Lactobacillus plantarum strain NZ-4 and its application

Abstract

The present invention discloses a strain of Lactobacillus plantarum NZ-4, which was deposited with the Guangdong Provincial Microbial Culture Collection Center on December 14, 2023, with a deposit number of GDMCC NO: 64145. The advantages of the present invention are: Lactobacillus plantarum NZ-4 has high lactic acid production, strong acid and bile salt resistance, and the ability to inhibit pathogens. It has broad application prospects in extending the shelf life of food and regulating intestinal flora in food or health products.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to a strain of Lactobacillus plantarum with high lactic acid production and application thereof. Background Art

[0002] Yunnan's unique geographical environment and diverse cultural advantages have created a rich resource of fermented foods, and these traditional fermented foods contain a wealth of microbial resources. Related research has shown that Yunnan's traditional fermented foods contain a variety of lactic acid bacteria, including certain species of Lactobacillus plantarum, which have certain immunomodulatory effects, inhibit certain pathogens, maintain intestinal flora balance, and inhibit the formation of tumor cells.

[0003] When humans consume common organic acids such as lactic acid, acetic acid, citric acid, malic acid, oxalic acid, and tartaric acid, they can regulate the balance of intestinal flora through competitive inhibition, thereby reducing the incidence of gastrointestinal diseases. Furthermore, studies have shown that the primary target of organic acids against bacteria is the cell wall, and their inhibitory mechanism involves disruption of pathogen cell structure, the outflow of intracellular substances, and cell death. Therefore, obtaining probiotics that can produce high levels of organic acids such as lactic acid is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a plant lactobacillus with high lactic acid production.

[0005] The present invention provides a Lactobacillus plantarum NZ-4. The Lactobacillus plantarum NZ-4 (Lactiplantibacillus plantarum NZ-4) has been deposited in Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with a deposit number of GDMCC NO: 64145.

[0006] The present invention provides a bacterial agent comprising Lactobacillus plantarum NZ-4.

[0007] The present invention provides application of Lactobacillus plantarum NZ-4 or a bacterial agent in the preparation of an antibacterial product.

[0008] Furthermore, the product is a preservative, antistaling agent or food additive.

[0009] The present invention provides application of Lactobacillus plantarum NZ-4 or a bacterial agent in preparing a product for regulating intestinal flora.

[0010] Furthermore, the product is a food or a health product.

[0011] Beneficial effects of the present invention:

[0012] The present invention isolates and screens Lactobacillus plantarum NZ-4 from milk residue samples in Shangri-La, Yunnan Province. The strain has high lactic acid production, strong acid and bile salt resistance, and the ability to inhibit pathogens. Experimental results show that the survival rate of Lactobacillus plantarum NZ-4 after 3-hour acid treatment is 97.16%, and its survival rate after 3-hour bile salt treatment is 98.58%. The lactic acid content in the fermentation broth after 24 hours is 21.3218 g / L. The strain has the best inhibitory effect on Escherichia coli, with an inhibition zone size of 53 mm. It also has relatively good inhibitory effects on Staphylococcus aureus and Candida albicans, with an inhibition zone size of 20 mm for Staphylococcus aureus and 17 mm for Candida albicans. Therefore, the Lactobacillus plantarum of the present invention has broad application prospects in extending the shelf life of food and regulating intestinal flora in food or health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the colony morphology of Lactobacillus plantarum NZ-4;

[0014] Figure 2 This is the morphological picture of Lactobacillus plantarum NZ-4 under a microscope;

[0015] Figure 3 is the phylogenetic tree of Lactobacillus plantarum NZ-4;

[0016] Figure 4 The graph shows the antibacterial effects of Lactobacillus plantarum NZ-4 on Escherichia coli, Staphylococcus aureus, and Candida albicans from left to right. DETAILED DESCRIPTION

[0017] The following is a further description of specific embodiments of the present invention in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0018] The present invention provides a Lactobacillus plantarum NZ-4, which is isolated from milk residue in Shangri-La, Yunnan Province. Lactobacillus plantarum NZ-4 (Lactiplantibacillus plantarum NZ-4) has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with a deposit number of GDMCC NO: 64145; the deposit address is: 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; Contact number: 020-87137633.

