Lactobacillus plantarum NZ-4 and application thereof

Abstract

The invention discloses a lactobacillus plantarum NZ-4, which is preserved in Guangdong Microbial Culture Collection Center on December 14, 2023, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 64145. The lactobacillus plantarum NZ-4 has the advantages that the lactobacillus plantarum NZ-4 produces lactic acid at high yield, has strong acid resistance, cholate resistance and pathogenic bacterium inhibition capacity, and has wide application prospects in food or health care products for prolonging the shelf life of food and regulating intestinal flora.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a plant lactobacillus strain 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 in Yunnan, and these traditional fermented foods contain rich microbial resources. Relevant studies have shown that there are a variety of lactic acid bacteria in Yunnan's traditional fermented foods, among which some plant lactobacilli have certain immunomodulatory effects, have inhibitory effects on certain pathogenic bacteria, can maintain the balance of intestinal flora and inhibit the formation of tumor cells.

[0003] After the human body consumes common organic acids such as lactic acid, acetic acid, citric acid, malic acid, oxalic acid, and tartaric acid, it can regulate the balance of intestinal flora through competitive inhibition, thereby reducing the incidence of gastrointestinal diseases. In addition, studies have shown that the target of organic acids on bacteria is mainly the cell wall, and its inhibitory mechanism is to destroy the cell structure of pathogens, and the outflow of intracellular substances leads to cell death. Therefore, it is of great significance to obtain probiotics that can produce high levels of organic acids such as lactic acid. Summary of the invention

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

[0005] The present invention provides a Lactobacillus plantarum NZ-4, wherein 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 invention provides a bacterial agent comprising Lactobacillus plantarum NZ-4.

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

[0008] Furthermore, the product is a preservative, a freshness-preserving agent or a 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 separates and screens plant lactobacillus NZ-4 from a milk residue sample in Shangri-La, Yunnan Province. The plant lactobacillus NZ-4 has high lactic acid production, strong acid resistance, bile salt resistance and pathogen inhibition capabilities. Experimental results show that the survival rate of plant lactobacillus NZ-4 after acid treatment for 3 hours is 97.16%, and the survival rate of bile salt treatment for 3 hours is 98.58%; the lactic acid content in the fermentation liquid after 24 hours is 21.3218 g / L; the plant lactobacillus has the best inhibitory effect on Escherichia coli, with an inhibition zone size of 53 mm, and has good inhibitory effects on Staphylococcus aureus and Candida albicans, wherein the inhibition zone size on Staphylococcus aureus is 20 mm; and the inhibition zone size on Candida albicans is 17 mm. Therefore, the plant lactobacillus of the present invention has a wide application prospect in extending the shelf life of food and regulating intestinal flora 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 antibacterial results of Lactobacillus plantarum NZ-4 against Escherichia coli, Staphylococcus aureus, and Candida albicans from left to right. DETAILED DESCRIPTION

[0017] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but 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 can be combined with each other as long as they do not conflict with each other.

[0018] The present invention provides a strain of 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 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 invention provides a bacterial agent comprising plant lactobacillus NZ-4 and other auxiliary materials.

[0020] The invention provides application of Lactobacillus plantarum NZ-4 or a bacterial agent in preparing 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 a strain of Lactobacillus plantarum NZ-4 and its application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] Isolation, purification, identification and storage of Lactobacillus plantarum NZ-4

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

[0028] Lactobacillus plantarum NZ-4 was isolated from milk residue samples in Shangri-La, Yunnan Province. The method is as follows: accurately weigh 1.0g of milk residue sample in 5mL of physiological saline under sterile conditions, fully shake and elute the attached microorganisms, take 20μL of the eluate and inoculate it into 5mL of MRS liquid culture 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 physiological saline to 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 culture 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 culture was completed, a sterilized toothpick was used to select purified colonies with obvious calcium dissolving rings, smooth surface, middle ridge, detailed edge features, and white or milky white color from each plate for Gram staining and catalase test. After preliminary identification, molecular biological identification was performed, and finally 9 lactic acid bacteria strains with obvious calcium dissolving rings were screened out. It was preliminarily determined that these 9 strains had the ability to produce acid, namely: 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 strains

[0035] The isolated NZ-4 strain was inoculated into MRS solid medium and cultured at 37°C for 24 h. 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 The results of Gram staining showed that NZ-4 was a Gram-positive bacterium, and the results of catalase test were negative.

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

[0037] 3. Molecular Biological Identification of Strain

[0038] The target strains screened were inoculated with MRS liquid culture medium and cultured at 37℃, 180r / min shaking for 24h. The genome of the strain was extracted using a bacterial DNA extraction kit, and the extracted genome was 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 was: BGI2×Super PCR Mix 25μL, forward primer 1μL, reverse primer 1μL, template 1μL, ddH2O2 2μL. The amplification conditions were: 94℃ pre-denaturation for 4min; 94℃ denaturation for 30s, 60℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; 72℃ final extension for 5min. The amplified products were subjected to 1.5% agarose gel electrophoresis and sent to 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 3 As shown, the sequence homology between NZ-4 and the standard strain of Lactobacillus plantarum was greater than 97%, and strain NZ-4 was determined 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 100mL MRS liquid culture medium at a rate of 4‰. They were anaerobically cultured at 37°C for 24h, 5mL of bacterial solution was disrupted by a cell disruptor, 10ml of 0.5% H3PO4 was added, ultrasonic extraction was performed for 15min, the solution was cooled to room temperature, the volume was made up to 25ml with 0.5% H3PO4, filtered through a 0.45μm filter membrane, and the filtrate was taken to determine its pH and lactic acid content by high performance liquid chromatography (HPLC), thereby screening out high lactic acid producing strains.

