Bacillus velezensis GGL2 and application thereof

Abstract

The invention belongs to the field of microorganisms, and discloses a bacillus velezensis GGL2 and application thereof, the bacillus velezensis GGL2 is separated and screened from spoilage soy sauce, is named as bacillus velezensis by taxonomy, is preserved in Guangdong Microbial Culture Collection Center on February 22, 2024, and has a preservation number of GDMCC No.63987. The invention further discloses a preparation method of the bacillus velezensis GGL2. The invention further discloses a preparation method of the bacillus velezensis GGL2. The strain can survive under the condition of 12% NaCl, and when the strain is inoculated into low-salt soy sauce according to the inoculum size of 105 CFU / mL, the low-salt soy sauce is spoilage; the strain is resistant to various common food preservatives, can be used as a detection indicator bacterium for the production quality of finished soy sauce, and can also be used as a strain resource for production enterprises and scientific research institutions to develop novel preservatives or natural preservatives and research, development and formulation of effective soy sauce sterilization and bacteriostasis methods.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a strain of Bacillus Velezii GGL2 and an application thereof. Background Art

[0002] Soy sauce, also known as "clear sauce" or "sauce", is a seasoning liquid with special color, aroma, taste and body, made from vegetable protein and carbohydrates from soybeans and wheat through the action of microbial enzymes or cells. Although the salt in soy sauce has a bactericidal and inhibitory effect, excluding salt-intolerant wild bacteria and harmful microorganisms that are insensitive to salt, there are still a variety of salt-tolerant microbial flora. At the same time, soy sauce is a fermentation system containing a variety of sufficient nutrients. Therefore, soy sauce is easily contaminated and deteriorated during the fermentation, production and storage process. If the sterilization operation is not perfect during the production process, some microbial flora that can cause deterioration are retained. Under the influence of these spoilage bacteria, the soy sauce will become sour and produce various harmful substances, and the surface will appear swollen bags, precipitation, turbidity, and blooming. Not only does the deteriorated soy sauce reduce its original nutritional value, but the harmful substances in it may also cause consumers to suffer from food poisoning to varying degrees.

[0003] As the public pays more and more attention to food safety issues, food quality and safety issues caused by microbial contamination have become the main risk, and the microbial contamination of soy sauce will become a research hotspot in the future. Currently reported bacteria that can cause soy sauce spoilage include Bacillus licheniformis, Bacillus halodurans, Bacillus megaterium, Bacillus subtilis, Staphylococcus carnosus, Lactobacillus acidipiscis, Enterobacter aerogenes, Enterococcus faecium, etc. Further expansion of the isolation and identification of soy sauce spoilage bacteria and research on their physiological and biochemical characteristics and causes of soy sauce spoilage will help manufacturers find out the source of contamination and reduce the risk of soy sauce spoilage caused by contaminating microorganisms by improving the sterilization process, which is of great significance to soy sauce companies and consumers. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a strain of Bacillus Velezii GGL2.

[0005] Another object of the present invention is to provide an application of the above-mentioned Bacillus Velez GGL2 strain. The strain is separated from spoiled soy sauce, has high salt tolerance and strong tolerance to a variety of common food preservatives, can be used as an indicator bacteria for detecting the production quality of finished soy sauce, and can also be used as a bacterial strain resource for production enterprises and scientific research institutions to develop new preservatives or natural preservatives and to develop and formulate effective soy sauce sterilization and antibacterial methods.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A strain of Bacillus velezensis GGL2, the taxonomic name of which is Bacillus velezensis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on February 22, 2024, with the deposit number GDMCC No. 63987.

[0008] Preferably, the above-mentioned Bacillus Velezii GGL2 strain is isolated from spoiled soy sauce and obtained by separation and screening on a high-salt LB plate;

[0009] Preferably, the obtained 16S rDNA gene sequence is used to analyze and identify the strain as Bacillus velezensis. The 16S rDNA sequence of Bacillus velezensis GGL2 is shown in SEQ ID No. 1, which has been submitted to the NCBI GenBank nucleic acid database with accession number OR875392.

[0010] Preferably, the Bacillus Velezii GGL2 strain is salt-tolerant, can survive under a salt concentration of 12%, and has a strong salt tolerance.

[0011] Preferably, the above-mentioned Bacillus Velez GGL2 strain is resistant to food preservatives and is insensitive to a variety of common food preservatives such as potassium sorbate, sodium benzoate, sodium acetate, sodium diacetate, sodium dehydroacetate, natamycin, nisin, EDTA, ε-polylysine hydrochloride (according to the maximum addition concentration of food additives in soy sauce in GB 2760-2014).

