Weissella virida 38-3 and application thereof

Abstract

The invention discloses a Weissella virida 38-3 strain and an application thereof. Relates to the technical field of microorganisms. The invention provides a strain and application thereof. The strain is safe in separation source, high in growth speed and excellent in fermentation performance; the strain has the advantages of good biological safety, no hemolytic activity and no generation of bioamine, is sensitive to antibiotics such as ampicillin, erythromycin, kanamycin, streptomycin, tetracycline, vancomycin, chloramphenicol, gentamicin, clindamycin, ciprofloxacin, cefixime and the like, has good probiotic performance, has good gastric juice and intestinal juice tolerance and strong cell surface adhesion, and can be used for preparing antibiotics. Good pathogenic bacterium inhibiting and cholesterol lowering capabilities are realized. The strain disclosed by the invention can be used as a probiotic to prepare a food leavening agent, multifunctional medicines, functional foods or health-care foods, and has wide market prospects and application values.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and more specifically to a strain of Weissella viridis 38-3 and application thereof. Background Art

[0002] Probiotics are defined as a type of live microbial supplements that, when taken in sufficient amounts, will supplement the host with normal bacteria, bring health benefits, and thus improve the dynamic balance of the host's flora. A large number of clinical studies have shown that probiotics can provide the body with physiological functions that other flora cannot provide, such as inhibiting the growth and reproduction of pathogens, improving the structure of intestinal flora, enhancing the body's immunity, stimulating immune response, reducing the incidence of tumors, inhibiting the growth of cancer cells, delaying the aging of the body, removing free radicals from the body, regulating the body's blood lipids, reducing cholesterol levels, alleviating lactose intolerance, anti-oxidation, and enhancing human immunity. They are deeply involved in the management of host health.

[0003] As people pay more and more attention to health in their diet, the categories of probiotics on the market continue to expand. From yogurt to chocolate, dietary supplements, to personal care products, probiotics are becoming more and more known and accepted by consumers and have huge commercial value.

[0004] Common probiotics include Bacillus subtilis, Lactobacillus, Bacillus licheniformis, yeast, etc. Among them, lactic acid bacteria have a long history of use as food fermentation agents and have multiple physiological functions. Weissella belongs to lactic acid bacteria and is widely distributed in fermented foods such as sausages, kimchi, soy sauce, and fermented black beans. Most strains play an important role in the fermentation process of food and play an important role in the synthesis of flavor substances such as esters, organic acids, and short-chain fatty acids in food. In recent years, studies have found that Weissella has good probiotic potential and has great development value.

[0005] On the other hand, probiotics, as common strains in the environment, may become an important reservoir of antibiotic resistance genes, and these resistance genes can be spread through the food chain, posing a threat to human health. Studies have found that the problem of probiotic resistance is becoming more and more serious, and some probiotic products have transferable resistance genes, which poses a serious safety hazard.

[0006] Therefore, exploring probiotic resources with good biosafety is an urgent problem that needs to be solved by technicians in this field. Summary of the invention

[0007] In view of this, the present invention provides a green Weissella 38-3 and its application. The strain has a fast growth rate, good tolerance to gastric juice and intestinal juice, strong cell surface adhesion, can inhibit pathogens, reduce cholesterol, scavenge free radicals, and is sensitive to common antibiotics.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The preservation information is: Weissella viridescens 38-3, deposited in the General Microbiology Center of China Culture Collection Administration on October 14, 2024, with the preservation number CGMCC No.32210, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] A strain of Weissella viridescens 38-3, with a deposit number of CGMCC No.32210.

[0011] The present invention also provides a fermentation product comprising the viridis bacteria 38-3.

[0012] The present invention also provides a bacterial agent comprising the viridis bacteria 38-3.

[0013] The present invention also provides food containing the viridis bacteria 38-3.

[0014] The present invention also provides a health product containing the viridis bacteria 38-3.

[0015] The present invention also provides a medicine comprising the viridis bacteria 38-3.

