A strain of Lactobacillus brevis ZG-7 and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus brevis ZG-7 and its application. The Lactobacillus brevis ZG-7 has been preserved in the Guangdong Provincial Microbial Culture Collection Center on June 14, 2023, with a preservation number of GDMCC No.63562. The Lactobacillus brevis ZG-7 of the present invention has a strong inhibitory ability against Salmonella typhimurium, and has good acid and alkali resistance and bile salt resistance, and the inhibitory property has good thermal stability and ultraviolet stability. The Lactobacillus brevis ZG-7 of the present invention can be used to regulate the microecological balance in the intestine of animals, enhance the nonspecific immune function of the body to prevent diseases, and can also provide nutritional factors, promote the digestion and absorption of nutrients, promote animal growth and improve feed conversion rate.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus brevis ZG-7 and an application thereof. Background Art

[0002] Lactobacillus brevis belongs to the Lactobacilales, Lactobacillaceae, and Lactobacillus brevis. Its optimal growth temperature is 30-37°C, its optimal pH is 6.5, its genome length is 2.91-3.70Mb, and its GC accounts for 44.2%-45.1%. This bacterium is widely distributed and is used in fruit and vegetable production, feed processing, and fermentation of dairy and meat products to inhibit the growth of spoilage bacteria, improve food flavor, improve chronic metabolic diseases, lower cholesterol, improve immunity, and resist oxidation.

[0003] For example, patent CN101144064A discloses a Lactobacillus brevis BT0898 with anti-mutagenic activity and production of exopolysaccharides and its use in fermented foods. However, the existing Lactobacillus brevis still has poor stability problems such as acid-base stability, thermal stability, and ultraviolet stability, which shortens the shelf life of the products produced. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus brevis ZG-7 with stable antibacterial properties and application thereof.

[0005] The present invention is achieved in that:

[0006] First, the present invention provides a strain of Lactobacillus brevis (Levilactobacillus brevis) ZG-7, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on June 14, 2023. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 63562.

[0007] Secondly, the present invention provides the application of the Lactobacillus brevis ZG-7.

[0008] Furthermore, the present invention provides the use of the Lactobacillus brevis ZG-7 in the preparation of a drug for inhibiting pathogenic bacteria.

[0009] Furthermore, the pathogenic bacteria include Salmonella typhimurium.

[0010] Furthermore, the present invention provides the use of the Lactobacillus brevis ZG-7 in preparing animal feed.

[0011] Furthermore, the present invention provides the use of the Lactobacillus brevis ZG-7 in preparing a starter, for example, as a fermentation bacterium for preparing yogurt.

[0012] In addition, the extracellular polysaccharide of Lactobacillus brevis ZG-7 has a good effect in improving immunity and can also be used as an adjuvant for animal vaccines.

[0013] The invention has the following advantages: the Lactobacillus brevis ZG-7 of the invention has a strong inhibitory ability against Salmonella typhimurium, has good acid and alkali resistance and bile salt resistance, and has good thermal stability and ultraviolet stability. The Lactobacillus brevis ZG-7 of the invention can be used to regulate the microecological balance in the intestine of animals, has the effect of enhancing nonspecific immune function to prevent diseases, and can also provide nutritional factors, promote digestion and absorption of nutrients, promote animal growth and improve feed conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0015] Figure 1 This is a graph showing the changes in the copy number of Lactobacillus brevis ZG-7 at different growth times.

[0016] Figure 2 This is a scanning electron micrograph of the antibacterial activity of Lactobacillus brevis ZG-7. A: Control group: Salmonella typhimurium; B: Salmonella typhimurium treated with the supernatant of Lactobacillus brevis ZG-7. The arrows in the treatment group showed obvious bacterial deformation and a layer of granular material.

[0017] Figure 3 This is a graph showing the changes in diarrhea rate in mice caused by the strain inhibiting Salmonella typhimurium.

[0018] Figure 4 The strain inhibits the change in the mortality rate of mice caused by Salmonella typhimurium. DETAILED DESCRIPTION

[0019] 1 Method

[0020] 1.1 Materials

[0021] Lactobacillus brevis BNCC1853929 (BNC) was purchased from Beina Biotechnology, and Bacillus and Lactobacillus brevis YXJ0032 (YMJ) were purchased from Shanghai Yingxin Laboratory.