[0019] The present invention provides a bacterial agent comprising Lactobacillus plantarum NZ-4 and other auxiliary materials.

[0020] The present invention provides application of Lactobacillus plantarum NZ-4 or a bacterial agent in the preparation of an antibacterial product.

[0021] Preferably, the product is a preservative, a freshness-preserving agent or a food additive.

[0022] The present invention provides application of Lactobacillus plantarum NZ-4 or a bacterial agent in preparing a product for regulating intestinal flora.

[0023] Preferably, the product is a food or a health product.

[0024] The following is a detailed description of the strain Lactobacillus plantarum NZ-4 and its applications provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Isolation, Purification, Identification and Preservation of Lactobacillus plantarum NZ-4

[0027] 1. Isolation, purification and initial screening of acid production capacity of strains

[0028] Lactobacillus plantarum NZ-4 was isolated from milk dregs samples in Shangri-La, Yunnan Province. The method is as follows: under sterile conditions, accurately weigh 1.0g of milk dregs sample in 5mL of normal saline, shake thoroughly to elute attached microorganisms, take 20μL of the eluate and inoculate it into 5mL of MRS liquid medium, and incubate anaerobically in a 37℃ incubator for 24 hours to enrich the microorganisms. Then, take 100μL of the enriched bacterial solution and dilute it in 10-fold gradients with 0.9% sterile normal saline until it reaches 10 -6 Take 100 μL and dilute it to 10 -4 , 10 -5 , 10 -6 The dilutions were spread on MRS modified agar medium and cultured anaerobically at 37℃ for 48h.

[0029] The formulas of the MRS liquid culture medium and the MRS modified agar culture medium are as follows:

[0030] MRS liquid medium (Oxoid LTD, pH 6.2±0.2): 10 g protein Chen, 8 g beef powder, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 1000 mL distilled water were mixed, the pH was adjusted to 6.2±0.2, and sterilized at 115°C for 20 min.

[0031] MRS modified agar medium: Add 15 g agar, 10 g CaCO3 and 0.004% (w / v) bromocresol purple to each liter of MRS liquid medium and sterilize at 115°C for 20 min.

[0032] After the incubation period, sterilized toothpicks were used to select pure white or milky colonies from each plate. These colonies displayed a distinct calcium-solubility ring, a smooth surface, a raised center, and distinct edges. Gram staining and catalase testing were performed. Following preliminary identification, molecular biological analysis was performed, ultimately resulting in the selection of nine lactic acid bacteria strains with distinct calcium-solubility rings. These nine strains were preliminarily determined to possess acid-producing abilities: YM-5-1, NZ-4, LC-1-2, LC-2-3, LC-8-6, GJ-5-1, GJ-6-2, ZT-1-1, and ZT-1-2.

[0033] The above 9 single colonies were inoculated into 5 mL of sterilized MRS liquid culture medium, cultured at 37°C for 48 h, and then stored in a -80°C refrigerator with 50% glycerol for later use.

[0034] 2. Morphological Identification of Strain

[0035] The isolated NZ-4 strain was inoculated into MRS solid medium and cultured at 37°C for 24 hours. The colony characteristics of the NZ-4 strain were as follows: round, convex in the middle, smooth on the surface, white and occasionally light yellow. Figure 1 Gram staining results showed that NZ-4 was round-ended and straight, single, paired or short chain, usually lacking flagella, but able to move, such as Figure 2 Gram staining results showed that NZ-4 was a Gram-positive bacterium, and catalase test results were negative.

[0036] MRS solid medium: 10 g peptone, 8 g beef powder, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar; dilute to 1000 mL with distilled water, adjust the pH to 6.2 ± 0.2, and sterilize at 115°C for 20 min.