[0016] The present invention also provides application of the green Weissella 38-3 in fermentation production.

[0017] The present invention also provides the use of the viridis 38-3 in the preparation of a bacterial agent or medicine for regulating intestinal flora, improving intestinal function, inhibiting pathogens or lowering cholesterol.

[0018] Through the above technical scheme, it can be known that compared with the prior art, the present invention discloses a green Weissella 38-3 and its application, and the technical effect achieved is that the strain has good biological safety, no hemolytic activity, no bioamine, and is sensitive to antibiotics such as ampicillin, erythromycin, kanamycin, streptomycin, tetracycline, vancomycin, chloramphenicol, gentamicin, clindamycin, ciprofloxacin, and cefixime. The hydrophobicity of the strain cell surface is 58.41%, and the self-polymerization reaches 53.63% after 2 hours, and 64.92% can be reached in 4 hours, which is significantly higher than the commercial strain Lactobacillus rhamnosus LGG, indicating that the strain has good surface adhesion and great colonization potential in the intestine. The strain also has good ability to inhibit pathogens and lower cholesterol, is an effective supplement to the current probiotic library, can be used to prepare various foods, health products or medicines that regulate intestinal flora, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure is a colony morphology diagram of the strain 38-3 provided by the present invention.

[0021] Figure 2 The accompanying figure is a Gram staining microscopic image of strain 38-3;

[0022] Figure 3 The attached figure is a phylogenetic tree of strain 38-3.

[0023] Figure 4 The attached figure is the growth curve of strain 38-3

[0024] Figure 5 The attached figure is a biosafety diagram of strain 38-3, wherein the left figure is a diagram showing the results of a biogenic amine experiment; the right figure is a diagram showing the results of a hemolysis experiment.

[0025] Figure 6 The attached drawings are diagrams of the probiotic performance of strain 38-3, wherein A: number of surviving cells in simulated gastric fluid (CFU / mL); B: number of surviving cells in simulated intestinal fluid (CFU / mL); C: cell hydrophobicity; D: Caco-2 cell adsorption rate; E: cholesterol removal rate; and F: cell self-aggregation rate. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The embodiment of the invention discloses a Weissella viridis 38-3 strain and application thereof.

[0028] Example 1

[0029] Isolation, Screening and Identification of Weissella

[0030] 1. Isolation and purification of strains: Take 0.5 g of hand-fermented vegetables from Xi'an, Shaanxi, cut the sample into 0.3 × 0.3 cm small pieces with sterile scissors, add 10 ml of sterile physiological saline, vortex and oscillate for 3 min, and dilute to 10 -7 , absorb 10－4 ~10 －7 100 μL of the dilution was spread on MRS solid medium with 2% calcium carbonate and cultured at 37°C for 48 h. The colony morphology of strain 38-3 was Figure 1 ; Pick the colonies with calcium dissolution rings, streak them on MRS solid medium, purify them by Gram staining and observe them under a microscope. Among them, the Gram staining of strain 38-3 showed Figure 2 ; Select Gram-positive strains with short rod-shaped microscopic morphology, freeze them in 25% glycerol at -80℃ for later use.

[0031] 2. Strain screening and identification: The DNA of the isolated strains was extracted using a bacterial genome extraction kit (TaKaRa MiniBEST Bacterial Genomic DNA Extraction kit ver2.0), and PCR amplification was performed using bacterial 16S rDNA universal primers: upstream primer 27F: AGAG TTTG ATCM TGGC TCAG; downstream primer 1492R: GGTT ACCT TGTTACGA CTT. PCR amplification reaction system (50 μL): 5.0 μL 10× Taq enzyme buffer, 0.4 μmol / L primer, 4 μL 200 mmol / L dNTP, 40 ng DNA template, 1U Taq enzyme (TaKaRa), 34 μL ddH2O. The PCR reaction was amplified according to the following procedure: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 1 min, 72°C extension for 1 min, 35 cycles; and finally 72°C extension for 5 min. The reaction was carried out on a Bio-Rad PCR instrument, and the 16S rDNA amplified fragment was detected by 1% agarose gel electrophoresis and sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. The sequencing results were compared with the sequences in GenBank by Blast, and Weissella was selected and inoculated into MRS liquid culture medium at a 1% inoculation rate, placed in a shaker at 37°C for 24 hours, and the OD of the strain culture medium was measured. 600 nm values, the results are shown in Tables 1 and 2 below.