[0022] 1.2 Strain isolation and identification

[0023] 1.2.1 Isolation and screening of Lactobacillus brevis ZG-7

[0024] In July 2021, strains were extracted from Zuogong herdsmen's kimchi, and strain isolation and subsequent experiments were carried out in the laboratory of the Institute of Animal Husbandry and Veterinary Medicine of Fujian Academy of Agricultural Sciences. In the sterile operation room, sterile tweezers were used to take a sterilized 10μL TIP head, and a small amount of sterile water was dipped in the TIP tip. Then, the colony was scraped with the TIP tip and placed in a centrifuge tube containing 1.5mL MRS culture medium. The tube was sealed with a sealing film and placed in a constant temperature shaking incubator at 35℃ and 190r / min for 2h. After standing in a 35℃ incubator for 30min, the colonies were diluted into 10 –2 , 10 –3 and 10 –4 80 μL of suspension of different concentrations was taken and spread on MRS medium plate. The temperature was 35℃ and the humidity was 45% in a constant temperature incubator for 3 days. Different single colonies were picked and streaked for purification.

[0025] 1.2.2 Identification of Lactobacillus brevis ZG-7

[0026] (1) Observation of morphological characteristics

[0027] The strains on the MRS medium plate were picked and streaked onto the MRS plate again, cultured at 35°C for 3 days, and the colony morphology was observed.

[0028] (2) Molecular Biological Identification

[0029] The culture medium was removed by washing with PBS, and the genomic DNA of the strain was extracted by using a bacterial genomic DNA extraction kit. The extracted genomic DNA was used as a template, and the universal primer 5′-GGGGCAACCCAGCAGTTTTA-3′ for fungal amplification, downstream primer R: 5′-TAATGAGATGTTTCAGTTCACAGCG-3′, and the product size was 91bp. The gene was amplified by PCR. The PCR reaction system: 2×TaqPCRMix 12.5μL, F primer (10μmol / L) 1.0μL, R primer (10μmol / L) 1.0μL, DNA template 1.5μL, ddH2O 9.5μL. PCR reaction conditions: 98℃5min; 95℃30s, 55℃30s, 72℃1min, 35 cycles; 72℃10min. The PCR amplification product was sent to Boshan Biotechnology Co., Ltd. for purification and sequencing.

[0030] 1.2.3 Growth characteristics analysis

[0031] Take the previously isolated and verified Lactobacillus brevis ZG-7 and inoculate it on the MRS plate for 2 to 3 activations to make the effective viable count 10 8CFU / mL. Inoculate into MRS medium at 2% inoculum and culture at 35℃, 190r / min shaking for 7 days. Take out the culture every 12 hours for counting. Draw the growth curve of the strain with time as the horizontal axis and the number of bacteria as the vertical axis.

[0032] 1.2.4 Determination of the antibacterial spectrum of the supernatant of Lactobacillus brevis ZG-7

[0033] The agar diffusion method was used to determine the antibacterial activity of the strain. First, prepare 200 mL of LB nutrient agar medium, sterilize at 121°C for 20 minutes, and after cooling slightly, pour 20 mL of the medium onto a 10 cm pre-sterilized plate, place it on a sterile operating table to solidify and blow dry the condensed water, and then place it in a refrigerator at 4°C overnight. Take 0.5 mL of newly proliferated Salmonella typhimurium 10 every other day. 7 CFU / mL, add LB nutrient agar plate, and then use a sterilized glass push rod to evenly spread the bacterial liquid on the surface of the plate. It is best if there is no liquid flowing on the plate. Then use a sterilized hole puncher to vertically punch 6 holes on the prepared plate. The 6 holes should be evenly distributed and 15mm away from the edge. Use a sterilized toothpick to pick out the agar block. Then use flame sealing to bake each hole under the flame of an alcohol lamp for 10s to seal the bottom. Then inject 20μL of the supernatant of Lactobacillus brevis ZG-7 fermentation liquid into each hole, incubate at 35℃ for 6h, observe and measure the changes in the diameter of the inhibition zone with a vernier caliper.