[0037] 3. Molecular Biological Identification of Strains

[0038] The selected target strain was inoculated into MRS liquid culture medium and cultured at 37°C with a shaker at 180 rpm for 24 hours. The strain genome was extracted using a bacterial DNA extraction kit and used as a template for PCR amplification. The forward primer was 27F (5'-AGAGTTTGATCCTGGCTAG-3') and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3'). The reaction system consisted of 25 μL of BGI2×Super PCR Mix, 1 μL of forward primer, 1 μL of reverse primer, 1 μL of template, and 2 μL of ddH2O2. Amplification conditions were: initial denaturation at 94°C for 4 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 5 minutes. The amplified products were subjected to 1.5% agarose gel electrophoresis and sent to a bioengineering company (Shanghai) for sequencing. The sequences were compared with the NCBI database and a phylogenetic tree was constructed using the phylogenetic tree software MEGA 7.0. The phylogenetic tree is shown in Figure 2. Figure 3 As shown, the sequence homology between NZ-4 and the standard strain of Lactobacillus plantarum was greater than 97%, and the strain NZ-4 was confirmed to be Lactobacillus plantarum.

[0039] 4. Rescreening of strains with high lactic acid production capacity

[0040] The 9 acid-producing strains obtained in the initial screening were inoculated into sterilized MRS liquid culture medium, activated for two generations at 37°C, and then inoculated into 100 mL of MRS liquid culture medium at a 4‰ inoculum size. The culture was anaerobically cultured at 37°C for 24 h. 5 mL of the bacterial solution was disrupted with a cell disruptor, and 10 ml of 0.5% H3PO4 was added. Ultrasonic extraction was performed for 15 min, and the culture was allowed to cool to room temperature. The volume was adjusted to 25 ml with 0.5% H3PO4, and the culture was filtered through a 0.45 μm filter membrane. The filtrate was taken and its pH and lactic acid content were determined by high-performance liquid chromatography (HPLC), thereby screening out high-lactic acid-producing strains.

[0041] HPLC conditions: Agilent C18 column (5 m, 250×4.6 mm); mobile phase: 0.05% H 3 PO 4 : methanol (90:10); detection wavelength: 210 nm; flow rate: 1.0 ml / min; column temperature: 40° C.; injection volume: 10 μl.

[0042] Standard curve drawing: Use external standard method for quantification, dilute the prepared lactic acid standard solution into mixed standard solutions of 5, 20, 40, 80, and 120 mg / L, inject into liquid phase analysis, and draw the standard curve with peak area as the horizontal axis and concentration as the vertical axis.

[0043] The results of the lactic acid content are shown in the following table:

[0044]

[0045]

[0046] The results showed that Lactobacillus plantarum NZ-4 had the highest lactic acid content of 21.3218 g / L, while the production of the other strains generally ranged from 4.1 to 11.9 g / L, indicating that there were large differences in lactic acid content among the strains, indicating that NZ-4 has the ability to produce high lactic acid.

[0047] 3. Strain Preservation

[0048] Lactobacillus plantarum NZ-4 was deposited in the Guangdong Provincial Microbial Culture Collection Center on December 14, 2023. The address is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; Tel: 020-87137633.

[0049] Example 2

[0050] Analysis of acid and bile salt tolerance of Lactobacillus plantarum

[0051] 1. Acid resistance test

[0052] The strain was inoculated into MRS liquid medium at an inoculum size of 4‰ and activated for two generations at 37°C. The activated bacterial solution was inoculated into acidic MRS liquid medium with a pH of 6.2±0.2 (blank control group) and pH of 3, respectively, and cultured at 37°C for 3 hours. The viable bacteria were counted at 0 and 3 hours. The survival rate of the strain was calculated according to the following formula. The formula for calculating the survival rate of the strain is as follows:

[0053] Strain survival rate / % = N1 / N0×100

[0054] Where N1 is the number of viable bacteria after acid treatment for 3 h; N0 is the initial number of viable bacteria of the strain.

