A recombinant Lactobacillus plantarum and its application

Abstract

This invention discloses a recombinant *Lactobacillus plantarum* and its applications, belonging to the field of bioengineering technology. The purpose of this invention is to improve the cold tolerance and antioxidant capacity of *Lactobacillus plantarum*, and to increase its ability to produce lactic acid and degrade nitrite. This invention provides a recombinant *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Using *Lactobacillus plantarum* as the starting strain, the nucleic acid molecule shown in SEQ ID NO.1 is overexpressed. It is used in fermentation products in the food industry.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a recombinant plant lactobacillus and its applications. Background Technology

[0002] Low-temperature fermented foods are of higher quality, with a softer, more delicious texture and no off-flavors. However, the microorganisms in the fermentation system are often affected by low-temperature stress, which reduces their fermentation performance and thus affects product quality. Therefore, this invention provides a method for constructing recombinant *Lactobacillus plantarum* overexpressing amino acid metabolism genes, and using it in a low-temperature environment to improve its growth, fermentation acid production, and nitrite degradation capabilities, aiming to enhance the flavor and quality of low-temperature fermented foods such as sauerkraut, while shortening the fermentation time.

[0003] In the food industry, many fermented products (such as yogurt, fermented milk, and kimchi) require storage and transportation at low temperatures to extend shelf life and inhibit the growth of harmful microorganisms. If *Lactobacillus plantarum* possesses good cold resistance, it can maintain its activity at low temperatures, helping to preserve the fermentation characteristics, flavor, and safety of the product. Cold-resistant *Lactobacillus plantarum* can be applied to more fermentation or health food production processes requiring low-temperature environments, such as refrigerated fermented foods and low-temperature preserved probiotic preparations, expanding its industrial application scope.

[0004] The accumulation of lactic acid by *Lactobacillus plantarum* during fermentation not only lowers the pH value but also increases lactic acid production, contributing to improved quality and consistency of fermented foods. Furthermore, microbial fermentation can efficiently produce lactic acid, an industrial chemical, thereby reducing environmental pollution and resource waste and facilitating industrial applications.

[0005] In the food industry, the antioxidant properties of *Lactobacillus plantarum* can delay food oxidation and spoilage, extending shelf life. For example, its fermentation products can inhibit oil oxidation and enhance the antioxidant properties of fermented foods (such as yogurt and fermented black beans). There is a pressing need for *Lactobacillus plantarum* strains possessing these capabilities. Summary of the Invention

[0006] The purpose of this invention is to improve the cold resistance and antioxidant capacity of *Lactobacillus plantarum*, and to increase its ability to produce lactic acid and degrade nitrite.

[0007] This invention provides a recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum Using *Lactobacillus plantarum* SC-MDJ as the starting strain, the nucleic acid molecule shown in SEQ ID NO.1 was overexpressed; the *Lactobacillus plantarum* SC-MDJ, with accession number CGMCC NO.28112, is deposited at the China General Microbiological Culture Collection Center on August 7, 2023, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The present invention provides a microbial preparation containing the above-mentioned recombinant Lactobacillus plantarum.

[0009] This invention provides a method for improving the antioxidant capacity of Lactobacillus plantarum by overexpressing the nucleic acid molecule shown in SEQ ID NO.1 in Lactobacillus plantarum SC-MDJ.

[0010] This invention provides a method for improving the cold resistance of Lactobacillus plantarum by overexpressing the nucleic acid molecule shown in SEQ ID NO.1 in Lactobacillus plantarum SC-MDJ.

[0011] A breeding method to enhance the ability of Lactobacillus plantarum to degrade nitrite involves overexpressing the gene shown in SEQ ID NO.1 in Lactobacillus plantarum SC-MDJ.

[0012] This invention provides the application of the above-mentioned recombinant Lactobacillus plantarum in cold-resistant fermentation, nitrite-reducing fermentation, antioxidant fermentation, or lactic acid-producing fermentation.

