreading, the plates were placed in a 37°C incubator for 48 hours. Single colonies with typical spore-forming fungi colony characteristics were observed and selected for repeated streaking purification to obtain pure strains.

[0042] Streak the plate on LB solid medium and incubate at 37℃ for 48h to observe the colony morphology: colony size, color, shape, colony edge, protrusion, etc. Figure 1 .

[0043] The picked colonies are white in color; irregular in shape, with uneven edges, easy to pick up, and smooth on the surface.

[0044] Microscopic morphology of Bacillus subtilis Figure 2 ; For the growth curve of Bacillus subtilis, see Figure 4 .

[0045] Example 2

[0046] Molecular Biological Identification of Bacillus subtilis:

[0047] The isolated Bacillus subtilis WQ1 was subjected to 18S DNA sequencing, and after comparing the sequencing results, it was determined that the strain was Bacillus subtilis.

[0048] Pick up a millet-sized colony (clone diameter is 0.1-0.3 cm) and put it into the TE solution. Try to avoid picking up any solid culture medium (mixed culture medium will affect the subsequent genome extraction effect);

[0049] 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; add 100 μL LYS solution (preheated at 55°C) and mix by gently pipetting;

[0050] Add 20 μL of proteinase K solution and mix thoroughly; incubate at 55°C for 30 minutes, add 220 μL of EXT solution, invert several times until mixed, and centrifuge at 10,000 × g for 5 minutes;

[0051] 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, 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.

[0052] Reload the DNA centrifuge column into a 2 mL collection tube and repeat this step; centrifuge at 12,000 rpm for 2 minutes; reload the DNA centrifuge column into a clean 1.5 mL EP tube and air dry at room temperature for 5 minutes; add 50-100 μL Elution Buffer or water to the DNA centrifuge column; let stand at room temperature for 3-5 minutes, centrifuge at 12,000 rpm for 2 minutes to collect genomic DNA.

[0053] DNA was stored at -20℃, and the universal primers were ITS1:TCCGTAGGTGAACCTGCGG; ITS4:TCCTCCGCTTATTGATATGC. The extracted DNA sample was diluted appropriately and used as a PCR template, and amplified with Qingke 1×TSE101 Gold Mix. The sequencing results were spliced ​​with ContigExpress, and the inaccurate parts at both ends were removed. The spliced ​​sequence was compared in the NCBI database (blast.ncbi.nlm.nih.gov), and the identification result was Bacillus subtilis.

[0054] The nucleotide sequence of Bacillus subtilis in the present invention (SEQ ID No. 1) is:

[0055]

[0056] Example 3

[0057] Determination of Bacillus subtilis stress resistance:

[0058] Pour 2 mL of bacterial solution frozen at -80°C into 100 mL of LB liquid medium (0.5% yeast powder, 1% tryptone, 2% NaCl), 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 37°C with a shaker at 120 rpm until OD600 = 0.8, which is considered to be completed.

[0059] The activated strain was inoculated in batches with 2% inoculation into LB liquid culture medium containing 0%, 5%, 10%, 15%, and 20% NaCl (w / w); LB liquid culture medium with pH values ​​of 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7. The culture was carried out at 37°C for 48 hours, and the fermentation broth was taken and its absorbance value (OD600nm) was measured at a wavelength of 600nm. The blank LB liquid culture medium with the corresponding pH value and ethanol value was used as a control, and the measurement was repeated three times in parallel. The measurement results are shown in the following table. Figure 3 shown.

[0060] The NaCl and pH tolerance limit thresholds were found to be 15% (w / w) and pH 4.5, respectively.

[0061] Example 4

[0062] Determination of protease production ability of Bacillus subtilis:

[0063] Casein culture medium: 0.036% sodium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.0014% zinc chloride, 0.107% disodium hydrogen phosphate heptahydrate, 0.016% sodium chloride, 0.0002% calcium chloride, 0.4% casein, 0.005% hydrolyzed casein, 1.5% agar; adjust pH to 5.5, 1kg / cm 2 Sterilize for 40 minutes.

