Capsaicin-tolerant lactobacillus plantarum and application of capsaicin-tolerant lactobacillus plantarum in pickled pepper fermentation

Abstract

The invention discloses capsaicin-tolerant lactobacillus plantarum and application thereof in pickled pepper fermentation, and belongs to the technical field of microorganisms. The lactobacillus plantarum MYL-6 provided by the invention is preserved in the China Center for Type Culture Collection on August 10, 2023, and the preservation number of the lactobacillus plantarum MYL-6 is CCTCC (China Center for Type Culture Collection) M 20231407. According to the invention, a fusion strain MYL-6 is constructed through inactivation of parents and protoplast fusion. Experimental results prove that the prepared lactobacillus plantarum strain MYL-6 not only has the capacity of producing lactic acid at high yield, but also has the characteristic of capsaicin tolerance, after the lactobacillus plantarum provided by the invention is used for fermenting pickled peppers, the lactic acid content is increased, the acid fragrance is improved, the fermentation period of the pickled peppers is shortened, the quality of the pickled peppers can be improved, and the production cost is reduced. The method has huge application potential in the pickled pepper manufacturing industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a capsaicin-tolerant Lactobacillus plantarum and application thereof in pickled pepper fermentation. Background Art

[0002] As a traditional food seasoning in my country, pickled peppers are widely loved by people in southwest my country. Modern biological science research shows that the fermentation process of pickled peppers is mainly participated by lactic acid bacteria. Lactic acid bacteria fermentation can produce a large amount of lactic acid and beneficial organic acids, which are the key substances for the formation of pickled pepper flavor. However, the traditional process mainly relies on natural environment fermentation production. Lactic acid bacteria are greatly affected by external factors, fermentation conditions are difficult to control, and the quality of pickled pepper products is unstable. Therefore, more and more industrial pickled peppers are currently being produced by artificial inoculation of probiotic lactic acid bacteria for enhanced fermentation. This method can not only increase the fermentation speed and greatly shorten the fermentation cycle, but also the rapidly formed acidic environment can inhibit the growth of miscellaneous bacteria, which can effectively ensure the stability of product quality, thereby solving many problems in the traditional fermentation production of pickled peppers. However, due to the antibacterial properties of capsaicin alkaloids, in the early stage of fermentation of pickled peppers, lactic acid bacteria with excellent performance are often inhibited by capsaicin in the raw materials and cannot grow rapidly, and cannot form an effective acidic environment to inhibit miscellaneous bacteria, and thus their own good fermentation performance cannot be effectively exerted. Therefore, modifying the capsaicin tolerance of lactic acid bacteria with good fermentation performance so that they can quickly exert their excellent acid production performance in the presence of higher concentrations of capsaicin is the key to producing high-quality pickled peppers, and is of great significance for improving the overall quality of fermented peppers and enhancing functionality and flavor components. Summary of the invention

[0003] The purpose of the present invention is to provide a capsaicin-tolerant Lactobacillus plantarum and application thereof in pickled pepper fermentation to solve the problems existing in the above-mentioned prior art. The Lactobacillus plantarum MYL-6 provided by the present invention has a high tolerance to capsaicin and has good lactic acid production capacity.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a Lactobacillus plantarum MYL-6, the Lactobacillus plantarum MYL-6 has a preservation number of CCTCC M 20231407, and has been deposited in the China Center for Type Culture Collection on August 10, 2023, with a preservation address of Wuhan University, Wuhan, China.

[0006] The invention provides application of the Lactobacillus plantarum MYL-6 in preparing a fermentation agent.

[0007] The present invention also provides a fermentation bacterial agent, comprising the Lactobacillus plantarum MYL-6.

[0008] The present invention also provides a method for preparing the fermentation agent, comprising the following steps: activating the plant lactobacillus MYL-6 to obtain an activated bacterial solution;

[0009] The activated bacterial liquid was inoculated into MRS liquid culture medium, cultured at 37°C for 15 h, centrifuged to precipitate the bacteria, washed, and resuspended to prepare a 10 7 CFU / mL of bacterial suspension is obtained to obtain the fermentation agent.

[0010] Preferably, in the inoculation, the volume ratio of the activated bacterial liquid to the MRS liquid culture medium is 6:1000.

[0011] The present invention also provides application of the plant lactobacillus MYL-6 or the fermentation bacterial agent in preparing pickled peppers.

[0012] The invention also provides a method for preparing pickled peppers, comprising the step of fermenting chili peppers by using the plant lactobacillus MYL-6 or the fermentation agent.

