Recombinant plant lactobacillus and application thereof

Abstract

The invention discloses recombinant plant lactobacillus and application thereof, and belongs to the technical field of bioengineering. The invention aims to improve the cold-resistant growth capability and the oxidation resistance of the plant lactobacillus, and increase the capability of producing lactic acid and degrading nitrite. The invention provides a recombinant lactobacillus plantarum, which is characterized in that the lactobacillus plantarum is used as an original strain, and a nucleic acid molecule as shown in SEQ ID NO.1 is overexpressed. The method is used for fermenting products in food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Lactobacillus plantarum and an application thereof. Background Art

[0002] Low-temperature fermented foods are of high quality, with a softer, more delicious taste and no odor. However, the microorganisms in the fermentation system are often affected by low-temperature stress, which reduces the fermentation performance of the microorganisms and thus affects the product quality. Therefore, the present invention provides a method for constructing a recombinant Lactobacillus plantarum that overexpresses amino acid metabolism genes, and is used to improve growth, fermentation acid production and nitrite degradation capabilities under low-temperature conditions, in order to improve 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 tolerance, it can remain active at low temperatures, helping to maintain the fermentation characteristics, flavor, and safety of the product. Cold-tolerant Lactobacillus plantarum can be applied to a wider range of fermentation or health food production processes requiring low temperatures, such as refrigerated fermented foods and probiotic preparations stored at low temperatures, expanding its industrial application potential.

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

[0005] In the food industry, the antioxidant properties of Lactobacillus plantarum can delay food oxidative spoilage and extend 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. Lactobacillus plantarum strains with these capabilities are urgently needed. Summary of the Invention

[0006] The purpose of the invention is to improve the cold-resistant growth ability and antioxidant capacity of Lactobacillus plantarum and increase the ability to produce lactic acid and degrade nitrite.

[0007] The present invention provides a recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum ), using Lactobacillus plantarum SC-MDJ as the starting strain, overexpressing the nucleic acid molecule shown in SEQ ID NO.1; the Lactobacillus plantarum SC-MDJ has the accession number CGMCC NO.28112, is deposited in the China General Microorganism Collection Center, the deposit date is August 7, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

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

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

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

[0011] A breeding method for improving the nitrite degradation ability of Lactobacillus plantarum, comprising overexpressing the gene shown in SEQ ID NO. 1 in Lactobacillus plantarum SC-MDJ.

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

[0013] Beneficial effect: Inoculation with overexpression of amino acid metabolism genes L. plantarum Fermentation of SC-MDJ-NC8_RS11250 under low temperature conditions can increase the OD 600nm The pH value of the fermentation system was increased to 1.895, which was 33.17% higher than that of the control group. At the same time, the pH value of the fermentation system was reduced to 4.51, which was 9.26% lower than that of the control group. The antioxidant capacity was 21.86 U / mg protein, which was 81.86% higher than that of the control group. Therefore, the above results show 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 environment endow the starter with unique physiological functions and improve the strain's ability to resist low temperature oxidative stress; Inoculation with overexpression of amino acid metabolism genes L. plantarum Fermentation of 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, and achieving a nitrite degradation rate of 98.00%. This increase in total acid content enhances the taste and flavor of food, thereby promoting the development of a favorable flavor profile. Inoculation with overexpression of amino acid metabolism genes 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. This increase in lactic acid content can enhance the unique sour taste of foods, thereby promoting the development of a favorable flavor.

[0014] [Biological preservation information]: Lactobacillus plantarum is named Lactobacillus plantarum SC-MDJ, the preservation number is CGMCCNO.28112, and it is deposited in the China General Microorganism Collection Center. The preservation date is August 7, 2023. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The construction process of amino acid metabolism gene overexpression plasmid; Figure 2 Overexpression of amino acid metabolism genes L. plantarum PCR identification results; Figure 3 This is the result graph of the relative expression level of NC8_RS11250 gene mRNA. DETAILED DESCRIPTION

[0016] MRS broth medium: peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH = 5.7 ± 0.2.

[0017] LB broth medium: 10.0 g tryptone, 5.0 g yeast extract powder, 10.0 g sodium chloride, 1000 mL distilled water, pH = 7.0 ± 0.1.