[0032] Table 1. Weissella isolated from fermented vegetables in Shaanxi Province

[0033]

[0034] Table 2. pH value after 24h fermentation of Weissella

[0035]

[0036] The results showed that strain 38-3 grew fastest, with OD 600The similarity between the 38-316SrDNA gene sequence of strain 1 and the W.viridescens strain 6996 gene sequence was the highest, at 99.86%. The phylogenetic tree was constructed using MEGA-X software ( Figure 3 ), it was found that the strain was Weissella viridescens, which was deposited under patent procedure.

[0037] 3. Growth curve

[0038] The strain 38-3 was inoculated into MRS liquid medium at a 1% inoculum (commercial strain Lactobacillus rhamnosus GG strain NBRC 3425 was used as a control) and cultured in a 37°C shaker for 20 h. Three replicates were taken every 2 h to measure the OD value of the culture medium. 600 nm value and pH value, with culture time as the horizontal axis and OD 600 The growth curve of the strain was drawn with nm value and pH as the ordinate. Figure 4 It can be seen that the growth rate of strain 38-3 in the early stage of fermentation was significantly higher than that of the control strain LGG, the growth rate slowed down after 16 hours, and the bacterial density was basically the same as that of the control strain LGG at 20 hours.

[0039] Example 2

[0040] Safety performance of strain 38-3

[0041] 1. Antibiotic sensitivity

[0042] The experiment was conducted on a 96-well plate, and the antibiotics were gradiently diluted to make the concentrations of the diluted antibiotics 1024μg / ml, 512μg / ml, 256μg / ml, 128μg / ml, 64μg / ml, 32μg / ml, 16μg / ml, 8μg / ml, 4μg / ml, and 2μg / ml. 40μl of gradient diluted antibiotic solution, 158μl of MRS liquid culture medium, and 2μl of 068-1 bacterial solution after two generations of activation were added to the wells respectively; the experiment set up a negative control (sterile water instead of bacterial solution) and a positive control (sterile water instead of antibiotics). After constant temperature culture at 37℃ for 24h, the OD was detected using an enzyme marker. 600 Absorbance values ​​are shown in Table 3.

[0043] Table 3. Antibiotic susceptibility of LGG and 38-3

[0044]

[0045] Note: Drug resistance refers to breakpoint values ​​(Bories G2008; EFSA2012). R, drug resistance.

[0046] The strain 38-3 of the present invention has lower resistance to 11 antibiotics including ampicillin, erythromycin, kanamycin, streptomycin, tetracycline, vancomycin, chloramphenicol, gentamicin, clindamycin, ciprofloxacin and cefixime than the EFSA 2012 regulations (European Food Safety Authority 2012, Weissella 38-3 selected Leuconostoc as a reference), and LGG is resistant to kanamycin.

[0047] 2. Hemolysis test

[0048] After activating strain 38-3 for two generations, use an inoculation loop to streak on a blood plate, incubate at 37°C for 48 hours, and observe whether there is a hemolytic zone. No transparent zone means no β-hemolytic activity, that is, no effect on hemolysis or no hemolysis. The commercial strain LGG was used as a negative control, and the strain S. paratyphi B CMCC 50094 with β-hemolytic activity was used as a positive control.

[0049] The results showed that S. aureus B produced a clearing zone on the blood agar plate and had β-hemolytic activity; 38-3 (see Appendix Figure 5 ) and LGG bacteria have no transparent zone around them and therefore have no β-hemolytic activity.

