Probiotics separated from pickled peppers and having fermentation characteristic as well as preparation method and application of probiotics

Abstract

The invention relates to the field of preparation of edible probiotics, and particularly discloses probiotics with fermentation characteristics separated from pickled peppers as well as a preparation method and application of the probiotics, and the probiotics are Lactobacillus plantarum TNBB-001 and are preserved in the China Center for Type Culture Collection. The TNBB-001 probiotics prepared through strain culture, purification, dyeing selection, detection and the like are sensitive to amoxicillin, penicillin G, erythromycin and chloramphenicol, which indicates that the TNBB-001 probiotics are a safe strain. And the strain has relatively strong acid production capacity and relatively strong gastrointestinal fluid endurance capacity. According to the present invention, the gene coding the resistance group and the transferable antibiotic drug resistance gene in the L. plantarum TNBB-001 is not found, such that the strain is safe, does not have the drug resistance gene, and can be used for pickled pepper fermentation and yogurt fermentation.

Description

Technical Field

[0001] The present invention relates to the field of edible probiotic preparation, and in particular to a probiotic with fermentation characteristics separated from pickled peppers, a preparation method and an application thereof. Background Art

[0002] Pickled peppers, a traditional fermented food in my country, are highly sought after for their rich organic acids and numerous beneficial effects, including maintaining a balanced intestinal flora, lowering cholesterol, enhancing immunity, and providing antioxidant properties. Currently, industrial production of pickled peppers relies primarily on natural fermentation of the raw material's own microbial flora and environmental microorganisms. Due to the complex and diverse strains involved in natural fermentation, the fermentation process is difficult to control, resulting in varying quality and flavor between batches and making consistent quality difficult to achieve. Therefore, artificially incorporating probiotic strains with fermentative advantages during fermentation, replacing traditional natural fermentation, can lead to the development of more stable products and greater suitability for large-scale, standardized production. Identifying strains with fermentative advantages is a prerequisite for achieving enhanced fermentation. While excellent lactic acid bacteria strains have been screened from kimchi, few reports have identified superior lactic acid bacteria suitable for pickled pepper fermentation. This is due to significant differences between the microbial flora of kimchi and pickled peppers. While the primary inhibitory factor for microbial growth in kimchi is the high salt environment, pickled peppers, in addition to high salt content, also have a pungent odor caused by capsaicinoids, which inhibits the growth of related microorganisms. In addition to being acid- and salt-tolerant, the lactic acid bacteria isolated from pickled peppers are also hypothesized to exhibit a certain degree of resistance to capsaicinoids and capsaicinoids. Therefore, isolating and screening superior strains with strong acid production, safety, and excellent fermentation properties from regions rich in pickled pepper resources has important theoretical and practical implications for the pickled pepper production industry. Summary of the Invention

[0003] The purpose of the present invention is to provide a probiotic with fermentation characteristics separated from pickled peppers, a preparation method and an application thereof, so as to screen out excellent strains with strong acid production, safety and good fermentation characteristics.

[0004] The strain is deposited in the China Center for Type Culture Collection (CCTCC, address: 299 Bayi Road, Wuhan University, China Center for Type Culture Collection, zip code 430072). The strain deposit number of Lactobacillus plantarum TNBB-001 is CCTCC M2025807, the deposit date is April 16, 2025, the classification name is Lactobacillus plantarum, and the strain name is TNBB-001.

[0005] The technical solution for achieving the purpose of the present invention is as follows: A preparation method for isolating probiotics with fermentation properties from pickled peppers, comprising the following steps:

[0006] Strain culture method

[0007] The collected pickled pepper samples were placed in sterile saline, shaken evenly, and then diluted with sterile saline 0.9% NaCl gradient and evenly coated in culture medium for cultivation;

[0008] Select a dilution factor of 10 -6 ,10 -7 and 10 -8 Use a sterile inoculating loop to pick a single white and smooth colony from the coated plate, perform Gram staining under a microscope, and streak the colony onto a new sterile agar medium. Incubate at 37°C for 48 hours, and repeat the process until no other bacteria are found under the microscope.

[0009] The purified single colonies, i.e., the single colonies without mixed bacteria, were inoculated into sterile liquid culture medium, cultured at 37°C for 16-18 hours, centrifuged at 3,500 rpm for 5 minutes, and the fermentation liquid of the strain was removed. Then, new sterile liquid culture medium was added to the bacterial slurry, and the mixture was shaken evenly to obtain a uniform bacterial solution. The bacterial solution was mixed with an equal volume of glycerol and stored in a -80°C refrigerator for future use.

[0010] Strain staining:

[0011] Gram staining was used to stain the strains. An oil immersion lens was used for microscopic examination to observe the bacteria. The Gram staining results of the bacteria were judged. Bacteria stained blue-purple were Gram-positive bacteria, and those stained red were Gram-negative bacteria. Only Gram-positive bacteria were selected.

[0012] Gram-positive Lactobacillus plantarum TNBB-001 was inoculated into MRS liquid culture medium and cultured at 37° C. for 18 hours to obtain the probiotics of the present application.

[0013] Preferably, the gradient dilution factor is 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 ,10 -8 ,10 -9 .