[0064] Pour the casein medium into a plate, wait for it to solidify, and then spot 2 μL of Bacillus subtilis culture medium with an OD between 0.6 and 0.8 on the center of the casein plate; culture in a 37°C incubator for 96 hours and observe the transparent zone.

[0065] like Figure 5 As shown, the Bacillus subtilis of the present invention produces a transparent circle on the casein plate, the colony growth diameter is 11.00 mm, and the transparent circle diameter is 44.00 mm, which shows that the protease activity of Bacillus subtilis is relatively high. This feature is beneficial to promote better decomposition of protein in unfermented fish sauce and increase the content of amino acid nitrogen and total nitrogen in fish sauce.

[0066] Example 5

[0067] Changes in pH value after fish sauce fermentation with different strains inoculated:

[0068] (1) Preparation method of fish sauce fermented by "blight": use LB liquid culture medium to inoculate Bacillus subtilis WQ1, culture in a shaking incubator at 37°C, and culture the strain to a concentration of 10 8 CFU / mL; 1 mL of bacterial solution was centrifuged and added to 100 mL of fish juice, and fermented in an incubator at 30°C for 100 days to obtain "dry" fermented fish sauce.

[0069] (2) Preparation method of fish sauce fermented by "bacillus + gelatin": use LB liquid culture medium to inoculate Bacillus subtilis, culture in a shaking incubator at 37°C, and culture the strain to a concentration of 10 8 CFU / mL;

[0070] YPD liquid medium was used to inoculate red yeast (GT_317, the same strain as in CN2023101642796, the same below), and cultured in a shaking incubator at 30°C until the strain concentration reached 10 8 CFU / mL;

[0071] 1 mL of the bacterial solution and 1 mL of the above-mentioned Bacillus subtilis bacterial solution were respectively added to 100 mL of fish juice by centrifugation, and fermented in an incubator at 30° C. for 100 days to obtain "bacillus + gelatin" fermented fish sauce.

[0072] (3) Preparation method of fish sauce fermented by "glue": Use YPD liquid culture medium to inoculate glue red yeast (GT_317), culture in a shaking incubator at 30°C, and culture the strain to a concentration of 10 8 CFU / mL;

[0073] Take 1 mL of bacterial solution by centrifugation and add it to 100 mL of fish juice, and ferment it in an incubator at 30°C for 100 days.

[0074] (4) Preparation of fish sauce without inoculation: The fish sauce without adding bacterial strains was directly fermented in an incubator at 30°C for 100 days.

[0075] 5.0 ml of fish sauce after different fermentation forms were taken into 10 ml test tubes respectively and measured using a pH meter.

[0076] like Figure 6As shown in the figure, the pH of fish sauce decreased after fermentation with "ku" and "ku + glue", while the pH of fish sauce with "glue" fermentation increased to 6.24. It is generally believed that a pH value above 6 indicates an increase in corruption during the fermentation process. An acidic environment is not conducive to the growth of many pathogenic microorganisms and corrupt microorganisms, thereby improving the safety of the product. Relevant literature mentions that Bacillus subtilis produces antibacterial substances during its growth, which has a good inhibitory effect on the growth of bacteria and fungi. It is speculated that the addition of Bacillus subtilis reduces the adverse effect of glue red yeast on the pH value of fish sauce, maintains the pH of fish sauce in an acidic environment below 6, and is conducive to improving the safety of the product.

[0077] Example 6

[0078] Changes in amino acid nitrogen and total nitrogen content after Bacillus subtilis fermentation:

[0079] Pour 2 mL of bacterial solution frozen at -80°C into 100 mL of LB liquid culture medium, and culture at 37°C with a shaker at 120 rpm for 24 h. Then, inoculate the cultured bacterial solution into 100 mL of fresh LB liquid at a ratio of 2%, and culture at 37°C with a shaker at 120 rpm until OD600 = 0.6-0.7, which is considered to be activated.

[0080] The activated strain was inoculated into unfermented fish sauce at a 2% (v / v) inoculation amount and cultured at 30° C. for 100 days.