[0050] 3. Test for biogenic amine production

[0051] Histidine monohydrochloride, lysine monohydrochloride, ornithine monohydrochloride and tyrosine (free radical) were used as precursor amino acids for histamine, cadaverine, putrescine and tyramine (all purchased from McLean, Shanghai, China). The test medium was MRS agar containing 0.006% bromocresol purple, 0.005% pyridoxal-5-phosphate and 1% of each amino acid tested, and the pH of the medium was adjusted to 5.3 by adding 1M HCl. The ability of Weissella strains to produce biogenic amines (BA) was tested: the strains were passaged twice in MRS culture medium containing 0.1% precursor amino acids and 0.005% pyridoxal-5-phosphate. Overnight cell cultures were streaked on the test medium and, after incubation at 37°C for 4 days, the presence of purple around the colonies on the plates was assessed. The presence of purple around the colonies indicated the production of biogenic amines. Culture dishes without amino acids were used as controls. The analysis was performed in triplicate.

[0052] See also Figure 5 , the results showed that there was no purple color around the colonies, eliminating the risk of biogenic amines produced during the growth of 38-3.

[0053] Example 3

[0054] Probiotic properties of strain 38-3

[0055] 1. Gastrointestinal fluid tolerance of strain 38-3

[0056] The strain 38-3 stored at -80℃ was activated for two generations and inoculated into MRS liquid medium at 1% (v / v) inoculation volume. The culture was kept at 37℃ for 18h, and the cells were collected by centrifugation (8000×g, 4℃, 10min), resuspended in PBS, and the cell concentration was adjusted to 10 9 cfu / ml. Take 1ml of the above bacterial suspension and add 9mL artificial gastric juice (3g / L pepsin, pH 3.0) or artificial intestinal juice (1g / L trypsin, 0.3% ox bile salt, pH 8.0), and incubate at 37℃ for 3h. Take samples during the process, centrifuge and discard the supernatant, dilute with 0.85% sterile saline, and then spread on MRS plates for counting. The initial viable count without treatment with gastrointestinal simulated fluid is recorded as N0, and the viable count after treatment is recorded as Nt. Three parallels are used in each group [commercial strain Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG strain NBRC 3425) is used as the control]. The survival rate is calculated as follows:

[0057] Survival rate % = (log cfu / ml N t ) / (log cfu / ml N0)×100

[0058] The results show that (see Appendix Figure 6 In A and B), the survival rate of 38-3 bacteria was above 91.55% after 3 h in simulated gastric fluid, and the number of viable bacteria was above 10 7 cfu / ml; in simulated intestinal fluid, the survival rate of 38-3 was 92.03% at 3h, and the number of viable bacteria was 10 7 cfu / ml or more. The tolerance of strain 38-3 to simulated gastrointestinal fluid was better than that of LGG.

[0059] 2. Cell surface adhesion characteristics

[0060] (1) Cell hydrophobicity: Lactobacillus strains stored at -80°C were activated for two generations and inoculated into MRS liquid medium at a 1% (v / v) inoculum. The cells were cultured at 37°C for 18 h and collected by centrifugation (8000 g, 4°C, 10 min). The cells were washed twice with PBS (pH 7.4) and resuspended in PBS. The OD 600 to 0.4±0.02, marked as A0. 3mL of the above cell suspension was mixed with 1mL of chloroform, vortexed for 30s, and then allowed to stand at 37℃ for 30min. The upper aqueous phase was taken and the absorbance at 600nm was measured and marked as A. t .

[0061] Surface hydrophobicity (A%) calculation formula: A% = (A0-A t ) / A0×100%,

[0062] (2) Caco-2 cell adsorption rate: The cells were seeded into a 24-well cell culture plate and cultured in a 37°C incubator with 5% CO2 for 15 days to allow the cells to differentiate. 1 ml of DMEM (1×10 8 CFU / mL) resuspended Weissella suspension was incubated with Caco-2 monolayer for 1 hour (37°C, 5% CO2), and then washed twice with pre-warmed DPBS (pH 7.2, without Ca and Mg) (Procell, China) to remove unattached Weissella cells. Adherent bacteria were separated with DPBS containing 1% (v / v) TritonX-100. Adhesion capacity was determined by serial dilution plate counting.

