A Lactobacillus plantarum producing 5-methyltetrahydrofolic acid, its fermentation method and application

Abstract

This application relates to the field of food microbiology, and specifically discloses a Lactobacillus plantarum capable of producing 5-methyltetrahydrofolic acid, its fermentation method and application. A Lactobacillus plantarum inm30-LP capable of producing 5-methyltetrahydrofolic acid, with a preservation date of December 26, 2022, and the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is CGMCC No. 26278. This application is based on the milk curd in the Gannan Tibetan area as the food source, isolates probiotics, and screens out Lactobacillus plantarum with high folic acid production from them. Moreover, the strain has obvious advantages over other lactic acid bacteria in terms of acid resistance and bile salt resistance, is suitable for growing in the gastrointestinal environment, has the ability to proliferate, has no antibiotic resistance, has antibacterial activity, and can produce bacteriocin.

Description

Technical Field

[0001] The present application relates to the field of food microorganisms, and particularly relates to a Lactobacillus plantarum producing 5-methyltetrahydrofolate, its fermentation method and application. Background Art

[0002] Folic acid is a water-soluble vitamin with important physiological functions. The earliest reason for its popularity and attention is the impact of folic acid deficiency on the nervous system development of fetuses and infants. Research has found that folic acid also has functions such as anti-tumor, prevention and treatment of cardiovascular and cerebrovascular diseases, and intestinal diseases. Since humans lack the corresponding folic acid synthesis genes and cannot synthesize the folic acid they need, they can only obtain it from the outside through diet or by absorbing folic acid produced by the decomposition and metabolism of intestinal flora.

[0003] Folic acid has important physiological functions, but the potential hazards and side effects of its chemical synthesis are obvious. Therefore, the synthesis of folic acid by lactic acid bacteria has become a research hotspot. At present, most of the strains reported in the literature belong to Bifidobacterium, Streptococcus, Lactobacillus, Lactococcus, etc. Although lactic acid bacteria of the Bifidobacterium genus and Streptococcus genus can produce folic acid, the relative yield of folic acid is lower than that of other lactic acid bacteria.

[0004] Judging from the current research results, the amount of folic acid synthesized by lactic acid bacteria is not enough to meet all the folic acid needs of the human body. Even for foods fermented by high-folic acid-producing lactic acid bacteria, their folic acid yields still cannot reach the recommended daily intake for adults. Summary of the Invention

[0005] In order to improve the folic acid-producing ability of lactic acid bacteria, the present application provides a Lactobacillus plantarum producing 5-methyltetrahydrofolate, its fermentation method and application.

[0006] In the first aspect, the Lactobacillus plantarum producing 5-methyltetrahydrofolate provided by the present application adopts the following technical solution: The preservation date of a Lactobacillus plantarum inm30-LP producing 5-methyltetrahydrofolate is December 26, 2022. The preservation unit is the China General Microbiological Culture Collection Center, the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 26278.

[0007] In the second aspect, a fermentation method provided by the present application adopts the following technical solution:

[0008] A fermentation method, inoculating Lactobacillus plantarum inm30-LP in a first culture medium, culturing for 36-48 h, then transferring to a second culture medium with an inoculation amount of 1-3% (v / v), the initial pH is 4-6, the fermentation temperature is 25-37 °C, and the fermentation time is 20-36 h to obtain a fermentation broth.

[0009] Optionally, 8 - 25 mg / L of p-aminobenzoic acid is also added to the second culture medium.

[0010] Optionally, the addition amount of p-aminobenzoic acid is 20 mg / L.

[0011] Optionally, the inoculation amount is 3%.

[0012] Optionally, the initial pH of fermentation is 5.

[0013] By adopting the above technical solution, the culture conditions and technological process of Lactobacillus plantarum inm30-LP are optimized to improve its folic acid yield.

[0014] In a third aspect, the present application provides an application of Lactobacillus plantarum in the preparation of probiotic products.

[0015] Optionally, the probiotic products include functional dairy products, functional beverages, fermenting agents, bacterial powders, and bacteriocins.