[0018] Example 1. Method for constructing recombinant Lactobacillus plantarum (1) Extraction of genomic DNA and acquisition of amino acid metabolism genes Lactobacillus plantarum ( Lactiplantibacillus plantarum SC-MDJ was inoculated into MRS broth at a 2% inoculum volume and cultured at 30 °C, 200 rpm until the logarithmic growth phase and passaged twice. Then, genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit. L. plantarum Reference genes NC8_RS11250 Sequence design primers were used for PCR amplification. After the PCR product was detected by 1% agarose gel electrophoresis, the target gene fragment was cut out and recovered using a spin column type ordinary agarose gel DNA recovery kit and stored in a -20°C refrigerator until use. NC8_RS11250 ) 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.

[0019] Table 1 Primers and sequences used

[0020] Table 2 PCR amplification reaction system

[0021] Table 3 PCR amplification reaction program

[0022] (2) Plasmid extraction and linearization Escherichia coli ( Escherichia coli) was inoculated into LB broth containing erythromycin at a final concentration of 600 μg / mL at a 2% inoculum volume, cultured at 37 °C, 200 rpm until the logarithmic growth phase and passaged twice, and then the plasmid was extracted using a plasmid extraction kit. Xba I and Hind The pMG36e plasmid was double-digested with the restriction endonuclease III. The digestion reaction was carried out in a 37°C water bath for 20 min. After completion of the reaction, the fragment was detected by 1% agarose gel electrophoresis. The target fragment was recovered using a spin column-based agarose gel DNA recovery kit and stored at -20°C until use. The double-digestion reaction system is shown in Table 4.

[0023] Table 4 Double enzyme digestion reaction system

[0024] (3) Ligation and transformation of target gene and plasmid The purified target gene was ligated with the linearized pMG36e plasmid using a one-step cloning kit, reacted in a 37°C water bath for 30 min, and immediately placed on ice after the reaction. E. coli DH5α competent cells were placed on ice for slowing down. 100 μL competent cells were gently mixed with 10 μL plasmid and placed on ice for 30 min. The ligated plasmid was transformed into E. coli Finally, the DH5α competent cells were plated on LB agar medium containing 600 µg / mL erythromycin. After colonies grew on the plate, transformants were screened and verified.

[0025] (4) Screening and verification of E. coli transformants After picking individual colonies with a sterile pipette tip, transformants were verified by PCR using primers pMG36e-F and pMG36e-R. Correctly identified colonies were inoculated into LB broth containing a final concentration of 600 µg / mL erythromycin and cultured at 37°C, 200 rpm, until the logarithmic growth phase. The bacterial suspension was aspirated and washed twice with sterile saline (0.85% NaCl, w / v) before DNA sequencing. The E. coli transformant suspension was stored in a glycerol freezer at -80°C, completing the construction of the recombinant plasmid pMG36e-NC8_RS11250. The PCR verification reaction system for transformants is shown in Table 5, and the amplification reaction procedure is shown in Table 6.

[0026] Table 5 Transformant PCR verification reaction system

[0027] Table 6 PCR amplification reaction program

[0028] (5) Extraction and transformation of recombinant plasmid The recombinant plasmid pMG36e-NC8_RS11250 was E. coli DH5α strain was inoculated into LB broth with a final concentration of 600 μg / mL erythromycin at a 2% inoculum volume and cultured at 37 °C, 200 rpm until the logarithmic growth phase. The recombinant plasmid was extracted using a plasmid extraction kit. 10 μL of plasmid was mixed with 100 μL of L. plantarum Gently mix the SC-MDJ competent cells and incubate on ice for 5 minutes. Then, use an electroporator to transform the plasmid into the competent cells. Using a 1 mm gap cuvette, adjust the voltage to 1.25 kV and perform two consecutive electroporations. Immediately add 890 μL of pre-chilled MRS broth and incubate at 30°C for 2.5 hours. After completion, concentrate the culture solution by centrifugation and spread it on MRS agar containing 100 μg / mL erythromycin. Incubate at 30°C until colonies emerge for screening and verification.

[0029] (6) Screening and verification of gene overexpression strains A single colony was inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin using a sterile pipette tip and cultured at 30 °C, 200 rpm until the logarithmic growth phase. The bacterial liquid was aspirated and washed twice with sterile saline (0.85% NaCl, w / v). PCR verification was performed using pMG36e-F and pMG36e-R primers. The verified bacterial liquid was re-inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin and cultured at 30 °C, 200 rpm until the logarithmic growth phase. The bacterial liquid was stored in a -80 °C freezer using the glycerol preservation method. L. plantarum The construction is completed and named L. plantarum SC-MDJ-NC8_RS11250. The PCR verification reaction system is the same as in Table 5, and the amplification reaction procedure is the same as in Table 6.