[0063] (3) Self-aggregation ability: The lactic acid bacteria strains stored at -80°C were activated for two generations and inoculated into MRS liquid medium at a 1% (v / v) inoculum. The cells were collected by centrifugation (8000 g, 4°C, 10 min); washed twice with PBS (pH 7.4) and resuspended in PBS. The bacterial solution concentration was adjusted to 10 8 cfu / mL, take 50μl of bacterial suspension, add it to 150μl PBS, mix it, measure the absorbance at 600nm, record it as A0; 4mL of the same amount of cell suspension is placed in a 5mL centrifuge tube, mix it and let it stand at 37℃ for 6h. Take 50μl of the upper bacterial suspension, add it to 150μl PBS, mix it, measure the absorbance at 600nm, record it as A t .

[0064] Agglutination rate (A%) calculation formula: A% = (A0-A t ) / A0×100%

[0065] The results are as follows Figure 6 As shown in C, D, and F, the cell surface hydrophobicity of strain 38-3 is 58.41%, and the cell adsorption rate is lower than that of the reference strain LGG. The self-aggregation rate reaches 53.63% after 2 hours and 64.92% after 4 hours, which is significantly higher than the commercial strain Lactobacillus rhamnosus LGG (2 hours, 3.34%; 4 hours, 5.55%), indicating that the strain has good surface adhesion and great potential for colonization in the intestine.

[0066] 3. Antagonistic performance of strain 38-3

[0067] The plate diffusion method was used to determine the antibacterial activity of the strains. Seven indicator bacteria (Salmonella paratyphi CMCC50094, Shigella flexneri CMCC51574, Staphylococcus aureus subspecies CGMCC 1.0089, Listeria monocytogenes CICC21635, Enterococcus faecalis CICC 10396, Enterococcus faecium CGMCC 1.101, Escherichia coli CMCC44102.) and four indicator fungi (Aspergillus flavus CICC 40375, Rhizoctonia solani CICC 40529, Fusarium oxysporum CICC 2532, Candida albicans GGMCC2.2086) were used for the experiment. The bacterial culture conditions were all 37℃, 180rpm shaking culture, and after two generations of activation, they were used for antibacterial test: strains 38-3 and LGG (control) were activated for two generations, centrifuged at 8000rpm, 4℃, 5min, and the supernatant was taken. Take 50 μl of indicator bacteria suspension and add it to 200 ml of MRS agar, mix it thoroughly and pour it into a culture dish. Use a hole puncher to punch holes with a diameter of 8 mm on the plate, add 80 μl of the supernatant of the test strain to each hole, place the plate at 37 ° C and incubate overnight, observe the inhibition zone and measure its diameter (mm) with an electronic vernier caliper. The antifungal activity of Weissella strains was evaluated by soft agar overlay method. After growing for 16 hours at 37 ° C, the Weissella strain was inoculated on an MRS agar plate with a row spacing of 2 cm and continued to be cultured at 37 ° C for 24 hours. The fungus was cultured on a potato dextrose agar (PDA) plate at 25 ° C for 5-7 days. Add 15 ml of sterile ultrapure water to collect the spore suspension and quantify it with a hemocytometer. Then cover the plate with a soft (0.7% agar) PDA medium containing a fungal spore suspension (10 spores per ml) and culture it at 28 ° C for 3 days. Antifungal activity was assessed as a clear zone of inhibition around bacterial smears.

[0068] Table 4

[0069]

[0070]

[0071] Note: “-” means no inhibition; “+” means the inhibition zone is between 0 and 3 mm; “++” means the inhibition zone is between 3 and 6 mm; “+++” means the inhibition zone is greater than 6 mm. The diameter of the hole (8 mm) has been deducted.

