CN117903998A - Lactobacillus plantarum ST10-12 for high-yield phenyllactic acid and application thereof - Google Patents

Abstract

The invention discloses a lactobacillus plantarum ST10-12 for high-yield phenyllactic acid, which is preserved in the microorganism strain preservation center of Guangdong province in 12 months and 14 days of 2023, wherein the preservation number is GDMCCNo:63843. The invention has the advantages that: the screened lactobacillus plantarum ST10-12 can effectively inhibit the growth of various bacteria and fungi, is high in phenyllactic acid yield, sensitive to various antibiotics, safe, and has good application prospect in the preparation of antibacterial products, food preservatives, antistaling agents, health-care products, food additives and feed additives.

Description

A strain of Lactobacillus plantarum ST10-12 with high phenyllactic acid production and its application

Technical Field

The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus plantarum ST10-12 with high phenyllactic acid production and application thereof.

Background technique

Phenyllactic acid (PLA) is a natural small molecule organic acid. PLA has a very wide antibacterial spectrum and has good antibacterial effects on 11 bacteria such as thermophilic Streptococcus and Salmonella, and 15 fungi such as Aspergillus flavus and Fusarium. Its excellent antibacterial effect, safety and non-toxicity, easy solubility in water, high temperature and acid-base stability make it very promising for food preservation and fruit and vegetable preservation. Other studies have shown that PLA also has application prospects in the pharmaceutical and feed industries. In medicine, PLA is a derivative of danshensu and has similar pharmacological effects to danshensu. PLA can also be used to synthesize non-protein amino acids and make hypoglycemic agents. In the feed industry, it is found that adding PLA to laying hen feed can improve the immunity of laying hens, and also improve the quality and yield of eggs.

At present, studies have found that Lactobacillus plantarum can produce PLA, and PLA-producing lactic acid bacteria can be isolated from sourdough, kimchi, and traditional fermented vegetables. Although lactic acid bacteria have the ability to produce PLA, their synthesis ability is generally weak, and there are large differences between strains. Therefore, screening lactic acid bacteria resources with high PLA production has important research value and application prospects.

Summary of the invention

The technical problem to be solved by the present invention is to provide a plant lactobacillus strain with high phenyllactic acid production.

The present invention provides a strain of Lactobacillus plantarum ST10-12 with high phenyllactic acid production. The Lactobacillus plantarum ST10-12 (Lactobacillus plantarum ST10-12) has been deposited in Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with a deposit number of GDMCC No: 63843.

The invention provides a bacterial agent comprising Lactobacillus plantarum ST10-12.

The invention provides a fermentation product obtained by fermenting Lactobacillus plantarum ST10-12 or a bacterial agent.

The invention provides an application of plant lactobacillus ST10-12 or a bacterial agent or a fermentation product in the preparation of an antibacterial product, a food preservative, a freshness-preserving agent, a food additive, a feed additive, a food, a medicine and a health product.

Beneficial effects of the present invention:

The strain ST10-12 isolated from Guizhou Kaili sour soup can effectively inhibit the growth of multiple bacteria and fungi, has high PLA production, is sensitive to multiple antibiotics, is safe, and has good application prospects in the preparation of antibacterial products, food preservatives, preservatives, health products, food additives, and feed additives. ST10-12 can adapt well to the fermentation environment of sour soup, significantly increase the phenyllactic acid content in sour soup, improve the flavor and quality of sour soup, and the whole fermentation process does not need to add preservatives, making the sour soup safer and more delicious, and providing a new strain for the fermentation of sour soup.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the antibacterial results of Lactobacillus plantarum ST10-12 against Escherichia coli, Staphylococcus aureus, and Candida albicans from left to right.

Fig. 2 is a colony morphology diagram of Lactobacillus plantarum ST10-12;

FIG. 3 is a phylogenetic tree of Lactobacillus plantarum ST10-12.

Detailed ways

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.

The present invention provides a strain of Lactobacillus plantarum ST10-12 with high phenyllactic acid production, which is isolated from Kaili sour soup in Guizhou Province. Lactobacillus plantarum ST10-12 has been deposited in Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with a deposit number of GDMCC No: 63843. The deposit address is: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; Contact number: 020-87137633.

The invention provides a bacterial agent comprising Lactobacillus plantarum ST10-12.

The invention provides a fermentation product obtained by fermenting Lactobacillus plantarum ST10-12 or a bacterial agent.