[0081] With reference to the national standard of soy sauce, according to the various standard requirements of GB18186-2000 "Brewed Soy Sauce", the fermented soy sauce product of the method of the present invention was tested for soy sauce related indicators.

[0082] The test objects are: fish sauce in the early stage of fermentation and fish sauce fermented by the strain of the present invention.

[0083] Control group: fish sauce in the early stage of fermentation;

[0084] Experimental group: added with standard preserved strain Bacillus subtilis (CCTCC NO: M2024356, China Center for Type Culture Collection).

[0085] Table 1 Standard grades of fish sauce

[0086]

[0087] The amino acid nitrogen content of fish sauce samples was determined by formaldehyde method; the total nitrogen was determined by Kjeldahl method.

[0088] As shown in Table 1, Figure 7 and Figure 8As shown in the figures, the Bacillus subtilis of the invention improves the fermentation process of soy sauce and promotes a more thorough decomposition of macromolecular proteins and starch substances, and can effectively increase the amino acid nitrogen content and total nitrogen level in the original brewed soy sauce without adding any MSG. This strategy is beneficial to deepening the flavor level of soy sauce and comprehensively improving its quality characteristics.

[0089] Example 7

[0090] Changes in organic acid content after Bacillus subtilis fermentation:

[0091] Take 1.0 ml of sample (the "dry + gum" sample in Example 5), add 19.0 ml of sterile water, mix well and let stand for 20 minutes, centrifuge at 8000r for 5 minutes, and filter the supernatant through a 0.22μm filter membrane before loading onto the machine.

[0092] Chromatographic conditions: C18 column (5 μm, 4.6 mm×250 mm): flow rate 0.5 mL / min; mobile phase A: potassium dihydrogen phosphate (0.01 molL, pH=2.8), B: methanol; isocratic elution: A:B=95:5; injection volume 10: detection time: 15 min.

[0093] like Fig. 9 As shown in the figure, the fish sauces without inoculation and after inoculation (Bacillus subtilis + Rhodotorula glutinosus) were tested for organic acids, including oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, propionic acid, and fumaric acid, a total of ten kinds of organic acids. The results showed that only formic acid was detected in the fish sauce of the uninoculated fermentation group.

[0094] After inoculation and fermentation, the six organic acids in fish sauce were significantly increased, and the formic acid content was significantly reduced. The total content of ten organic acids in fish sauce after inoculation and fermentation changed from 136.809 mg / ml to 129.098 mg / ml. Acetic acid and succinic acid were not detected in both fermented fish sauces. Lactic acid is the main organic acid in fish meat, which can provide fish meat products with a special sour taste and richness. Lactic acid usually comes from the reduction reaction of pyruvic acid, which is produced by glucose metabolism under the action of lactate dehydrogenase. Lactic acid can enhance the contribution of L-amino acids to taste.

[0095] Example 8

[0096] Application of Bacillus subtilis in improving flavor in fermented fish sauce:

[0097] Headspace solid phase microextraction and gas chromatography-mass spectrometry were used to detect the volatile flavor compounds in the experimental group and the control group.

[0098] Take 3 mL of the fermented sample (the "depleted + gum" sample in Example 5) and place it in a headspace bottle, and add 30 uL of 20 ppm 2-octanol standard. After sealing the headspace bottle, place it on a heated magnetic stirrer and equilibrate it in a 60°C water bath for 20 minutes.

[0099] After equilibrium, extract the headspace for 30 minutes at 60°C, and desorb at 250°C for 15 minutes for GC-MS injection. GC conditions: start at 40°C for 3 minutes, increase to 150°C at 4°C / min and maintain for 1 minute, then increase to 250°C at 8°C / min and maintain for 6 minutes. Helium is used as carrier gas, the injection port temperature is 250°C, and the injection time is 1 minute. MS conditions: ion source temperature is 200°C, interface temperature is 220°C, solvent delay time is 1.5 minutes, ionization mode is EI, 70eV scanning mass range is 33-500m / z, and scanning rate is 3.00s cans / s.