[0012] The above-mentioned Bacillus Velez GGL2 strain can deteriorate 8-12% NaCl low-salt soy sauce.

[0013] The above-mentioned Bacillus Velez GGL2 is used as an indicator bacteria in the quality detection of finished soy sauce.

[0014] The application of the above-mentioned Bacillus Velez GGL2 in the development of preservatives for soy sauce.

[0015] Preferably, the application comprises the following steps:

[0016] Activate and shake the GGL2 bacterial suspension until OD 600 =04-0.6, mixed with LB solid culture medium dissolved and kept at 55°C in a volume ratio of 1:5, poured into a flat dish with a thickness of 0.4-0.6cm, and set aside after solidification; soak the sterilized filter paper discs in preservative liquids of different concentrations, dry them, and spread them on the above-mentioned LB solid culture medium containing GGL2 with sterile tweezers, and culture them at 37°C for 24h to observe whether there is an inhibition zone.

[0017] The application of the above-mentioned Bacillus Velez GGL2 in the sterilization and antibacterial effect of soy sauce.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The strain is salt-tolerant and can survive under a salt concentration of 12%, with strong salt tolerance. The strain is resistant to food preservatives and can be used as an indicator of the quality of finished soy sauce production. It can also be used as a reference strain for production companies and scientific research institutions to develop new preservatives or natural preservatives, providing strain resources for production companies and scientific research institutions to develop and formulate effective soy sauce sterilization and antibacterial methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a characteristic diagram of the single colony morphology of the Bacillus Velezii GGL2 strain in the example of the present invention.

[0021] Figure 2 It is the salt tolerance of the Bacillus Velez GGL2 strain in the embodiment of the present invention, wherein No. 7 is the Bacillus Velez GDL5 strain, No. 8 is the Bacillus Velez GGL2 strain, and No. 9 is the Staphylococcus hominis GGL1 strain.

[0022] Figure 3 Growth curve of Bacillus Velezii GGL2 strain in low-salt soy sauce in the example of the present invention.

[0023] Figure 4 The Bacillus Velez GGL2 strain in the examples of the present invention causes the quality of low-salt soy sauce to change. DETAILED DESCRIPTION

[0024] In order to make the invention purpose, technical scheme and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, not for limiting the present invention. The experimental materials and reagents used, unless otherwise specified, are all consumables and reagents that can be obtained from commercial channels.

[0025] Embodiment 1:

[0026] The GGL2 strain is Bacillus Velezii, which was isolated from the final fermentation sample of Cantonese crude soy sauce provided by Guangdong Delicious Fresh Seasoning Food Co., Ltd. (pasteurization has been completed and no preservatives have been added to the sample). The specific isolation process is as follows: the soy sauce sample is stored at 37°C until it is corrupted, 10ml of the corrupted soy sauce is added to 90mL of sterile 8% sodium chloride solution, shake well, and then transfer 1mL of the soy sauce sample to 9mL of 8% sodium chloride solution for 10-fold serial dilution, and so on; -1 , 10 -2 , 10 -3 , 10 -4 0.1 mL of the diluted bacterial suspension of the dilution gradient was spread on an LB plate with 8% salinity and cultured at 37° C. for 24 hours. A GGL2 strain was obtained by the above separation method, and the strain was identified as Bacillus velezensis by analyzing the 16S rDNA gene sequence of the strain.

[0027] The Bacillus velezensis GGL2 strain was deposited in the Guangdong Microbiological Culture Collection Center on February 22, 2024. The deposit address is: Guangdong Microbiological Culture Collection Center, 5th Floor, Dayuan Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, GDMCC No. 63987.

[0028] PCR amplification and sequencing of the 16S rRNA gene of Bacillus velezensis strain GGL2.

[0029] The strain was inoculated into liquid LB medium, shaken at 200rpm and 37℃ for 12h, and the bacteria were collected by centrifugation. The genomic DNA was extracted using the bacterial extraction kit of Guangzhou Dingguo Biotechnology Co., Ltd., and then the 16SrDNA gene was amplified by PCR using the forward primer 27F (5'-AGA GTTTGA TCC TGG CTC AG-3') and the reverse primer 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). The amplification of the 16S rRNA fragment was observed by gel electrophoresis and the PCR stock solution was sent for sequencing. The 16SrRNA PCR conditions were: 95℃, 5min; 94℃, 45sec; 53℃, 45sec; 72℃, 1min; 30cycles; 72℃, 10min, and stored at 4℃. The length of the 16S rDNA partial gene sequence is about 1500bp, and the result is shown in the sequence table SEQ ID No.1.