The invention provides an application of plant lactobacillus ST10-12 or a bacterial agent or a fermentation product in the preparation of an antibacterial product, a food preservative, a freshness-preserving agent, a food additive, a feed additive, a food, a medicine and a health product.

The following is a detailed description of a strain of Lactobacillus plantarum ST10-12 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.

Example 1

Isolation, purification, identification and storage of Lactobacillus plantarum ST10-12

1. Isolation and Purification of Strains

Lactobacillus plantarum ST10-12 was isolated from Kaili sour soup in Guizhou. The isolation method was as follows: a test tube containing 5 mL of MRS liquid culture medium was sterilized by high-pressure steam at 115°C for 20 min, an appropriate amount of sour soup sample was quickly inoculated into the test tube, and a rubber stopper was immediately plugged, and the test tube was placed in a 37°C incubator for static culture for 24 h. In the sterile operating table, 100 μL of the cultured and mixed bacterial solution for 24 hours was taken and added to the 900 uL of sterile physiological saline that had been packaged for gradient dilution. Then 100 μL of 10 ⁵ , 10 ⁶ , and 10 ⁷ dilutions were evenly spread on the MRS solid medium. Each gradient was repeated three times. The coated MRS solid medium was placed in a 37°C incubator for 24 hours. After a single colony grew, a sterilized toothpick was used to pick a single colony and streaked on the MRS solid medium. It was placed in a 37°C incubator for 24 hours. The toothpick was used to pick a single colony and streaked 2-3 times until a purified single colony grew. Single colonies were selected according to the morphological characteristics of the colony, such as morphology, color, size, whether it was raised, transparency, and whether the edge was regular. A total of 15 strains of bacteria were selected from the sour soup. The 15 single colonies were inoculated into 5 mL of sterilized MRS liquid culture medium, incubated at 37°C for 48 h, and then stored in a -80°C refrigerator with 50% glycerol for later use.

The MRS liquid culture medium (g/L) is composed of: 10 g of peptone, 5 g of yeast extract, 10 g of beef extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 0.58 g of magnesium sulfate, 0.05 g of manganese sulfate, and 2 g of dipotassium hydrogen phosphate. 1000 mL of water is added and mixed, and sterilized at 115° C. for 20 min.

The MRS solid culture medium (g/L) is composed of: 10 g of peptone, 5 g of yeast extract, 10 g of beef extract, 20 g of glucose, 5 g of sodium acetate, 2 g of diammonium citrate, 1 mL of Tween 80, 0.58 g of magnesium sulfate, 0.05 g of manganese sulfate, 2 g of dipotassium hydrogen phosphate, 15 g of agar, 1000 mL of water is added, mixed, and sterilized at 115° C. for 20 min.

2. Preliminary screening of lactic acid bacteria with high phenyllactic acid production

The antibacterial activity of probiotics was determined by the Oxford cup method: about 10 mL of MRS solid culture medium was evenly poured on the bottom of the plate. After drying, an Oxford cup was placed in the middle of the plate, and 10 ⁸ CFU/mL of isolated and preserved strains were added to the Oxford cup. When the culture medium corresponding to the pathogens cooled to 40°C, three appropriate amounts of pathogens were added to the culture medium to make the final concentration of the pathogens 10 ⁶ CFU/mL. After complete cooling and solidification, it was placed at 37°C for 24 hours, and the diameters of the inhibition zones of the 15 selected strains against the three different pathogens were measured. All experiments were repeated 3 times in parallel.

The pathogenic bacteria were Escherichia coli (the culture medium was LB solid culture medium, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.), Staphylococcus aureus (the culture medium was BHI solid culture medium, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.) and Candida albicans (the culture medium was Sabouraud culture medium, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.).

The antibacterial experiment showed that 10 of the 15 selected strains had antibacterial effects on the three pathogens at the same time, among which ST10-12 had the strongest antibacterial effect on Escherichia coli, with an inhibition zone of 50 mm, and also had a high antibacterial effect on Staphylococcus aureus and Candida albicans. The antibacterial results are shown in Figure 1. ST9-1, ST8-5, and ST9-7 also had good inhibitory effects on the three pathogens.