[0100] Compounds with similarity ≥ 90% 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.

[0101] Each group was tested three times in parallel, and the flavor substances with significant differences were screened. The Flavornet database (http: / / flavornet.org / flavornet.html) and standard substances were referred to to screen out the caramel sauce-flavor substances with significant differences.

[0102] Table 2 Differential flavor components of soy sauce caramel aroma

[0103]

[0104] The caramel flavor components of fish sauce are shown in Table 2. Compared with the control group, the caramel flavor substances in the fish sauce fermented with Bacillus subtilis WQ1 of the present invention are significantly increased. A total of 10 volatile flavor components were detected in the inoculated and uninoculated fish sauces, mainly aldehydes, ketones and other compounds. The volatile flavor substances in the inoculated group (9 kinds) increased compared with the uninoculated group (8 kinds).

[0105] Example 9

[0106] Bacillus subtilis antibacterial effect in improving soil environment:

[0107] Before the test, the concentration of Salmonella enterica, Staphylococcus aureus and Candida albicans was adjusted to 10 8 CFU / mL, take 100 μL of bacterial solution respectively, spread it evenly on LB agar medium, then take out the sterile Oxford cup and place it on the medium that has been coated with indicator bacteria, and place 4 evenly on each medium.

[0108] 100 μL of the bacterial solution to be tested was respectively taken and added into the Oxford cup. After 24 hours of incubation at 37°C, the diameter of the inhibition zone was measured. The test was repeated twice and the average value was taken.

[0109] Table 3 Bacillus subtilis antibacterial test results

[0110]

[0111] As shown in Table 3 and Fig.10 As shown in the figure, the Bacillus subtilis of the invention has obvious transparent circles against Staphylococcus aureus and Salmonella enterica compared with the negative control, indicating that it has a good inhibitory effect on the indicator bacteria, but there is no obvious transparent circle against Candida albicans. It is preliminarily judged that Bacillus subtilis has no inhibitory effect on Candida albicans or the inhibitory effect is not obvious. This feature is beneficial to inhibit the reproduction of spoilage microorganisms in the soil, reduce fermentation interference factors, and make it more controllable.

[0112] 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 subtilis ( Bacillus subtilis )WQ1, deposited in China Center for Type Culture Collection on February 29, 2024, and named Bacillus subtilis WQ1, the deposit number is CCTCC NO: M 2024356, and the deposit address is Wuhan University, Wuhan, China.

2. The Bacillus subtilis according to claim 1, wherein: The Bacillus subtilis grows in a pH = 4.5, 15wt% NaCl environment and has a strong stress resistance.

3. The Bacillus subtilis according to claim 1 or 2 ( Bacillus subtilis )Application of WQ1 in the preparation of fish sauce.

4. The use according to claim 3, characterized in that: The bacillus subtilis increases the amino acid nitrogen and total nitrogen in the fish sauce.

5. The use according to claim 3, characterized in that: The Bacillus subtilis fermented fish sauce changes the contents of eight organic acids.

6. The use according to claim 3, characterized in that: The Bacillus subtilis produces high amounts of ketone compounds and aldehyde derivatives thereof with caramel aroma.

7. The use according to claim 6, characterized in that: The ketone compounds with caramel sauce aroma and aldehyde derivatives thereof include 2,3-butanedione, 3-hydroxy-2-butanone, 3-methylbutyraldehyde, methylthioaldehyde, benzaldehyde, heptadecanal, nonanal, phenylacetaldehyde and decanal.

8. The use according to claim 6, characterized in that: The 2,3-butanedione content reached 28.112 μg / L, and the 3-hydroxy-2-butanone content reached 107.142 μg / L.

9. The use according to claim 6, characterized in that: The Bacillus subtilis weakens the influence of miscellaneous bacteria on the flavor of fish sauce and inhibits the growth of spoilage microorganisms during the fermentation process.

10. Use of Bacillus subtilis in agricultural control as claimed in claim 1.

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