[0072] The results showed (Table 4) that strain 38-3 and commercial strain LGG showed good antagonistic activity against 8 of the 12 indicator bacteria, but the antibacterial spectra were different. Compared with LGG, 38-1 also showed significant inhibitory effects against Salmonella paratyphi B CMCC 50094 among bacteria and Aspergillus flavus 3.395 among fungi.

[0073] 4. Cholesterol-lowering ability

[0074] Preparation of cholesterol micelle MRS: weigh 0.1g cholesterol, 0.2g ox bile salt, 0.1g sucrose ester, add to 1mL Tween-80, stir evenly, add 5mL glacial acetic acid and heat to 60℃ to dissolve, and the solution is ultrasonically treated (130w, 20kHz, 60% ultrasonic crushing for 20 cycles, 8 times each cycle, ultrasonic 2s, 3s interval) to obtain a cholesterol micelle solution. The solution is quickly added to the MRS liquid culture medium, stirred continuously to form a uniform and stable colloidal solution, fixed to 1L, and sterilized at 121℃ for 15min, where the cholesterol micelle concentration is 1mg / mL. Add 0.3% ox bile powder and water-soluble cholesterol (150μg / ml, filter sterilized) to the fresh MRS broth culture medium.

[0075] Culture and determination:

[0076] Probiotics were inoculated at a rate of 1%, and cultured at 37°C for 20 hours; centrifuged at 10,000 rpm for 15 minutes. 1 ml of supernatant was mixed with 1 ml KOH (33% w / v) and 2 ml 96% ethanol, vortexed for 2 minutes, and incubated in a 60°C water bath for 15 minutes. Cooled to room temperature, 2 ml of distilled water and 3 ml of n-hexane were added, and vortexed for 1 minute. 1 ml of n-hexane was quickly transferred to a glass tube and evaporated in an 80°C water bath. The residue was quickly dissolved in 2 ml of o-phthalaldehyde, and 0.5 ml of concentrated sulfuric acid was added, and vortexed for 1 minute. The above mixture was reacted at room temperature for 30 minutes, and the absorbance at 550 nm was measured. The absorbance at 0h OD550 is recorded as C0; the OD at 20h is recorded as C0. 550 Absorbance value, recorded as C t , the calculation formula is as follows:

[0077] Cholesterol removal rate (C%) calculation formula: C% = (C0-C t ) / C0×100%

[0078] The experimental results showed that the cholesterol removal rate of 38-3 was 16.67%, which was higher than that of the reference strain LGG, which was 15.48%. Figure 6 Middle E. This indicates that 38-3 has good cholesterol scavenging ability.

[0079] In summary, the green Weissella viridescens 38-3 provided by the present invention has good biological safety, no hemolytic activity, no bioamine production, and is sensitive to antibiotics such as ampicillin, erythromycin, kanamycin, streptomycin, tetracycline, vancomycin, chloramphenicol, gentamicin, clindamycin, ciprofloxacin, and cefixime. At the same time, the strain has a fast fermentation start-up speed, good probiotic performance, and its self-polymerization ability is significantly better than that of the commercial strain Lactobacillus rhamnosus LGG. It can be used as a new dietary supplement to regulate intestinal flora, improve intestinal function, and is applied to the fields of food, health products, and medicines.

[0080] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0081] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Weissella viridescens 38-3, characterized in that: The deposit number is CGMCC No.32210.

2. The fermentation product of Weissella viridis 38-3 according to claim 1.

3. A bacterial agent comprising the viridis bacteria 38-3 according to claim 1.

4. A food comprising the viridis bacteria 38-3 of claim 1.

5. A health product comprising the viridis bacteria 38-3 according to claim 1.

6. A medicine comprising the Weissella viridis 38-3 according to claim 1.

7. Use of the viridis bacteria 38-3 according to claim 1 in fermentation production.

8. Use of Weissella viridis 38-3 as described in claim 1 in the preparation of a bacterial agent or medicine for regulating intestinal flora, improving intestinal function, inhibiting pathogens or lowering cholesterol.

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