3. Rescreening of lactic acid bacteria with high phenyllactic acid production

The single colonies ST10-12, ST9-1, ST8-5, and ST9-7 with larger inhibition zones were selected and cultured in MRS liquid medium at 37°C for 48 hours. The obtained fermentation broth was centrifuged at 8000r/min for 10 minutes, and the supernatant was taken. The supernatant was filtered with a 0.22μm filter membrane for standby use. The PLA yield in the fermentation supernatant of ST10-12, ST9-1, ST8-5, and ST9-7 in MRS liquid medium was determined by high performance liquid chromatography, thereby screening out high-yield phenyllactic acid strains. The PLA yield results are shown in the following table:

Strain number PLA yield (mg/L) ST9-1 70 ST8-5 16.6 ST9-7 98.5 ST10-12 180

HPLC working conditions: chromatographic column Agilent ZORBAX SB-C18 (4.6×12.5 mm, 5 μm), mobile phase 0.05% trifluoroacetic acid-water solution (A) and 0.05% trifluoroacetic acid-methanol solution (B), elution program: 80% A; detection wavelength: 210 nm; flow rate: 1.0 mL/min; injection volume: 20 uL; column temperature: 30°C.

Preparation of standard: accurately weigh 0.001g PLA standard and dissolve it in 1mL ultrapure water, shake it thoroughly to dissolve it, and prepare 1mg/mL standard stock solution; dilute the stock solution to 200ug/mL working solution, filter it into a liquid phase bottle with a 0.22um organic phase filter, analyze different volumes (5, 10, 20, 30, 40, 50uL) on the machine, and draw a standard curve with PLA content as the horizontal axis and peak area as the vertical axis. Repeat 3 times for each concentration and take the average value.

Mobile phase A: 0.05% trifluoroacetic acid-water solution, add 500 μL of chromatography-grade trifluoroacetic acid to a 1L volumetric flask, and dilute to 1L with ultrapure water. Filter the organic phase twice with a membrane filter, and remove bubbles by ultrasonication for 20 minutes.

Mobile phase B: 0.05% trifluoroacetic acid-methanol solution, add 500 μL of chromatography-grade trifluoroacetic acid to a 1L volumetric flask, and dilute to 1L with chromatography-grade methanol. Filter the organic phase twice with a membrane filter and remove bubbles by ultrasonication for 20 minutes.

The results showed that the PLA production of ST10-12 was the highest, at 180 mg/L, and the production of the other three strains was generally between 16.6 mg/L and 99 mg/L, indicating that there were large differences in PLA production among the strains. ST10-12 was subsequently selected as the experimental object.

4. Morphological identification of strains

The colony morphology of Lactobacillus plantarum ST10-12 on the plate is milky white, round, convex, smooth, opaque, moist, and easy to pick, as shown in Figure 2. Under a microscope, the bacterial morphology is long rod-shaped, arranged alone, in pairs, or in short chains. It is preliminarily judged to be a Lactobacillus plantarum based on the colony morphology and bacterial morphology.

5. Molecular Biological Identification of Strains

ST10-12 was inoculated into the liquid culture medium of MRS and placed in a 37℃ constant temperature incubator for activation culture. Then the genomic DNA of the strain was extracted (DNA extraction was performed using the QIAamp genomic DNA and RNA kits), and the 16S rRNA gene of the isolated strain was amplified by PCR using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') of the bacterial 16S rRNA gene. Reaction system: 2×TaqPCR Master Mix 10μL, upstream and downstream primers 0.5μL each, DNA template 1μL, ddH2O 8μL; reaction program: 95℃5min; 94℃1min, 55-58℃1min, 72℃90s, 30 cycles; 72℃10min. The PCR products of 16S rRNA of the strains isolated and cultured in the samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequence determination, and then the measured 16S rRNA gene sequences were compared by Blast in GenBank of the NCBI database, and the bacterial standard strain with the highest similarity to the isolated strain was selected to determine the strain type, and MEGA7.0 software was used to construct a phylogenetic tree. The phylogenetic tree is shown in Figure 3, and ST10-12 was determined to be Lactobacillus plantarum.

6. Identification results

Through morphological identification, molecular biological identification, 16S rRNA gene sequence comparison, and construction of phylogenetic tree, ST10-12 was identified as Lactobacillus plantarum.

7. Strain preservation

Lactobacillus plantarum ST10-12 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with the collection number GDMCCNo: 63843; the collection address is: 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; Tel: 020-87137633.