[0013] Beneficial effects: Inoculation with genes overexpressing amino acid metabolism L. plantarum Fermentation of SC-MDJ-NC8_RS11250 under low-temperature conditions can increase the OD of the strain. 600nm The pH of the protein was increased to 1.895, a 33.17% improvement over the control group; simultaneously, the pH of the fermentation system was reduced to 4.51, a 9.26% decrease compared to the control group; and the antioxidant capacity reached 21.86 U / mg protein, an 81.86% increase compared to the control group. Therefore, these results demonstrate that overexpression of amino acid metabolism genes can significantly improve… L. plants The growth, acid production, and antioxidant capacity of SC-MDJ in low-temperature environments endow the fermentation agent with unique physiological functions and enhance the strain's ability to resist low-temperature oxidative stress.

[0014] Inoculation with genes expressing amino acid metabolism L. plantarum Fermentation of SC-MDJ-NC8_RS11250 at 15 °C can increase the total acid content in the fermentation system to 12.27 mg / mL, while reducing the sodium nitrite content to 0.020 mg / mL, achieving a nitrite degradation rate of 98.00%. The increased total acid content enhances the texture and flavor of food, thus contributing to the formation of a pleasant taste.

[0015] Inoculation with genes expressing amino acid metabolism L. plantarumFermentation of SC-MDJ-NC8_RS11250 at 15 °C increased the lactic acid content in the fermentation system to 11.11 mg / mL, a 35.16% increase compared to the control group. This increased lactic acid content enhances the unique sour taste of the food, thus contributing to the development of a pleasant flavor.

[0016] [Biological Preservation Information]: The name of Lactobacillus plantarum is Lactobacillus plantarum SC-MDJ, with accession number CGMCC NO.28112. It is deposited in the China General Microbiological Culture Collection Center on August 7, 2023. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0017] Figure 1 The construction process of plasmids for overexpression of amino acid metabolism genes;

[0018] Figure 2 For overexpression of amino acid metabolism genes L. plantarum PCR identification results;

[0019] Figure 3 The graph shows the relative expression levels of NC8_RS11250 gene mRNA. Detailed Implementation

[0020] MRS broth medium: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, 1000 mL distilled water, pH=5.7 ± 0.2.

[0021] LB broth medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g sodium chloride, 1000 mL distilled water, pH=7.0 ± 0.1.

[0022] Example 1. Method for constructing recombinant Lactobacillus plantarum

[0023] (1) Extraction of genomic DNA and acquisition of amino acid metabolism genes

[0024] Lactobacillus plantarum ( Lactiplantibacillus plantarumSC-MDJ was inoculated into MRS broth at a 2% inoculum and cultured at 30 °C and 200 rpm until the logarithmic growth phase, and then passaged twice. Genomic DNA was then extracted from the strain using a bacterial genomic DNA extraction kit. The DNA was obtained from the National Center for Biotechnology Information (NCBI) database. L. plantarum Reference genes NC8_RS11250 Sequence primers were designed for PCR amplification. After PCR product detection by 1% agarose gel electrophoresis, the target gene fragment was excised and recovered using a standard agarose gel DNA recovery kit. The fragment was stored at -20°C for later use. Amino acid metabolism gene ( NC8_RS11250 The sequence is (SEQ ID NO.1). The primers and sequences used are shown in Table 1, the PCR amplification reaction system is shown in Table 2, and the amplification reaction procedure is shown in Table 3.