[0034] 1.2.5 Stability test of Lactobacillus brevis ZG-7 supernatant against Salmonella typhimurium

[0035] (1) Acid-base stability

[0036] Take 15mL of the supernatant of the three types of Lactobacillus brevis, adjust the pH value with 1mol / L HCl and 1mol / L NaOH, respectively. After reacting for 12h, use Salmonella typhimurium as the indicator bacteria and the supernatant of Lactobacillus brevis ZG-7 as the control (pH=3.3 is the pH value of the original solution). Refer to the agar diffusion method in the previous section to determine the antibacterial activity of the strains. Repeat 3 times and evaluate the acid-base stability of the supernatant of Lactobacillus brevis ZG-7 according to the change in the diameter of the inhibition zone.

[0037] (2) Thermal stability

[0038] Take 15mL of the supernatant of three kinds of Lactobacillus brevis and treat them at 35, 50, 65, 80, 95, and 110℃ for 1h respectively, and use Salmonella typhimurium as the indicator bacteria and the fermentation stock solution (25℃) as the control. Refer to the agar diffusion method in the previous section to determine the antibacterial activity of the strains, and repeat it 3 times. The thermal stability is investigated according to the change of the diameter of the inhibition zone.

[0039] (3) UV stability

[0040] Take 15mL of the supernatant of three kinds of Lactobacillus brevis, place it under a 25W ultraviolet lamp at a distance of 35cm, and irradiate for 2, 4, 6, 8, 10, and 12 hours respectively. Use Salmonella typhimurium as the indicator bacteria and the fermentation stock solution as the control. Refer to the agar diffusion method in the previous section to determine the antibacterial activity of the strains, and repeat 3 times. Investigate its ultraviolet light stability based on the change in the diameter of the inhibition zone.

[0041] 1.2.6 Scanning electron microscopic observation of the inhibitory effect of Lactobacillus brevis ZG-7 supernatant on Salmonella typhimurium

[0042] The supernatant of the fermentation broth of the ZG-7 strain was added with newly grown Salmonella typhimurium (concentration 10 7 CFU / mL), and cultured at 37℃ and 180rpm for 6h. Then, 0.5mL of Salmonella typhimurium solution was taken from the control group and the supernatant treatment group, centrifuged and washed three times with PBS. The bacteria were fixed with 0.5mL of glutaraldehyde, and then the samples were fixed on graphite tape, and a thin gold coating of about 24.5nm was applied on each sample, and then high-resolution images were obtained under a scanning electron microscope.

[0043] 1.2.7 Inhibition of Salmonella typhimurium by supernatant of Lactobacillus brevis ZG-7

[0044] Fifty 7-day-old BALB / c mice were divided into five groups: blank group, Lactobacillus brevis ZG-7 treatment group, Lactobacillus brevis BNCC treatment group, Lactobacillus brevis YXJ treatment group and control group, with 10 mice in each group, half male and half female. The blank group was gavaged with an equal amount of supernatant of MRS culture medium. The control group was first gavaged with 0.5 mL of newly propagated Salmonella typhimurium (concentration 10 7 CFU / mL), and then 3 times of MRS culture supernatant, 1 mL each time, with an interval of 8 hours. The treatment group was first gavaged with 0.5 mL of newly propagated Salmonella typhimurium (concentration 10 7 CFU / mL), and then the mice were gavaged with 1 mL of supernatant of Lactobacillus brevis ZG-7, Lactobacillus brevis BNCC, and Lactobacillus brevis YXJ three times, with an interval of 8 hours. The diarrhea and mortality rates of the mice were observed and recorded.

[0045] 1.2.8 Data Analysis

[0046] Data analysis was performed using Excel 2017, SPSS 19.01, and GraphPad Prim 9.0.0 (121) software.