[0014] Preferably, the process of culturing after gradient dilution is to culture in sterile MRS medium at 37° C. for 16-18 hours.

[0015] Preferably, the glycerol is a mixture of glycerol and water in a volume ratio of 1:1.

[0016] Preferably, all strains need to be activated for two generations before use, and all operations of transplanting microbial strains should be performed under a sterile environment.

[0017] The probiotics with fermentation properties separated from pickled peppers are applied to the fermentation of pickled peppers and the fermentation of yogurt.

[0018] The present invention has the following beneficial effects: the TNBB-001 probiotic prepared by the present invention is sensitive to amoxicillin, penicillin G, erythromycin, and chloramphenicol, indicating that the TNBB-001 probiotic is a safe strain. Furthermore, after 24 hours of culture, the pH of the culture solution is 3.97±0.01, indicating that the bacteria has a strong acid-producing ability. The gastric juice survival rate of the strain TNBB-001 is 88.0±2.1%, and the intestinal juice survival rate is 84.21±8.9%, indicating that the strain has a strong ability to tolerate gastrointestinal juice. No genes encoding resistomes or transferable antibiotic resistance genes were found in the ResFinder and Disinfinder databases in L. plantarum TNBB-001, indicating that the strain is safe and does not contain resistance genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a Gram staining diagram during the preparation process of the strain of the present invention.

[0020] Figure 2 is the TNBB-001 phylogenetic tree.

[0021] Figure 3 is the general genome circular map of L. plantarum TNBB-001. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0023] A preparation method for isolating probiotics with fermentation properties from pickled peppers, comprising the following steps:

[0024] Strain culture method

[0025] The collected pickled pepper samples were placed in sterile saline, shaken evenly, and then diluted with sterile saline 0.9% NaCl gradient and evenly coated in culture medium for culture. The gradient dilution multiple was 10 -1,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 ,10 -8 ,10 -9 The process of culturing after gradient dilution is to culture in sterile MRS medium at 37° C. for 16-18 hours;

[0026] Select a dilution factor of 10 -6 ,10 -7 and 10 -8 Use a sterile inoculating loop to pick a single white and smooth colony from the coated plate, perform Gram staining under a microscope, and streak the colony onto a new sterile agar medium. Incubate at 37°C for 48 hours, and repeat the process until no other bacteria are found under the microscope.

[0027] The purified single colonies, i.e., the single colonies without mixed bacteria, were inoculated into sterile liquid culture medium, cultured at 37°C for 16-18 hours, centrifuged at 3,500 rpm for 5 minutes, and the fermentation broth of the strain was removed. New sterile liquid culture medium was then added to the bacterial slurry, and the mixture was shaken evenly to obtain a uniform bacterial solution. The bacterial solution was mixed with an equal volume of glycerol, which was a mixture of glycerol and water in a volume ratio of 1:1, and then stored in a -80°C refrigerator for later use.

[0028] Strain staining:

[0029] Gram staining was used to stain the strains. An oil immersion lens was used to observe the bacteria and determine the Gram staining results. Bacteria that were stained blue-purple were Gram-positive bacteria, and those that were stained red were Gram-negative bacteria. Figure 1 , only Gram-positive bacteria were selected;

[0030] Gram-positive Lactobacillus plantarum TNBB-001 was inoculated into MRS liquid culture medium and cultured at 37°C for 18 hours to obtain the probiotics of the present application. Shanghai Meiji Biological Company was commissioned to perform whole genome identification, and the identification result was Lactobacillus plantarum.

[0031] All strains must be activated for two generations before use, and all operations involving the transfer of microbial strains should be performed under a sterile environment.

[0032] Strain safety

[0033] To evaluate the antibiotic susceptibility of Lactobacillus plantarum TNBB-001.

[0034] Six antibiotic susceptibility discs (amoxicillin, penicillin G, erythromycin, and chloramphenicol) were placed in MRS solid medium inoculated with TNBB-001. The clear zones were then measured with a vernier caliper to determine antibiotic susceptibility (see Table 1).

[0035] Table 1 Antibiotic experiments

[0036]

[0037] a :CLSI, Clinical and Laboratory Standards Institute. The inhibition zone is evaluated according to the standard values provided by CLSI: sensitive > 20 mm, intermediate ≈ 15-19 mm, and resistant ≤ 14 mm (according to the CLSI 2012 standard).

[0038] b : Diameter of the inhibition zone (including the diameter of the drug-sensitive plate). The results are expressed as the mean ± standard deviation of three independent repeated experiments.

[0039] The safety test results showed that TNBB-001 was sensitive to amoxicillin, penicillin G, erythromycin and chloramphenicol, indicating that TNBB-001 is a safe strain.

[0040] Acid production capacity test

[0041] After the strain was activated, it was aseptically transferred into a test tube containing 5 mL of liquid culture medium at a 5% inoculum size. Each test tube was labeled with the strain number and time, and cultured at 30°C. The pH value was measured at 0, 4, 8, 12, and 24 h of culture time. The results are shown in Table 2, indicating that the bacteria has a strong acid-producing ability.