[0030] The specific composition of the liquid LB medium is: 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, and distilled water is added to make up to 1 L.

[0031] Embodiment 2:

[0032] Bacillus velezensis GGL2 strain has strong salt tolerance.

[0033] GGL2 was activated and shaken overnight, and the bacterial solution was streaked on LB solid plate medium with 0%, 4%, 8%, 12%, and 16% NaCl concentration (g / v) with an inoculation loop. The plate was inverted and cultured at 37°C for 24 hours, and its growth was observed. The results showed that the salt tolerance of GGL2 strain reached 12% (appended Figure 2 ), Figure 2 No. 8 in the middle is Bacillus velezensis GGL2.

[0034] Embodiment 3:

[0035] Referring to the Food Additives Use Standard GB 2760-2014, the national standard for the amount of food preservatives added in soy sauce, Bacillus velezensis GGL2 strain is resistant to many common food preservatives such as sodium benzoate, potassium sorbate, sodium acetate, sodium diacetate, sodium dehydroacetate, natamycin, nisin, ε-polylysine hydrochloride, etc. (see Table 1).

[0036] Activate and shake the GGL2 bacterial suspension until OD 600 =04-0.6, mixed with LB solid medium dissolved and kept warm to 55°C at a volume ratio of 1:5 and poured into a plate, with a thickness of 0.4-0.6cm, and set aside after solidification. Sterilized filter paper discs (diameter 8-10mm, punched with a hole puncher) were soaked in preservative liquids of different concentrations (0.1g / kg-254g / kg), dried and spread on the above LB solid medium containing GGL2 with sterile tweezers, and a blank control (filter paper discs soaked in sterile water and dried) was made, and cultured at 37°C for 24h to observe whether there was an inhibition zone. The results showed that the GGL2 strain had good resistance to a variety of common food preservatives such as sodium benzoate, EDTA, potassium sorbate, sodium acetate, sodium diacetate, sodium dehydroacetate, natamycin, and nisin. Among them, sodium benzoate, potassium sorbate, sodium acetate, and natamycin had no antibacterial effect on GGL2 strains; EDTA, sodium diacetate, sodium dehydroacetate, and nisin had a certain antibacterial effect on GGL2 strains, but their concentrations were at least 20 times higher than the maximum addition amount in the national standard. However, ε-polylysine hydrochloride had a certain antibacterial effect on GGL2 strains at 4 times the maximum addition amount in the national standard (Table 1).

[0037] Table 1 Antibacterial test of common food preservatives on Bacillus velezensis GGL2

[0038]

[0039] Embodiment 4:

[0040] Bacillus velezensis GGL2 has the potential to cause low-salt soy sauce with a salinity of 8-12% to spoil.

[0041] Activate and shake the GGL2 bacterial suspension until OD 600 =1, according to the inoculation volume 10 5 CFU / mL was inoculated into low-salt soy sauce (8-12% NaCl), and a blank control (no GGL2 strain) was used. The strain was placed in a 37°C incubator and the total colony count, salinity, pH, amino acid nitrogen, and total sugar were measured every 3 days for 24 consecutive days. The results showed that the growth curve of the GGL2 strain in low-salt soy sauce first decreased and then increased, eventually reaching 6.97lg CFU / mL (attached Figure 3 ); GGL2 strain can reduce the salinity of soy sauce by 18.5%, the pH value of soy sauce by 12.5%, the amino acid nitrogen of soy sauce by 7.6%, and the total sugar content by 42.1% (attached Figure 4 ), but increases the viscosity of soy sauce and is accompanied by a slight peculiar smell. Therefore, the Velezella GGL2 strain has the potential to cause low-salt soy sauce to spoil.

[0042] The determination methods of each indicator are as follows:

[0043] The total colony count is determined according to the national standard GB 4789.2-2022.

[0044] Amino acid nitrogen is determined according to the national standard GB 5009.235-2016.

[0045] Salinity determination: Pipette 5.0mL of soy sauce into a 200mL volumetric flask, add water to the mark, shake well, pipette 2mL of the diluent into a 250mL conical flask, add 100mL of water. If the pH is lower than 6.5, add sodium bicarbonate to titrate so that the pH is within the range of 6.5-7.2, then add two drops of potassium chromate solution and mix well, titrate with 0.1mol / L AgNO3 until an orange-red color appears, and perform a blank experiment at the same time. The solution should be shaken during the experiment, and the measurement should be performed three times in parallel to take the average value.