[0055] The calculation results showed that the initial concentration of Lactobacillus plantarum NZ-4 was 1.41×10^9 cfu / mL, and the final concentration of Lactobacillus fermentum after acid treatment for 3 hours was 1.37×10^9 cfu, and the calculated survival rate was 97.16%

[0056] 2. Bile salt tolerance test

[0057] After the strain is activated, it is inoculated into MRS liquid medium containing 0.3% and 0% (blank control) bile salts at an inoculum size of 4‰. After treatment at 37°C for 0 and 3 hours, the viable bacteria are counted on the plate at 0 and 3 hours. The survival rate is calculated as follows:

[0058] Strain survival rate / % = N1 / N0×100

[0059] Where N1 is the number of viable bacteria after culturing in MRS liquid medium containing bile salts for 3 hours; N0 is the initial number of viable bacteria of NZ-4 strain.

[0060] The results showed that the initial concentration of Lactobacillus fermentum was 1.41×10^9 cfu / mL, and the final concentration of Lactobacillus fermentum after 3 hours of bile salt treatment was 1.39×10^9 cfu. The calculated survival rate was 98.58%.

[0061] The experimental results show that Lactobacillus plantarum NZ-4 has strong probiotic properties such as acid resistance and bile salt resistance. Therefore, Lactobacillus plantarum NZ-4 can colonize in the intestines and participate in regulating the balance of intestinal microbial flora.

[0062] Example 3

[0063] Analysis of NZ-4's ability to produce other common organic acids

[0064] Lactobacillus plantarum NZ-4 was inoculated at a 4‰ inoculum into MRS liquid medium and activated for two generations at 37°C. After activation, the inoculum was inoculated at a 4‰ inoculum into 100 mL of MRS liquid medium and cultured for 24 hours. The fermentation supernatant was filtered through a 0.22 μm membrane and analyzed by high-performance liquid chromatography (HPLC).

[0065] Chromatographic conditions: chromatographic column: Aminex HPX-87H (300 mm × 7.8 mm), mobile phase: 5 mmol / L dilute sulfuric acid solution, isocratic elution, flow rate: 0.5 mL / min, detection wavelength: 210 nm, column temperature: 50 ° C, injection volume: 20 μL.

[0066] Organic acid standards (citric acid, tartaric acid, malic acid, and oxalic acid) with different concentrations of 1 to 500 μg / mL were prepared respectively. The peak areas were detected by HPLC. A standard curve of organic acids was drawn with the organic acid content as the abscissa and the peak area as the ordinate. The contents of citric acid, tartaric acid, malic acid, and oxalic acid in the fermentation broth were calculated based on the concentration and peak area of the standards. The results are shown in the table below:

[0067]

[0068] Results showed that the fermentation broth had a pH of 3.0, and Lactobacillus plantarum NZ-4, in addition to its high lactic acid production, also produced citric, oxalic, malic, and tartaric acids. Consumption of these organic acids can regulate the balance of intestinal flora through competitive inhibition, thereby reducing the incidence of gastrointestinal diseases. Furthermore, the accumulation of anions from the dissociation of organic acids in pathogens can cause cellular poisoning, interfere with glycolysis and nucleic acid synthesis, and ultimately lead to disruption of the membrane transduction system, inducing bacterial death.

[0069] Example 4

[0070] Analysis of the ability of Lactobacillus plantarum NZ-4 to inhibit foodborne pathogens

[0071] Antibacterial test

[0072] 1. The formulas of the culture media involved are as follows:

[0073] LB medium: peptone 10.0 g, yeast powder 5.0 g, sodium chloride 5.0 g, glucose 1.0 g, adjust the pH to 7.1 ± 0.1 (25°C), add distilled water to 1000 mL, and sterilize at 121°C for 15 min.

[0074] BHI medium: peptone 10.0 g, ox brain extract powder 12.5 g, ox heart extract powder 5.0 g, sodium chloride 5.0 g, glucose 2.0 g, disodium hydrogen phosphate 2.5 g, pH 7.4 ± 0.2 (25°C), dilute to 1000 mL with distilled water, and sterilize at 121°C for 15 min;

[0075] Sabouraud solid medium: peptone 10 g, agar 15 g, glucose 40 g, chloramphenicol 0.1 g, adjust the pH to 5.6 ± 0.2, add distilled water to 1000 mL, and sterilize at 115°C for 15 min.