Example 2

Drug sensitivity test of strains

The drug sensitivity paper agar diffusion method was used to detect the sensitivity of the strain to drugs. The specific steps are as follows: Cut the filter paper into small circular pieces with a diameter of 5 mm, sterilize at 121°C, and dry in an oven. Immerse the filter paper pieces in six different drug solutions (ampicillin, kanamycin, ofloxacin, chloramphenicol, norfloxacin, and penicillin) to prepare drug sensitivity paper pieces with standard drug content, and dry them for use. The strain was activated twice with MRS liquid culture medium. When the number of viable bacteria reached 1x10 ⁸ CFU/mL, 1mL of the bacterial solution was centrifuged to remove the supernatant, and the bacterial solution was washed twice with 0.9% sterile saline, and then resuspended. Take an appropriate amount of bacterial solution and evenly spread it on the MRS solid culture medium. After the culture medium solidifies, paste the homemade drug sensitivity paper pieces, with the spacing between the paper pieces not less than 24 mm and greater than 15 mm from the inner edge of the plate. After pasting, quickly put it in a constant temperature incubator, and measure the diameter of the inhibition zone after culturing at 37°C for 24 hours. The standard sensitive bacteria Staphylococcus aureus ATCC25923 was used as the control strain. The drug sensitivity results are shown in the following table:

R stands for resistance; S stands for sensitivity.

The results showed that ST10-12 had different sensitivities to various antibiotics. It was sensitive to ampicillin, kanamycin, chloramphenicol, and penicillin, but resistant to ofloxacin and norfloxacin. Judging from the results, ST10-12 was safer and could be used in subsequent food development.

Example 3

Sour soup fermentation test

Strain activation: The ST10-12 strain stored in a -80°C refrigerator was taken out, inoculated into 5 mL MRS liquid culture medium at 4‰ (v/v), and cultured at 37°C for 24 hours; the activated ST10-12 was inoculated into 50 mL MRS liquid culture medium at a ratio of 4‰ (v/v), and cultured at 37°C for 24 hours; the bacterial solution was mixed and poured into a sterilized 50 mL centrifuge tube, centrifuged at 8000 rpm/min, 4°C for 5 minutes, and the supernatant was removed; physiological saline was added, shaken and mixed, and then centrifuged at 8000 rpm/min, 4°C for 5 minutes, the supernatant was removed, and the washing was repeated three times. Finally, 5 mL of physiological saline was added and shaken and mixed for use.

Preparation of sour soup: Wash the same mass of cherry tomatoes and red peppers, soak them in 75% ethanol for 15 minutes, put them in a clean bench to dry, chop them, mix them and divide them into tissue culture bottles (50g/bottle). Each tissue culture bottle is inoculated with ST10-12 at 1×10 ⁶ CFU/g. All tissue culture bottles are divided into three parts, one without sodium chloride, one with 1% sodium chloride, and one with 3% sodium chloride. After fully shaking, they are cultured at 37℃ for 120 hours for sensory quality evaluation and pH measurement.

After the sour soup fermentation was completed, sensory evaluation was conducted on three groups of products with different sodium chloride addition amounts. The evaluation results are shown in the following table:

It can be seen that the group with 1% sodium chloride added scored higher. Artificially adding Lactobacillus plantarum ST10-12 to the fermentation sample can rapidly reduce the pH value during the fermentation process and inhibit the growth of spoilage bacteria. Through experiments, it can be seen that ST10-12 can adapt well to the fermentation environment of sour soup, and quickly produce acid, which can complete the fermentation of sour soup in a short time and significantly improve the product quality.

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 (4)

1. A strain of Lactobacillus plantarum ST10-12 with high phenyllactic acid production, characterized in that: the Lactobacillus plantarum ST10-12 (Lactobacillus plantarum ST10-12) has been deposited in Guangdong Provincial Microbiological Culture Collection Center on December 14, 2023, with a deposit number of GDMCC No: 63843. 2. A bacterial agent, characterized in that it comprises the Lactobacillus plantarum ST10-12 according to claim 1. 3. A fermentation product obtained by fermenting the plant lactobacillus ST10-12 according to claim 1 or the bacterial agent according to claim 2. 4. Use of the plant lactobacillus ST10-12 according to claim 1 or the bacterial agent according to claim 2 or the fermentation product according to claim 3 in preparing antibacterial products, food preservatives, preservatives, food additives, feed additives, foods, medicines and health products.