[0025] Table 1 Primers and sequences used

[0026]

[0027] Table 2 PCR amplification reaction system

[0028]

[0029] Table 3 PCR amplification reaction procedure

[0030]

[0031] (2) Plasmid extraction and linearization

[0032] E. coli containing pMG36e plasmid ( Escherichia coli The inoculum was 2% in LB broth containing 600 µg / mL erythromycin, and cultured at 37 °C and 200 rpm until the logarithmic growth phase. The broth was then passaged twice, and plasmids were extracted using a plasmid miniprep kit. Xba I and Hind The pMG36e plasmid was double-digested with restriction endonucleases III. The digestion reaction was carried out at 37 °C for 20 min. After the reaction, the results were detected by 1% agarose gel electrophoresis. The target fragment was recovered using a standard agarose gel DNA recovery kit and stored at -20 °C for later use. The double digestion reaction system is shown in Table 4.

[0033] Table 4. Double enzyme digestion reaction system

[0034]

[0035] (3) Ligation of the target gene with the plasmid and transformation

[0036] The purified target gene was ligated to the linearized pMG36e plasmid using a one-step cloning kit. The reaction was carried out in a 37°C water bath for 30 min, and immediately placed on ice after the reaction. Subsequently... E. coli DH5α competent cells were thawed on ice. 100 μL of competent cells were gently mixed with 10 μL of plasmid and incubated on ice for 30 min. The ligation plasmid was then transformed into DH5α using a heat shock transformation method. E. coli DH5α competent cells were finally plated on LB agar medium containing 600 µg / mL erythromycin. After the cells grew on the plates, transformants were screened and verified.

[0037] (4) Screening and validation of Escherichia coli transformants

[0038] After picking single colonies using a sterile pipette tip, PCR verification of the transformants was performed using pMG36e-F and pMG36e-R primers. The correctly verified colonies were inoculated into LB broth containing a final concentration of 600 µg / mL erythromycin and cultured at 37 °C and 200 rpm until the logarithmic growth phase. The bacterial culture was then aspirated and washed twice with sterile physiological saline (0.85% NaCl, w / v) before DNA sequencing verification. Simultaneously, the *E. coli* transformant culture was preserved at -80 °C using the glycerol preservation method. The recombinant plasmid pMG36e-NC8_RS11250 was then successfully constructed. The PCR verification reaction system for the transformants is shown in Table 5, and the amplification reaction procedure is shown in Table 6.

[0039] Table 5 Transformant PCR Validation Reaction System

[0040]

[0041] Table 6 PCR Amplification Reaction Procedure

[0042]

[0043] (5) Extraction and transformation of recombinant plasmids

[0044] The plasmid containing recombinant plasmid pMG36e-NC8_RS11250 was added. E. coli DH5α strain was inoculated at a 2% inoculum into LB broth with a final concentration of 600 µg / mL erythromycin and cultured at 37 °C and 200 rpm until the logarithmic growth phase. Recombinant plasmids were extracted using a plasmid miniprep kit. 10 µL of plasmid was then mixed with 100 µL of erythromycin... L. plantarum SC-MDJ competent cells were gently mixed and incubated on ice for 5 min. Then, plasmids were transformed into the competent cells using an electroporator with a 1 mm spacing. The voltage was adjusted to 1.25 kV, and two consecutive electroporations were performed. Immediately after electroporation, 890 μL of pre-chilled MRS broth was added, and the cells were incubated at 30 °C for 2.5 h for recovery. After recovery, the bacterial culture was centrifuged and concentrated, then spread onto MRS agar containing 100 µg / mL erythromycin. The cells were incubated statically at 30 °C until bacterial growth was observed, followed by screening and validation.

[0045] (6) Screening and validation of gene overexpression strains

[0046] Single colonies were inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL using a sterile pipette tip. The colonies were incubated at 30 °C and 200 rpm until the logarithmic growth phase. The bacterial culture was aspirated and the cells were washed twice with sterile physiological saline (0.85% NaCl, w / v). PCR verification was performed using pMG36e-F and pMG36e-R primers. The correctly verified bacterial culture was then re-inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL and incubated at 30 °C and 200 rpm until the logarithmic growth phase. The bacterial culture was then stored at -80 °C using the glycerol preservation method. The overexpression of amino acid metabolism genes... L. plantarum The construction is now complete, named L. plantarum SC-MDJ-NC8_RS11250. The PCR validation reaction system is the same as in Table 5, and the amplification reaction procedure is the same as in Table 6.