[0042] Table 2 Acid production performance of strains

[0043]

[0044] Simulated gastric fluid: Pepsin was dissolved in PBS (0.1 mol / L, pH = 7.3) and adjusted to a concentration of 3.0 g / L (pH = 2.5). The cells were then filtered through a sterile membrane (0.22 μm, Millex-GP). Simulated intestinal fluid: Trypsin was dissolved in PBS (0.1 mol / L, pH = 7.3) and adjusted to a concentration of 1.0 g / L (pH = 8.0). The cells were then filtered through a sterile membrane (0.22 μm, Millex-GP). The cells were incubated with simulated gastric fluid at a volume ratio of 1:9 at 37°C for 3 hours, followed by incubation with simulated intestinal fluid at the same volume ratio at 37°C for 8 hours. The cells were counted using the plate and colony counting methods. All operations were performed in a clean bench. LGG was used as a positive control, and the survival rate of the strain was calculated according to the following formula:

[0045] Strain survival rate (%) = (logN1 / logN0) × 100%

[0046] Where, N1: the number of viable bacteria in simulated gastric or intestinal fluid, N0: the initial number of viable bacteria of the strain.

[0047] Table 3 Gastrointestinal fluid tolerance of strains

[0048]

[0049] The above table shows that this strain has a strong tolerance to gastrointestinal fluid

[0050] Figure 2 This is the phylogenetic tree of TNBB-001. As can be seen from the figure, the strain TNBB-001 is Lactiplantibacillus plantarum.

[0051] The complete genome of L. plantarum TNBB-001 consists of a single circular chromosome of 3,300,704 bp in length with a G+C content of 44.45%. No genes encoding resistomes or transferable antibiotic resistance genes were found in L. plantarum TNBB-001 using the ResFinder and Disinfinder databases.

[0052] L. plantarum TNBB-001 contains 13 genes associated with bile salt resistance: yiaC, elaA, rimJ, paiA, yjaB, rimL, dnaK, and dnaJ; 7 genes associated with adhesion: srtA, dltD, dltA, lspA, tuf, gapA, and groEL; and 14 genes associated with oxidative stress tolerance: tpx, trxA, trxB, npr, nrdH, dps, recA, gshA, and arsC. These data suggest that TNBB-001 may possess strong bile salt tolerance, adhesion, and antioxidant abilities.

[0053] Table 4. Potential genes associated with probiotic properties and oxidative stress tolerance in L. plantarum TNBB-001

[0054]

[0055] The probiotics with fermentation properties separated from pickled peppers are applied to the fermentation of pickled peppers and the fermentation of yogurt.

[0056] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A probiotic with fermentation properties isolated from pickled peppers, characterized in that: The probiotic is Lactobacillus plantarum TNBB-001, which is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number being CCTCC M 2025807.

2. The method for separating probiotics with fermentation properties from pickled peppers according to claim 1, characterized in that: The steps include: Strain culture method The collected pickled pepper samples were placed in sterile saline, shaken evenly, and then diluted with sterile saline 0.9% NaCl gradient and evenly coated in culture medium for cultivation; Select a dilution factor of 10 -6 ,10 -7 and 10 -8 Use a sterile inoculating loop to pick a single white and smooth colony from the coated plate, perform Gram staining under a microscope, and streak the colony onto a new sterile agar medium. Incubate at 37°C for 48 hours, and repeat the process until no other bacteria are found under the microscope. The purified single colonies, i.e., the single colonies without mixed bacteria, were inoculated into sterile liquid culture medium, cultured at 37°C for 16-18 hours, centrifuged at 3,500 rpm for 5 minutes, and the fermentation liquid of the strain was removed. Then, new sterile liquid culture medium was added to the bacterial slurry, and the mixture was shaken evenly to obtain a uniform bacterial solution. The bacterial solution was mixed with an equal volume of glycerol and stored in a -80°C refrigerator for future use. Strain staining: Gram staining was used to stain the strains. During microscopy, an oil immersion lens was used to observe and judge the Gram staining results of the bacteria. Only Gram-positive bacteria were selected. Gram-positive Lactobacillus plantarum TNBB-001 was inoculated into MRS liquid culture medium and cultured at 37° C. for 18 hours to obtain the probiotics of the present application.

3. The method for separating probiotics with fermentation properties from pickled peppers according to claim 2, characterized in that: The gradient dilution factor is 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 ,10 -8 ,10 -9 .

4. The method for separating probiotics with fermentation properties from pickled peppers according to claim 2, characterized in that: The process of culturing after gradient dilution is to culture in sterile MRS medium at 37° C. for 16-18 hours.

5. The method for separating probiotics with fermentation properties from pickled peppers according to claim 2, characterized in that: The glycerol is prepared by mixing glycerol and water in a volume ratio of 1:

1.

6. The method for separating probiotics with fermentation properties from pickled peppers according to claim 2, characterized in that: All strains must be activated for two generations before use, and all operations involving the transfer of microbial strains should be performed under a sterile environment.

7. The probiotic having fermentation properties isolated from pickled peppers according to claim 1, characterized in that: The probiotics are used for the fermentation of pickled peppers and the fermentation of yogurt.

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