[0047] 2 Results

[0048] 2.1 Identification results of Lactobacillus brevis ZG-7 strain

[0049] 2.1.1 Gene sequence analysis of Lactobacillus brevis ZG-7

[0050] The genomic DNA of Lactobacillus brevis ZG-7 was extracted and used as a template to amplify and sequence its gene sequence using universal primers of bacteria to obtain a sequence of 1300bp in length and submit it to the GenBank database. After BLAST comparison in the NCBI database, it was found that the DNA gene sequence of the strain was 85% similar to that of the Lactobacillus brevis strain. Combined with morphological and physiological and biochemical characteristics, the strain was identified as a new Lactobacillus brevis strain.

[0051] 2.1.2 Analysis of growth characteristics of Lactobacillus brevis ZG-7

[0052] Lactobacillus brevis ZG-7 grew slowly in the initial 12 hours, and the bacterial content was low. After 24 hours, Lactobacillus brevis ZG-7 began to adapt to the growth environment, absorbing nutrients such as carbon sources in the culture medium, prompting rapid germination and growth, causing the amount of bacteria to increase exponentially. After nearly 80 hours of constant temperature shaking culture, the bacterial copy number reached a peak (see Figure 1 ). Then, as the culture time increases, the copy number begins to decrease.

[0053] Therefore, the fermentation broth after 3 days of cultivation was selected, centrifuged at 12000 r / min for 20 min, and then filtered to obtain the supernatant of the strain fermentation broth.

[0054] 2.2 Antibacterial test of strain supernatant

[0055] Previous studies have reported that Lactobacillus brevis ZG-7 can enhance human immune function, anti-oxidation, antibacterial and other medical values. In order to verify the inhibitory effect of the strain on common harmful bacteria in food, this study conducted a verification of the antibacterial effect of Lactobacillus brevis ZG-7 through agar expansion test.

[0056] The results showed that Lactobacillus brevis ZG-7 could inhibit Salmonella typhimurium, and the inhibition effect was the most obvious, with the inhibition zone reaching 18.9±1.5 a The inhibition zones of the common Lactobacillus brevis BNCC1853929 (BNC) (5.4±1.2 mm), Lactobacillus brevis YXJ0032 (YMJ) (6.9±1.3 mm) and Bacillus subtilis (3.2±1.1 b mm) (Table 1).

[0057] The supernatant of the fermentation liquid of ZG-7 strain was extracted and added to the newly cultured Salmonella typhimurium liquid. After 6 hours of culture, 1 mL of the bacterial liquid was taken for scanning electron microscopy observation. The results showed that the treatment group ( Figure 2 B) The area marked by the arrow showed obvious bacterial deformation and was covered with granular substances. Figure 2 The cell structure of the Salmonella typhimurium liquid in the middle (A) is complete, the outer layer of the cell is smooth, and the cell morphology is complete. This indicates that the supernatant of the strain has a significant inhibitory effect on the pathogenic Salmonella typhimurium ( Figure 2 ).

[0058] Table 1 Antagonistic effects of three different strains on Salmonella typhimurium

[0059]

[0060] Note: Different lowercase letters after the data indicate significant differences at the P<0.05 level.

[0061] 2.3 Antibacterial stability of strain supernatant

[0062] 2.3.1 Acid-base stability

[0063] As shown in Table 2, the fermentation supernatant of Lactobacillus brevis ZG-7 showed strong inhibitory activity after being treated with different pH acid and alkali. When the acidity was low, the supernatant of Lactobacillus brevis ZG-7 showed strong inhibitory activity, and the diameter of the inhibition zone was as high as 18.9±1.1, which was larger than that of the control group (supernatant when not treated). This may be due to the superposition of the inhibitory effect of acidity on Salmonella typhimurium. When the pH value was within the range of 5.3 to 7.3, the diameter of the inhibition zone did not change significantly compared with the untreated group. This shows that the supernatant of Lactobacillus brevis ZG-7 showed strong antibacterial effect under different pH conditions, and the antibacterial substance had a certain acid and alkali resistance.

[0064] Table 2 Acid-base stability of the supernatant of the strains against Salmonella typhimurium

[0065]

[0066] Note: The same shoulder or no letter in the same row of data in the table indicates no significant difference (P>0.05), and different lowercase letters indicate significant difference (P<0.05). The following tables are the same. PH=3.3 is the PH value of ZG-7 stock solution, which is used as a control here.