[0076] Sabouraud liquid medium: peptone 10 g, glucose 40 g, chloramphenicol 0.1 g, adjust the pH to 5.6 ± 0.2, add distilled water to 1000 mL, and sterilize at 115°C for 15 min.

[0077] Nutrient agar medium: peptone 10 g, beef extract powder 3 g, sodium chloride 5 g, agar 15 g, dilute to 1000 mL with distilled water, adjust the pH to 7.3 ± 0.2, and sterilize at 115°C for 20 min.

[0078] 2. Test methods

[0079] (1) Activate and passage Lactobacillus plantarum NZ-4 stored at -80℃: Activate the Lactobacillus plantarum NZ-4 at a concentration of 1×10 9 CFU / mL of Lactobacillus plantarum NZ-4 was inoculated into sterilized MRS liquid medium at a volume fraction of 4‰. After constant temperature static culture at 37℃ for 24h, the cultures were passaged and counted. The counted colony concentration of Lactobacillus plantarum NZ-4 was diluted to 1.0×10 9 CFU / mL and reserve for use.

[0080] (2) Foodborne pathogens Escherichia coli O157:H7, Staphylococcus aureus, and common pathogenic fungi Salmonella and Candida albicans stored at -80°C were removed and rapidly thawed. 5 mL of LB medium, BHI medium, and Sabouraud broth were inoculated at a volume fraction of 4‰. E. coli and Staphylococcus aureus were cultured in a shaker at 37°C, 150 rpm for 18 h, and Candida albicans was cultured in a fungal incubator at 28°C for 48-72 h. The cultured bacterial suspension was diluted and plate counted, followed by an antibacterial test.

[0081] (3) The antibacterial test was carried out using the Oxford cup method. 10 mL of nutrient agar medium was poured into the bottom plate. After it solidified, an Oxford cup was placed in the center of the plate. When the sterilized LB medium, BHI medium, and Sabouraud solid medium cooled to about 45-50 °C, Escherichia coli, Staphylococcus aureus, and Candida albicans were added (the final concentration of pathogens was 1.×10 6 CFU / mL), shake well and pour into the plate where the Oxford cup has been placed. After the upper culture medium solidifies, remove the Oxford cup and add 100 μL of the Lactobacillus plantarum NZ-4 bacterial solution prepared in step (1) to the holes left in the Oxford cup. Incubate in a 37°C incubator for 24 hours and measure the diameter of the inhibition zone with a vernier caliper. Perform three parallel experiments for each experiment. The results of the antibacterial tests on Escherichia coli, Staphylococcus aureus and Candida albicans are shown in order from left to right, as shown in Figure 2. Figure 4 shown.

[0082] The results showed that Lactobacillus plantarum NZ-4 exhibited excellent antibacterial activity against common pathogens, including Escherichia coli and Staphylococcus aureus. It exhibited the strongest inhibitory effect against Escherichia coli, with an inhibition zone of 53 mm, followed by Staphylococcus aureus, with an inhibition zone of 20 mm. Furthermore, NZ-4 also exhibited significant inhibitory activity against the fungus Candida albicans, with an inhibition zone of 17 mm. Therefore, Lactobacillus plantarum NZ-4 could be used as an excellent strain for food preservation after modification, with promising application prospects.

[0083] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations to these embodiments may be made without departing from the principles and spirit of the present invention, and these changes and modifications still fall within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum NZ-4, characterized by: The Lactobacillus plantarum ( Lactiplantibacillus plantarum ) NZ-4 was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with the deposit number GDMCC NO: 64145.

2. A bacterial agent, characterized in that: The invention comprises the Lactobacillus plantarum NZ-4 according to claim 1.

3. Use of the Lactobacillus plantarum NZ-4 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for inhibiting Escherichia coli, Staphylococcus aureus or Candida albicans.

4. The use according to claim 3, characterized in that The product is a preservative, antistaling agent or food additive.

5. Use of the plant lactobacillus NZ-4 according to claim 1 or the bacterial agent according to claim 2 in producing lactic acid, citric acid, oxalic acid, malic acid or tartaric acid.

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