[0047] Figure 1 This document outlines the construction process for overexpression plasmids of amino acid metabolism genes. Figure 2 For overexpression of amino acid metabolism genes L. plants PCR identification results. The successfully constructed recombinant plasmid pMG36e-NC8_RS11250 was extracted using a plasmid DNA extraction kit. E. coli Extracted from DH5α and converted to electroporation. L. plantarum In SC-MDJ, single colonies were picked from erythromycin-resistant MRS agar for PCR verification of transformants to check whether the recombinant plasmid had been successfully transformed into the target culture medium. L. plantarum In SC-MDJ, strains overexpressing amino acid metabolism genes were obtained. The results of 1% agarose gel electrophoresis are shown below. Figure 2 As shown, a single bright band appears at approximately 1245 bp, indicating good quality and successful transformation of the recombinant plasmid pMG36e-NC8_RS11250 to [the target region]. L. plantarum In SC-MDJ, strains overexpressing amino acid metabolism genes L. plantarum The SC-MDJ-NC8_RS11250 build is complete. Furthermore... L. plantarum SC-MDJ-NC8_RS11250 NC8_RS11250 The relative expression level of gene mRNA increased by 44.51 times, indicating successful gene overexpression. The results are shown in the figure below. Figure 3 As shown.

[0048] Example 2. Improvement L. plantarum Determination of the cold resistance of SC-MDJ-NC8_RS11250

[0049] (1) Preparation of fermentation agent

[0050] overexpression strains L. plantarumSC-MDJ-NC8_RS11250 (experimental group) and control strain L. plants SC-MDJ was inoculated at a 2% inoculum into MRS broth containing erythromycin at a final concentration of 10 µg / mL, cultured at 30 °C and 200 rpm until the logarithmic growth phase, and passaged twice. The cultures were then collected.

[0051] (2) Establishment of fermentation system

[0052] The culture was inoculated into MRS broth containing a final concentration of 10 µg / mL erythromycin for inoculation. L. plants The SC-MDJ fermentation system was used as a control group and inoculated. L. plantarum The fermentation system of SC-MDJ-NC8_RS11250 was used as the experimental group and fermented for 6 days at 15 °C and 200 rpm.

[0053] (3) Determination of fermentation agent growth, pH and antioxidant capacity

[0054] After collecting the cultures, the OD was measured using a spectrophotometer. 600nm To evaluate the growth capacity of the strain, the pH of the fermentation broth was measured using a pH meter to assess the acid production capacity of the strain, and the total antioxidant capacity (OD) of the strain was determined using a total antioxidant capacity assay kit. 600nm Antioxidant capacity = 0.8).

[0055] Table 7 L. plantarum OD of SC-MDJ-NC8_RS11250 600nm pH and antioxidant capacity

[0056]

[0057] Note: Different lowercase letters in the vertical column indicate significant differences between treatments. P <0.05).

[0058] Table 7 is... L. plantarum OD of SC-MDJ-NC8_RS11250 600nm Results of pH and antioxidant capacity assays were obtained. The experimental results showed that inoculation with organisms overexpressing amino acid metabolism genes... L. plantarum Fermentation of SC-MDJ-NC8_RS11250 under low-temperature conditions can increase the OD of the strain. 600nmThe pH of the protein was increased to 1.895, a 33.17% improvement over the control group; simultaneously, the pH of the fermentation system was reduced to 4.51, a 9.26% decrease compared to the control group; and the antioxidant capacity reached 21.86 U / mg protein, an 81.86% increase compared to the control group. Therefore, these results demonstrate that overexpression of amino acid metabolism genes can significantly improve… L. plantarum The growth, acid production, and antioxidant capabilities of SC-MDJ in low-temperature environments endow the starter culture with unique physiological functions and enhance the strain's ability to resist low-temperature oxidative stress. This not only helps to increase the fermentation speed of low-temperature fermented vegetable products such as sauerkraut, but also improves the flavor, quality, and safety of the products, and reduces resource waste in the food production process.