[0030] Figure 1 The process for constructing amino acid metabolism gene overexpression plasmids. Figure 2 Overexpression of amino acid metabolism genes L. plants The PCR identification results were obtained. The successfully constructed gene recombinant plasmid pMG36e-NC8_RS11250 was extracted from E. coli DH5α was extracted and transformed into 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 was successfully transformed into L. plantarumSC-MDJ was used to obtain amino acid metabolism gene overexpression strains. The results of 1% agarose gel electrophoresis were as follows: Figure 2 As shown, there is a single bright band at about 1245 bp, which is of good quality, indicating that the recombinant plasmid pMG36e-NC8_RS11250 was successfully transformed into L. plantarum SC-MDJ, amino acid metabolism gene overexpression strain L. plantarum SC-MDJ-NC8_RS11250 is built. In addition, L. plantarum SC-MDJ-NC8_RS11250 NC8_RS11250 The relative expression of gene mRNA increased by 44.51 times, which also shows that the gene was successfully overexpressed. The results are shown in the figure below. Figure 3 shown.

[0031] Example 2. Improvement L. plantarum Determination of cold resistance of SC-MDJ-NC8_RS11250 (1) Preparation of starter culture Overexpression strains L. plantarum SC-MDJ-NC8_RS11250 (experimental group) and control strains L. plants SC-MDJ were inoculated into MRS broth containing erythromycin at a final concentration of 10 μg / mL at a 2% inoculum volume, cultured at 30 °C, 200 rpm until the logarithmic growth phase, subcultured twice, and the cultures were collected.

[0032] (2) Establishment of fermentation system The culture was inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL to inoculate L. plants The SC-MDJ fermentation system was used as the control group and inoculated L. plantarum The fermentation system of SC-MDJ-NC8_RS11250 was used as the experimental group and fermented at 15 °C and 200 rpm for 6 days.

[0033] (3) Determination of starter culture growth, pH, and antioxidant capacity After the culture was collected, the OD was measured using a spectrophotometer. 600nm To evaluate the growth ability of the strain, the pH of the fermentation broth was measured using a pH meter to evaluate the acid production ability of the strain, and the total antioxidant capacity assay kit was used to determine the strain (OD 600nm =0.8) of antioxidant capacity.

[0034] Table 7 L. plantarum OD of SC-MDJ-NC8_RS11250 600nm , pH and antioxidant capacity

[0035] Note: Different lowercase letters in the vertical row indicate significant differences among treatments ( P <0.05).

[0036] Table 7 is L. plantarum OD of SC-MDJ-NC8_RS11250 600nm , pH and antioxidant capacity test results. The test results showed that L. plantarum Fermentation of SC-MDJ-NC8_RS11250 under low temperature conditions can increase the OD 600nm The pH value of the fermentation system was increased to 1.895, which was 33.17% higher than that of the control group. At the same time, the pH value of the fermentation system was reduced to 4.51, which was 9.26% lower than that of the control group. The antioxidant capacity was 21.86 U / mg protein, which was 81.86% higher than that of the control group. Therefore, the above results show that overexpression of amino acid metabolism genes can significantly improve L. plantarum SC-MDJ's growth, acid production and antioxidant capabilities in low-temperature environments give the fermentation agent unique physiological functions and improve the strain's ability to resist low-temperature oxidative stress. This not only helps to increase the fermentation rate 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.

[0037] Example 3. L. plantarum Determination of total acid and nitrite degradation levels in fermentation of SC-MDJ-NC8_RS11250 L. plantarum Determination of total acid produced by fermentation of SC-MDJ-NC8_RS11250: (1) Establishment of fermentation system 1 mg / mL sodium nitrite was added to MRS broth containing erythromycin at a final concentration of 10 μg / mL, and the culture medium without inoculation of starter culture was used as a control. L. plantarum SC-MDJ-NC8_RS11250 was the inoculated starter group, and fermentation was carried out at 15 °C and 200 rpm for 4 days to determine the total acid content in the fermentation system.

[0038] (2) Determination of total acid content Pipette 10.0 mL of fermentation broth and add purified water to a volume of 100 mL. Filter impurities through gauze. Collect 50 mL of filtrate and titrate with 0.1 mol / L NaOH. Simultaneously, add two drops of 10 g / L phenolphthalein indicator. Record the volume of NaOH solution when the solution reaches a slightly reddish color (V1) and the volume of NaOH solution consumed by replacing the sample solution with purified water (V2).