[0013] Preferably, the method comprises the following steps:

[0014] The sterilized chili peppers were mixed with the fermentation liquid, sterilized, inoculated with the fermentation agent, loaded into a fermentation tank, sealed with a breathable film, and fermented at a constant temperature of 30° C. for 5 days.

[0015] Preferably, the fermentation broth contains 1% by mass of crystal sugar and 8% by mass of salt;

[0016] The volume mass ratio of the fermentation liquid to the chili pepper is 8 mL:3 g;

[0017] The volume ratio of the fermentation agent to the fermentation liquid is 1:80.

[0018] The invention also provides pickled peppers prepared by the preparation method.

[0019] The present invention discloses the following technical effects:

[0020] The present invention is based on two parents of the same genus Lactobacillus plantarum, and adopts the method of fusing the protoplasts of the two parents after inactivation to obtain a fusion strain having the characteristics of both parents. The plant lactobacillus MYL-6 strain prepared by the present invention not only has the ability of high lactic acid production, but also has the characteristic of capsaicin tolerance. After the plant lactobacillus MYL-6 provided by the present invention is used for pickled pepper fermentation, the lactic acid content increases, the sour flavor is improved, the pickled pepper fermentation cycle is shortened, the quality of the pickled pepper can be improved, and the pickled pepper manufacturing industry has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 will be briefly introduced below. 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 paying creative work.

[0022] Figure 1 This is the colony morphology of Lactobacillus plantarum MYL-6;

[0023] Figure 2 is the Gram staining image of Lactobacillus plantarum MYL-6;

[0024] Figure 3 This is an electron micrograph of Lactobacillus plantarum MYL-6;

[0025] Figure 4 This is the growth curve of Lactobacillus plantarum MYL-6;

[0026] Figure 5 This is a graph showing the acid production capacity of Lactobacillus plantarum MYL-6. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The culture medium used in the present invention is as follows:

[0033] 40% PEG SMMD: Weigh 4 g of PEG 6000, dilute to 10 mL with SMM hypertonic buffer, and store at -4°C.

[0034] SMM hypertonic buffer: sucrose 17.5 g, MgCl2·6H2O 0.46 g, maleic acid 0.2336 g, ultrapure water 100 mL, pH 7.5.

[0035] Nascent calcium phosphate solution: K2HPO4 0.54 g, CaCl2·H2O 29.4 g, respectively dissolved in 100 mL of water and sterilized, mixed in equal volumes before use, ready for use.

[0036] MRS liquid culture medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, sodium acetate 5 g, diammonium hydrogen citrate 2 g, Tween-80 1 mL, K2HPO4 2 g, manganese sulfate 0.25 g, magnesium sulfate 0.58 g, agar 18 g, distilled water 1000 mL, pH (6.2-6.6), sterilization at 121°C for 20 min.

[0037] MRS solid culture medium: The preparation method is the same as that of liquid culture medium, and 1-2% agar powder is added.

[0038] CaCO3-MRS medium: The preparation method is the same as MRS solid medium, with the addition of 1-2% CaCO3.

[0039] Regeneration medium (RM): Add 0.02 mol / L MgCl2·6H2O, 0.6 mol / L mannitol and 0.02 mol / L maleic acid to MRS medium, pH 6.7.

[0040] The plant lactobacillus 1Y-35 used in the present invention has been disclosed in "Luo Lirong. Construction of lactic acid bacteria fusion and evaluation of fermented pickled pepper performance [D]. Guizhou University, 2023."; plant lactobacillus Y-6 has been disclosed in "Li Hongyang, Zhang Tonghua, Luo Lirong, et al. Screening of capsaicin-tolerant lactic acid bacteria in pickled pepper juice and research on their tolerance [J]. Food and Fermentation Industries, 2024, 50(04): 110-117.". The inventor promises to release the above strains to the public 20 years from the date of application of the present invention.

[0041] Example 1

[0042] (1) Preparation of two parental cells

[0043] The plant lactobacillus 1Y-35 with excellent lactic acid production performance and the capsaicin-tolerant plant lactobacillus Y-6 isolated and preserved in the previous laboratory were inoculated into MRS liquid culture medium and cultured until the cell concentration reached 10 7 -10 9 CFU / mL of bacterial suspension to obtain the two parental bacteria.