[0059] Example 3. L. plantarum Determination of total acid and degradation nitrite levels during fermentation using SC-MDJ-NC8_RS11250

[0060] L. plantarum Determination of total acidity in fermentation production using SC-MDJ-NC8_RS11250:

[0061] (1) Establishment of fermentation system

[0062] 1 mg / mL sodium nitrite was added to MRS broth containing 10 µg / mL erythromycin. The culture medium without inoculation with the fermenting agent was used as a control. The overexpressing strain... L. plantarum SC-MDJ-NC8_RS11250 was used as the inoculum for fermentation. Fermentation was carried out at 15 °C and 200 rpm for 4 days, and the total acid content in the fermentation system was measured.

[0063] (2) Determination of total acid content

[0064] Take 10.0 mL of fermentation broth, add purified water, and bring the volume to 100 mL. Filter the broth through gauze to remove impurities. Collect 50 mL of the filtrate and titrate with 0.1 mol / L NaOH, adding 2 drops of 10 g / L phenolphthalein indicator. Record the volume of NaOH solution (V1) when the solution turns slightly pink and the volume of NaOH solution consumed when using purified water instead of the sample solution (V2).

[0065] (3) The formula for calculating the total acid content (mg / mL) is as follows:

[0066]

[0067] Note: c: Concentration of sodium hydroxide standard titration solution, in mol / L; V1: Volume of sodium hydroxide standard titration solution consumed in titrating the test solution, in milliliters (mL); V2: Volume of sodium hydroxide standard titration solution consumed in the blank test, in milliliters (mL); k: Conversion factor for acid (calculated for lactic acid, 0.090); F: Dilution factor of the solution; m: Mass of the sample, in milliliters (mL); 1000: Conversion factor.

[0068] L. plantarum Determination of nitrite degradation by fermentation using SC-MDJ-NC8_RS11250:

[0069] (1) Establishment of fermentation system

[0070] 1 mg / mL sodium nitrite was added to MRS broth containing 10 µg / mL erythromycin. The culture medium without inoculation with the fermenting agent was used as a control. The overexpressing strain... L. plantarum SC-MDJ-NC8_RS11250 was used as the inoculum. Fermentation was carried out at 15 °C and 200 rpm for 4 days, and the sodium nitrite content in the fermentation system was measured.

[0071] (2) Determination of nitrite content

[0072] The nitrite content in water and soil was determined using a kit for determining nitrite content in water and soil (Grace, Suzhou, China).

[0073] (3) The formula for calculating the nitrite degradation rate is as follows:

[0074]

[0075] Note: X1 is the sodium nitrite content in the control culture medium, in milligrams per milliliter (mg / mL); X2 is the sodium nitrite content in the inoculated fermentation medium, in milligrams per milliliter (mg / mL).

[0076] Table 8 L. plantarum Total acid and sodium nitrite content in the SC-MDJ-NC8_RS11250 fermentation system

[0077]

[0078] Note: Different lowercase letters in the vertical column indicate significant differences between samples. P <0.05).

[0079] Table 8 is... L. plantarum Results of total acid and sodium nitrite content determination in the SC-MDJ-NC8_RS11250 fermentation system. The experimental results indicate that inoculation with a gene overexpressing amino acid metabolism... L. plantarumFermentation with SC-MDJ-NC8_RS11250 at 15°C increased the total acid content in the fermentation system to 12.27 mg / mL, while simultaneously reducing the sodium nitrite content to 0.020 mg / mL, achieving a nitrite degradation rate of 98.00%. The increased total acid content enhances the texture and flavor of the food, thus contributing to the development of a pleasant taste. Therefore, L. plantarum The SC-MDJ-NC8_RS11250 starter culture also enhances the flavor of fermented foods. Compared to medium-temperature fermentation, it can significantly improve the acid production and nitrite degradation capabilities of the starter culture at lower temperatures. This not only helps improve the quality and safety of low-temperature fermented vegetable foods such as sauerkraut, but also greatly increases the fermentation speed and reduces resource waste during the production process.