[0039] (3) The total acid content (mg / mL) is calculated as follows:

[0040] Note: c: concentration of sodium hydroxide standard titrant, in moles per liter (mol / L); V1: volume of sodium hydroxide standard titrant consumed in titrating the test solution, in milliliters (mL); V2: volume of sodium hydroxide standard titrant consumed in the blank test, in milliliters (mL); k: acid conversion factor (calculated based on lactic acid, 0.090); F: dilution factor of the solution; m: mass of the sample, in milliliters (mL); 1000: conversion factor.

[0041] L. plantarum Determination of nitrite degradation by fermentation of SC-MDJ-NC8_RS11250: (1) Establishment of fermentation system 1 mg / mL sodium nitrite was added to MRS broth containing erythromycin at a final concentration of 10 μg / mL, and the culture medium without inoculation of starter culture was used as a control. L. plantarum SC-MDJ-NC8_RS11250 was the inoculated starter group. Fermentation was carried out at 15 °C and 200 rpm for 4 days, and the sodium nitrite content in the fermentation system was measured.

[0042] (2) Determination of nitrite content Nitrite content was determined using a nitrite content assay kit for soil and water (Greis, Suzhou, China).

[0043] (3) The calculation formula of nitrite degradation rate is as follows:

[0044] 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 starter culture medium, in milligrams per milliliter (mg / mL).

[0045] Table 8 L. plantarum Total acid and sodium nitrite contents in SC-MDJ-NC8_RS11250 fermentation system

[0046] Note: Different lowercase letters in the vertical row indicate significant differences between samples ( P <0.05).

[0047] Table 8 is L. plantarumThe results of total acid and sodium nitrite content determination in the SC-MDJ-NC8_RS11250 fermentation system showed that the inoculated L. plantarum Fermentation of SC-MDJ-NC8_RS11250 at 15°C increased the total acid content in the fermentation system to 12.27 mg / mL, while reducing the sodium nitrite content to 0.020 mg / mL, and achieving a nitrite degradation rate of 98.00%. The increase in total acid content can enhance the taste and flavor of food, thereby promoting the formation of a good flavor in food. L. plantarum The SC-MDJ-NC8_RS11250 starter culture also enhances the flavor of fermented foods. Compared with medium-temperature fermentation, it can significantly improve the acid production and nitrite degradation capabilities of the starter culture under lower temperature conditions. 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 of the product and reduces resource waste in the production process.

[0048] Example 4. L. plantarum Lactate production capacity assay of SC-MDJ-NC8_RS11250 (1) Preparation of starter culture Overexpression strains L. plantarum SC-MDJ-NC8_RS11250 and control strains L. plantarum SC-MDJ were inoculated into MRS broth containing erythromycin at a final concentration of 10 μg / mL at a 2% inoculum volume, cultured at 30 °C, 200 rpm until the logarithmic growth phase, subcultured twice, and the cultures were collected.

[0049] (2) Establishment of fermentation system The culture was inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL to inoculate L. plants The SC-MDJ fermentation system was used as the control group and inoculated L. plantarum The fermentation system of SC-MDJ-NC8_RS11250 was used as the experimental group and fermented at 15 °C and 200 rpm for 6 days.

[0050] (3) Determination of lactic acid production capacity After the fermentation broth was collected, the lactic acid content in the fermentation broth was determined using a high performance liquid chromatography (HPLC) to evaluate the lactic acid production ability of the strain under low temperature conditions.

[0051] Table 9 L. plantarum Lactic acid production capacity of SC-MDJ-NC8_RS11250

[0052] Note: Different lowercase letters in the vertical row indicate significant differences among treatments ( P <0.05).

[0053] Table 9 is L. plantarum The results of the lactic acid content determination in the SC-MDJ-NC8_RS11250 fermentation system showed that the inoculated 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. The increase in lactic acid content can increase the unique sour taste of food, thereby promoting the formation of good food flavor. 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 foods like sauerkraut and promoting product maturation. Furthermore, since microbial fermentation can efficiently produce lactic acid, an industrial chemical, while also reducing environmental pollution and resource waste, it is promising for industrial applications.

Claims

1. A recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, Lactobacillus plantarum SC-MDJ is used as the starting strain to overexpress the nucleic acid molecule shown in SEQ ID NO.1; the Lactobacillus plantarum SC-MDJ has a deposit number of CGMCC NO.28112 and is deposited in the China General Microorganism Collection Center on August 7, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation containing the recombinant Lactobacillus plantarum according to 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 was overexpressed in Lactobacillus plantarum SC-MDJ.

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 was overexpressed in Lactobacillus plantarum SC-MDJ.

5. Use of the recombinant plant lactobacillus according to claim 1 in cold-resistant fermentation, nitrite-reducing fermentation, antioxidant fermentation or lactic acid-producing fermentation.

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