[0046] pH determination: pH value is determined using a Leiji pH meter or other brand pH meter. The calibration and use of the pH meter are operated according to the instructions. When determining the pH of soy sauce, take 10mL of soy sauce in a 50mL beaker and measure it with a pH meter. Perform parallel measurements 3 times and take the average value.

[0047] Determination of soluble solids: Directly use the handheld refractometer of Lichen Technology or other brands of refractometers. Open the prism cover of the handheld refractometer, gently wipe the refracting mirror with lens paper or soft flannel, drop a few drops of distilled water on the refracting mirror, close the cover, point the instrument toward the light, observe through the eyepiece, and turn the prism knob to divide the field of view into light and dark parts. Use a special screwdriver to turn the compensator knob to make the field of view black and white, and at the same time make the light and dark dividing line coincide with the "0" position on the ruler. Then open the cover and wipe off the moisture. Use a dropper to absorb the sample liquid, drop it on the detection mirror, and close the cover. Pay attention to prevent bubbles when covering the cover. Hold the refractometer level and point it toward the light source, adjust the eyepiece diopter circle, make the black and white dividing line of the field of view clearly visible, and read the scale reading, which is the soluble solid content in the sample liquid, expressed as a percentage. Repeat the measurement three times and calculate the average and standard deviation.

[0048] Total sugar determination: Anthrone colorimetric method is used. First, make a standard curve, accurately weigh 0.1g glucose (analytical grade), dissolve it and dilute it to 100mL with distilled water, then take out 1mL, 2mL, 3mL, 4mL, and 5mL respectively and add them to a 50 volumetric flask, and dilute it to 50mL with distilled water, and prepare the concentrations to 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL, and 100μg / mL respectively. Take 1mL of each in a test tube, and then add 3mL of anthrone reagent (dissolve 2g of anthrone in 1L of concentrated sulfuric acid (98% concentrated sulfuric acid), prepare and use on the same day.), quickly Quickly immerse in ice water to cool, wait for several test tubes to be evenly added, and immerse them together in a 100℃ constant temperature water bath. To prevent water evaporation, a glass ball or a stopper should be added to the mouth of the test tube. Start timing from the temperature rising to 100℃ again, accurately keep the temperature for 10 minutes, take it out, cool it with running water, and then balance it at room temperature for a while (about 10 minutes). On the spectrophotometer, at a wavelength of 620nm, use a 0.5cm thick colorimetric cup and use a blank tube as a control blank (the blank here refers to the anthrone reagent without glucose, and other reaction conditions are the same) to perform colorimetry. For the determination of total sugar in soy sauce, take 1mL of soy sauce in a test tube, add 3mL of anthrone reagent, and quickly immerse it in ice water to cool. The subsequent steps are the same as making the standard curve. Parallel determination is performed 3 times, and the total sugar content is calculated according to the standard curve, and the average value is taken.

[0049] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Bacillus velez GGL2, characterized in that: The Bacillus velezensis is Bacillus velezensis GGL2, whose taxonomic name is Bacillus velezensis. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on February 22, 2024, with the deposit number GDMCC No.63987.

2. The Bacillus Velezii GGL2 according to claim 1, characterized in that The Bacillus reis GGL2 strain is isolated from spoiled soy sauce and obtained by separation and screening on a high-salt LB plate.

3. The Bacillus Velezii GGL2 according to claim 1, characterized in that The 16S rDNA sequence of the Bacillus velezensis GGL2 is shown as SEQ ID No.

1.

4. The Bacillus Velezii GGL2 according to claim 1, characterized in that The Velez Bacillus GGL2 has salt tolerance and can survive under a salt concentration of 12%.

5. The Bacillus Velezii GGL2 according to claim 1, characterized in that The Velez bacillus is resistant to food preservatives, and the food preservatives are sodium benzoate, EDTA, potassium sorbate, sodium acetate, sodium diacetate, sodium dehydroacetate, natamycin, nisin, and ε-polylysine hydrochloride.

6. Use of the Bacillus Velez GGL2 described in claim 1 as an indicator bacteria in quality detection of finished soy sauce.

7. Use of the Bacillus Velez GGL2 according to claim 1 in the development of a preservative for soy sauce.

8. Application of the Bacillus Velez GGL2 according to claim 1 in sterilization and bacteriostasis of soy sauce.

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