[0067] 2.3.2 Thermal stability

[0068] As shown in Table 3, the fermentation liquid supernatant of Lactobacillus brevis ZG-7 is subjected to the heat treatment of different temperatures, and all shows stronger antibacterial effect. Along with the rising of the supernatant temperature of processing Lactobacillus brevis ZG-7, its antibacterial effect weakens slightly, and the supernatant of Lactobacillus brevis ZG-7 through 110 ℃ of heat treatments is 96.03% (reduction is 3.97%) of the control group to the inhibition circle diameter of Salmonella typhimurium. Therefore, it is explained that heat treatment has little influence on the antibacterial effect of the supernatant of Lactobacillus brevis ZG-7, and antibacterial substance has certain thermostability.

[0069] Table 3 Thermal stability of the inhibitory effect of Lactobacillus brevis ZG-7 supernatant on Salmonella typhimurium

[0070]

[0071] 2.3.3 UV stability

[0072] As shown in Table 4, after the fermentation supernatant of Lactobacillus brevis ZG-7 was irradiated with ultraviolet light for different time periods, its antibacterial effect slowly weakened as the irradiation time increased. This result confirmed to a certain extent that the supernatant of Lactobacillus brevis ZG-7 could maintain the antibacterial activity relatively stably under the condition of ultraviolet light irradiation.

[0073] Table 4 UV stability of the inhibitory effect of Lactobacillus brevis ZG-7 supernatant on Salmonella typhimurium

[0074]

[0075] 2.4 Strain supernatant reduces diarrhea rate and mortality rate in mice

[0076] In order to further verify the inhibitory effect of Lactobacillus brevis ZG-7 on Salmonella typhimurium, 50 7-day-old BALB / c mice were divided into 5 groups, namely blank group, Lactobacillus brevis ZG-7 treatment group, Lactobacillus brevis BNCC treatment group, Lactobacillus brevis YXJ treatment group and control group, with 10 mice in each group, half of which were male and half were female, and the mice were given the drug by gavage.

[0077] The results showed that the mice in the blank group did not have diarrhea or death, and their food and water intake were normal. In the Lactobacillus brevis ZG-7 treatment group, only one mouse showed mild diarrhea symptoms in the first 12 hours. With the continuous administration of the supernatant of Lactobacillus brevis ZG-7, the mice no longer had diarrhea and their mental state was normal. However, 2-3 mice in the Lactobacillus brevis BNCC treatment group and the Lactobacillus brevis YXJ treatment group showed symptoms of persistent diarrhea. The control group had severe diarrhea, which lasted until the end of the experiment ( Figure 3 ).

[0078] like Figure 4 As shown in the figure, from the results of the mouse mortality rate, it can be seen that only one mouse died in the Lactobacillus brevis ZG-7 treatment group, while six mice died in the control group at 84 hours of the experimental period, with a high mortality rate. One mouse died in the Lactobacillus brevis BNCC treatment group and the Lactobacillus brevis YXJ treatment group at 12 hours, 48 ​​hours, 60 hours and 90 hours respectively (a total of four mice died in the two groups), and the inhibitory effect of the Lactobacillus brevis ZG-7 treatment group was the most obvious.

[0079] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A strain of Lactobacillus brevis ZG-7, characterized in that: The Lactobacillus brevis ZG-7 is Lactobacillus brevis ( Levilactobacillus brevis ) ZG-7, was deposited in Guangdong Provincial Microbiological Culture Collection Center on June 14, 2023, with the deposit number GDMCC No.63562.

2. Use of Lactobacillus brevis ZG-7 as claimed in claim 1 in the preparation of a medicament for inhibiting pathogenic bacteria; the pathogenic bacteria include Salmonella typhimurium.

3. Use of Lactobacillus brevis ZG-7 as claimed in claim 1 in preparing animal feed.

4. Application of Lactobacillus brevis ZG-7 as claimed in claim 1 in preparing biological fermentation agent.

5. Use of Lactobacillus brevis ZG-7 as claimed in claim 1 in preparing extracellular polysaccharides.

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