[0080] Example 4. L. plantarum Determination of lactic acid production capacity of SC-MDJ-NC8_RS11250

[0081] (1) Preparation of fermentation agent

[0082] overexpression strains L. plantarum SC-MDJ-NC8_RS11250 and control strains L. plantarum SC-MDJ was inoculated at a 2% inoculum into MRS broth containing erythromycin at a final concentration of 10 µg / mL, cultured at 30 °C and 200 rpm until the logarithmic growth phase, and passaged twice. The cultures were then collected.

[0083] (2) Establishment of fermentation system

[0084] The culture was inoculated into MRS broth containing a final concentration of 10 µg / mL erythromycin for inoculation. L. plants The SC-MDJ fermentation system was used as a control group and inoculated. L. plantarum The fermentation system of SC-MDJ-NC8_RS11250 was used as the experimental group and fermented for 6 days at 15 °C and 200 rpm.

[0085] (3) Lactic acid production capacity determination

[0086] After collecting the fermentation broth, the lactic acid content in the fermentation broth was determined using high performance liquid chromatography (HPLC) to evaluate the lactic acid production capacity of the strain under low temperature conditions.

[0087] Table 9 L. plantarum Lactic acid production capacity of SC-MDJ-NC8_RS11250

[0088]

[0089] Note: Different lowercase letters in the vertical column indicate significant differences between treatments. P <0.05).

[0090] Table 9 is... L. plantarum Results of lactic acid content determination in the SC-MDJ-NC8_RS11250 fermentation system. The experimental results indicate that inoculation with a gene overexpressing amino acid metabolism... L. plantarum Fermentation of SC-MDJ-NC8_RS11250 at 15 °C increased the lactic acid content in the fermentation system to 11.11 mg / mL, a 35.16% increase compared to the control group. This increased lactic acid content enhances the unique sour taste of the food, thus contributing to the development of a pleasant flavor. Therefore, L. plants The SC-MDJ-NC8_RS11250 starter culture also enhances the flavor of fermented foods, helping to improve the quality and safety of low-temperature fermented vegetable products such as sauerkraut, and promoting product fermentation and maturation. Furthermore, given that microbial fermentation can efficiently produce the industrial chemical lactic acid while reducing environmental pollution and resource waste, it is conducive to industrial application.

Claims

1. A recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, Using *Lactobacillus plantarum* SC-MDJ as the starting strain, the nucleic acid molecule shown in SEQ ID NO.1 was overexpressed; the *Lactobacillus plantarum* SC-MDJ, with accession number CGMCC NO.28112, is deposited at the China General Microbiological Culture Collection Center on August 7, 2023, at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation containing the recombinant Lactobacillus plantarum as described in claim 1.

3. A breeding method for improving the antioxidant capacity of *Lactobacillus plantarum*, characterized in that, The nucleic acid molecule shown in SEQ ID NO.1 is overexpressed in *Lactobacillus plantarum* SC-MDJ according to claim 1.

4. A breeding method for improving the cold tolerance of *Lactobacillus plantarum*, characterized in that, The nucleic acid molecule shown in SEQ ID NO.1 is overexpressed in *Lactobacillus plantarum* SC-MDJ according to claim 1.

5. The application of the recombinant Lactobacillus plantarum according to claim 1 in cold-resistant fermentation, nitrite-reducing fermentation, antioxidant fermentation, or lactic acid-producing fermentation.

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