[0041] HPLC chromatographic conditions: Agilent C18 column (5m, 250×4.6mm); mobile phase: 0.05% H3PO4:methanol (90:10); detection wavelength: 210nm; flow rate: 1.0ml / min; column temperature: 40°C; injection volume: 10μl.

[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 the lactic acid content of Lactobacillus plantarum NZ-4 was the highest, at 21.3218 g / L, and the production of the other strains was generally between 4.1-11.9 g / L, indicating that the lactic acid content of each strain varied greatly, indicating that NZ-4 has the ability to produce high lactic acid.

[0047] 3. Strain preservation

[0048] Lactobacillus plantarum NZ-4 (Lactiplantibacillus plantarum NZ-4) was deposited in the Guangdong Provincial Microbiological 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 resistance of Lactobacillus plantarum

[0051] 1. Acid resistance test

[0052] The strain was inoculated in MRS liquid culture medium at an inoculum of 4‰ and activated for two generations at 37°C. The activated bacterial solution was inoculated in acidic MRS liquid culture medium with a pH of 6.2±0.2 (blank control group) and pH of 3, respectively, and cultured at 37°C for 3h. The viable bacteria were counted at 0 and 3h. 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] Wherein, N1 is the number of viable bacteria after 3 h of acid treatment and N0 is the initial number of viable bacteria of the strain.

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

[0056] 2. Bile salt tolerance test

[0057] After the strain was activated, it was inoculated into MRS liquid medium containing 0.3% and 0% (blank control group) bile salts at an inoculum volume of 4‰. After treatment at 37°C for 0 and 3h, the plate live bacteria were counted at 0 and 3h. The survival rate was 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 bile salt treatment for 3 hours 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 in MRS liquid medium at an inoculum size of 4‰, activated for two generations at 37°C, and then inoculated in 100 mL MRS liquid medium at an inoculum size of 4‰ for expansion culture for 24 hours. The fermentation supernatant was filtered through a 0.22 μm membrane and detected 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, oxalic acid) of different concentrations of 1 to 500 μg / mL were prepared respectively, and their peak areas were detected by HPLC. The standard curve of organic acid 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 according to the concentration and peak area of ​​the standard, and the results are shown in the following table:

[0067]

[0068] The results showed that the pH of the fermentation broth was 3.0. In addition to high lactic acid production, Lactobacillus plantarum NZ-4 can also produce citric acid, oxalic acid, malic acid and tartaric acid. After consuming these organic acids, the balance of intestinal flora can be adjusted through competitive inhibition, thereby reducing the incidence of gastrointestinal diseases. In addition, the dissociated anions of organic acids accumulate in large quantities in pathogenic bacteria, which can cause cell poisoning, interfere with glycolysis and nucleic acid synthesis, and ultimately lead to membrane transduction system disorders, inducing pathogenic bacteria to die.

[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 pH to 7.1 ± 0.1 (25 °C), add distilled water to 1000 mL, sterilize at 121 °C for 15 min;

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

[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 10g, beef extract powder 3g, sodium chloride 5g, agar 15g, distilled water to 1000mL, pH adjusted to 7.3±0.2, sterilized at 115℃ for 20min.

[0078] 2. Test methods

[0079] (1) Activate and subculture Lactobacillus plantarum NZ-4 stored at -80°C: Activate and subculture Lactobacillus plantarum NZ-4 at a concentration of 1×10 9 CFU / mL of Lactobacillus plantarum NZ-4 bacterial solution was inoculated into sterilized MRS liquid medium at a volume fraction of 4‰, and cultured at 37°C for 24 h before subculturing and counting. 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 taken out and thawed quickly. They were inoculated into 5 mL LB medium, BHI medium, and Sabouraud liquid medium at a volume fraction of 4‰, respectively. Escherichia coli and Staphylococcus aureus were cultured in a shaker at 37°C, 150 r / min for 18 h, and Candida albicans was cultured in a fungal incubator at 28°C for 48-72 hours. The cultured bacterial solution was diluted and plate counted, followed by an antibacterial test.

[0081] (3) The antibacterial experiment 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 were 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 with the Oxford cup already placed. After the upper culture medium solidifies, remove the Oxford cup, add 100 μL of 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 on each set. 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 had a good antibacterial effect. Lactobacillus plantarum NZ-4 had a good inhibitory ability against common pathogens such as Escherichia coli and Staphylococcus aureus. Among them, the inhibitory effect on Escherichia coli was the best, with an inhibition zone size of 53mm, followed by Staphylococcus aureus, with an inhibition zone size of 20mm; in addition, NZ-4 also showed a good inhibitory effect on the fungus Candida albicans, with an inhibition zone size of 17mm. Therefore, Lactobacillus plantarum NZ-4 can be used as an excellent strain for food preservation after modification, and has good 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. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum NZ-4, characterized in that: The Lactobacillus plantarum NZ-4 (Lactiplantibacillus plantarum NZ-4) was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with the collection 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 plant lactobacillus NZ-4 described in claim 1 or the bacterial agent described in claim 2 in the preparation of an antibacterial product.

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

5. Use of the plant lactobacillus NZ-4 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for regulating intestinal flora.

6. The use according to claim 5, characterized in that: The product is food or health product.

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