[0044] (2) Preparation of protoplasts from both parents

[0045] Take 1 mL of bacterial suspension from each of the two parent bacterial cultures in step (1), centrifuge at 4°C and 4000 rpm for 5 min, discard the supernatant, add SMM buffer to the precipitate, shake evenly, and centrifuge again. Repeat the above steps three times, and dilute the bacterial cells with SMM buffer to a concentration of 10 6 -10 7 1.5 mg / mL lysozyme was added to the 1Y-35 bacterial suspension and enzymolysis was performed at 37°C for 120 min; 1 mg / mL lysozyme was added to the Y-6 bacterial suspension and enzymolysis was performed at 37°C for 75 min; the two enzymolysis bacterial cells were immediately added with 0.1 mol / L EDTA solution preheated for 30 min to obtain 1Y-35 protoplasts and Y-6 protoplasts suspensions.

[0046] (3) Inactivation of protoplasts of both parents

[0047] The Y-6 protoplast suspension in step (2) was irradiated under a 15W ultraviolet lamp for 25 min (vertical irradiation distance was 30 cm) for ultraviolet inactivation, and the 1Y-35 protoplast suspension in step (2) was heat-inactivated in a 60°C water bath for 35 min.

[0048] (4) Fusion of protoplasts from two parents

[0049] Take 0.2 mL of the inactivated protoplasts of the two parent strains, mix them, centrifuge at 4000 rpm for 15 min, discard the supernatant, add 0.4 mL of SMMD containing 40% PEG (w / v) (add DNase to 5 μg / mL in SMM buffer, temporarily prepared for use) and 0.05 mL of fresh calcium phosphate solution to the precipitate, mix evenly, and keep warm at 37° C. for 10-20 min. Add SMM hypertonic buffer solution to the precipitate to restore the original volume and dilute appropriately, take 0.1 mL of the dilution and apply it to the regeneration medium, culture at 37° C. for 2-3 days, and wait for a single colony to grow on the medium.

[0050] (5) Screening of fusion products

[0051] The fusion colony grown in step (4) was transferred to MRS medium containing 1 mg / mL capsaicin and cultured at 37°C for 48 hours. Strains resistant to capsaicin were screened based on the increase in OD value, and the lactic acid content of the supernatant was determined by high-performance liquid chromatography. The conditions for determining lactic acid content by high-performance liquid chromatography are as follows:

[0052] Chromatographic column: C 18 Column 5 μm, 2504.6 mm;

[0053] pH range: 2-10;

[0054] Mobile phase: 2% H3PO4 in deionized water: methanol (v / v) = 99.5:0.5;

[0055] Detector wavelength: 210nm;

[0056] Flow rate: 0.3 mL / min;

[0057] After the fusion strain was cultured in the capsaicin-containing medium for 48 hours, the lactic acid content was detected by high performance liquid chromatography as shown in Table 1. Table 1 shows that the fusion strain numbered 6 produced the highest lactic acid of 10486.25 μg / mL in the capsaicin-containing environment, and the yield was close to that of the parent 1Y-35 in the MRS medium without capsaicin, which was a successfully constructed fusion strain. It was named MYL-6.

[0058] Table 1 Results of lactic acid content test of parent strains and fusion strains

[0059]

[0060] (7) Identification of fusion strain MYL-6

[0061] The 16S rDNA of the strain MYL-6 obtained in step (6) was cloned by PCR and sent to BGI for sequencing. The 16S rDNA sequence is shown in SEQ ID NO.1, with a length of 1469 bp. The sequence information was uploaded to NCBI for comparison, and it was found that it had a high degree of homology (≥99%) with the plant lactobacillus in the database. The colonies were milky white, round, convex, smooth and moist on the surface, with clear edges, and obvious calcium melting circles around the colonies (such as Figure 1 After Gram staining, the staining was positive (as shown in Figure 2 ); The bacteria were observed to be rod-shaped under electron microscope (as shown in Figure 3 Based on the above identification information, the fusion strain MYL-6 was determined to be Lactobacillus plantarum.

[0062] SEQ ID NO.1:

[0063]

[0064] Lactobacillus plantarum MYL-6 was deposited in the China Center for Type Culture Collection on August 10, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 20231407.

[0065] (8) Growth curve determination of fusion strain MYL-6

[0066] The final concentration of Lactobacillus plantarum MYL-6 strain was about 1.0×10 6 CFU / mL was inoculated into 600 mL of MRS liquid culture medium and cultured at 37°C for 24 h; 4 mL of bacterial solution was taken every 2 h for viable bacterial count analysis and pH determination;

[0067] Growth curve: The results are as follows Figure 4 As shown, Lactobacillus plantarum MYL-6 entered the logarithmic growth phase from the 4th hour, the stable growth phase from the 6th hour, and the plateau phase after 18 hours.

[0068] Acid production capacity: When inoculated into liquid MRS medium with an initial pH of 6.35, the pH dropped below 4 after 16 hours of fermentation, and the final pH reached about 3.7 (e.g. Figure 5 shown).

[0069] Example 2

[0070] According to the inoculum volume of 6‰ (v / v), Lactobacillus plantarum MYL-6 activated in MRS liquid medium for 15 h was inoculated into 100 mL MRS liquid medium, incubated at 37 °C for 15 h, centrifuged at 8000 rpm for 20 min to precipitate the bacteria, washed four times with sterile deionized water, and then added with sterile deionized water to make a concentration of 10 7 CFU / mL of bacterial suspension is the Lactobacillus plantarum MYL-6 fermentation agent;

[0071] 1 mL of MYL-6 fermentation agent was inoculated into a sterilized fermentation tank containing 80 mL of fermentation liquid containing 1% rock sugar and 8% salt and 30 g of sterilized chili pepper. The tank was sealed with a breathable membrane and fermented at a constant temperature of about 30°C for 5 days. The control was pickled pepper fermented without inoculation of the agent, and other conditions were consistent with the MYL-6 fermentation conditions.

[0072] Sensory evaluation: A sensory evaluation panel consisting of 10 graduate students with a food professional background was selected to conduct sensory evaluation on naturally fermented pickled peppers and MYL-6-inoculated fermented pickled peppers, and the overall score was calculated according to the following formula:

[0073] Overall score = color (10%) + appearance (10%) + aroma (20%) + texture (30%) + taste (30%).

[0074] The percentage in brackets in the formula represents the weight of a single sensory evaluation score in the overall score.

[0075] The scoring results are shown in Table 2. The kimchi fermented by inoculation with strain MYL-6 was significantly better than the naturally fermented kimchi in terms of aroma and taste (P < 0.05), and the overall sensory score was significantly higher than that of the naturally fermented kimchi (P < 0.05), indicating that capsaicin-resistant Lactobacillus plantarum MYL-6 can shorten the fermentation time and improve the sensory quality of kimchi.

[0076] Table 2 Pickled pepper sensory flavor evaluation score results

[0077]

[0078] Determination of lactic acid content in pickled peppers: The lactic acid content in pickled peppers fermented with MYL-6 was determined to be 3.12 mg / g, while the lactic acid content in pickled peppers fermented naturally was 1.73 mg / g. Fermentation with MYL-6 increased the lactic acid content in pickled peppers by 80.33%.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum MYL-6, characterized in that: The Lactobacillus plantarum MYL-6 has a deposit number of CCTCC M 20231407 and was deposited in the China Center for Type Culture Collection on August 10, 2023. The deposit address is Wuhan University, Wuhan, China.

2. Application of plant lactobacillus MYL-6 as claimed in claim 1 in preparing a fermentation agent.

3. A fermentation agent, characterized in that: The invention comprises the Lactobacillus plantarum MYL-6 as claimed in claim 1.

4. A method for preparing a fermentation agent as claimed in claim 3, characterized in that: The following steps are involved: activating the Lactobacillus plantarum MYL-6 to obtain an activated bacterial liquid; The activated bacterial liquid was inoculated into MRS liquid culture medium, cultured at 37°C for 15 h, centrifuged to precipitate the bacteria, washed, and resuspended to prepare a 10 7 CFU / mL of bacterial suspension is obtained to obtain the fermentation agent.

5. The preparation method according to claim 4, characterized in that: During the inoculation, the volume ratio of the activated bacterial solution to the MRS liquid culture medium is 6:1000.

6. Use of plant lactobacillus MYL-6 as claimed in claim 1 or the fermentation agent as claimed in claim 3 in the preparation of pickled peppers.

7. A method for preparing pickled peppers, characterized in that: The method comprises the step of fermenting chili pepper using the plant lactobacillus MYL-6 described in claim 1 or the fermentation agent described in claim 3.

8. The preparation method according to claim 7, characterized in that: The following steps are involved: The sterilized chili peppers were mixed with the fermentation liquid, sterilized, inoculated with the fermentation agent, loaded into a fermentation tank, sealed with a breathable film, and fermented at a constant temperature of 30° C. for 5 days.

9. The preparation method according to claim 8, characterized in that: The fermentation liquid contains 1% rock sugar and 8% salt by mass; The volume mass ratio of the fermentation liquid to the chili pepper is 8 mL:3 g; The volume ratio of the fermentation agent to the fermentation liquid is 1:

80.

10. Pickled peppers prepared by the preparation method according to any one of claims 7 to 9.

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