[0016] In summary, the present application includes at least one of the following beneficial technical effects:

[0017] 1. Based on the milk curd from Gannan Tibetan Autonomous Prefecture as the food source, the present application isolates probiotics, screens out Lactobacillus plantarum that produces 5-methyltetrahydrofolic acid therefrom, qualitatively and quantitatively screens the strains, measures the physiological characteristics of the strains, and optimizes its culture conditions and technological process to increase its yield, obtaining strains with high folic acid yield, laying a foundation for the development of probiotic products;

[0018] 2. The strains disclosed in the present application have obvious advantages over other lactic acid bacteria in terms of acid and bile salt tolerance, are suitable for growing in the gastrointestinal environment, have the ability to proliferate, have no antibiotic resistance, have antibacterial activity, and can produce bacteriocins. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the standard curve graph of determining folic acid in the examples of the present application;

[0020] Figure 2 is the HPLC chromatogram of the folic acid standard product in the examples of the present application;

[0021] Figure 3 is the comparison graph of folic acid yields of different strains in the examples of the present application;

[0022] Figure 4 is the colony morphology graph of Lactobacillus plantarum inm30-LP in the examples of the present application;

[0023] Figure 5 is the cell morphology graph of Lactobacillus plantarum inm30-LP stained by Gram staining in the examples of the present application;

[0024] Figure 6 This is an electrophoresis identification diagram of 16SrDNA of Lactobacillus plantarum inm30-LP in the embodiment of the present application;

[0025] Figure 7 This is a comparison chart of folic acid production of inm30-LP and LZ217 in the examples of the present application;

[0026] Figure 8 This is a comparison chart of the liquid chromatography peaks of inm30-LP and LZ217 in the examples of the present application.

[0027] Note: GQ1701 in the attached figure refers to Lactobacillus plantarum inm30-LP of the present application, and GQ1701 is a temporary name during the experiment. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can all be sourced from common commercial sources.

[0029] Folic acid-free culture medium (FACM) was purchased from Beijing Luqiao.

[0030] Description of biological deposit:

[0031] The preservation date of Lactobacillus plantarum inm30-LP is December 26, 2022. The preservation unit is the General Microbiology Center of China Culture Collection Administration. The preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC No.26278.

[0032] Example 1 Isolation and Identification of Lactobacillus plantarum inm30-LP (I) Isolation and Purification of Strain:

[0033] The strain originated from Naidu, Gannan Tibetan Autonomous Prefecture. 5 g of Naidu sample from Gannan Tibetan Autonomous Prefecture was collected in a sterile tube and immediately sent to the laboratory for strain isolation and purification.

[0034] When separating, take 1g of sample and put it into 9mL of MRS liquid culture medium. After vortex mixing, enrich and culture it at 37℃ for 48h. Then, take 1mL of culture medium in the clean bench and dilute it tenfold with sterile saline. Select three dilution gradients of 10-5, 10-6, and 10-7. Take 100μL of bacterial suspension from each dilution gradient and apply it on MRS solid culture medium, and culture it at 37℃ for 48h.

[0035] After the cultivation, select the plates with 30 - 300 single colonies grown on MRS solid medium, pick out the typical colonies, streak and isolate them on MRS agar plates multiple times until the colony morphology on the whole plate is consistent. Then pick out single colonies and transfer them to MRS liquid medium for subculture. The obtained strains are cryopreserved at -80 °C in MRS liquid medium containing 40% (w / v) glycerol.

[0036] (II) Qualitative screening of strains

[0037] Activation of S1: Dip an inoculation loop into the bacterial liquid obtained in step (I) and streak it on MRS solid medium, then culture it at 37 °C for 48 h. Pick out single colonies and inoculate them into MRS liquid medium, and culture it at 37 °C for 24 h. Repeat the activation 2 - 3 times; Qualitative screening of S2:

[0038] Add bromocresol purple as an indicator to the folic acid - free medium (FACM), with pH = 6.8. Take 1 - 2 drops of the activated liquid culture of the strain to be tested and inoculate it into the folic acid - free medium, and place it at 37 °C for 1 - 3 d. If the medium solution turns yellow, it indicates a positive result, meaning that the strain can synthesize folic acid; if there is no change, it can be inoculated 8 times continuously. Identify whether the strain can produce folic acid through the color - development experiment, and the results are shown in Table 1.

[0039] Table 1 Qualitative screening results of folic acid - producing strains

[0040]

[0041]

[0042] In this application, by testing a large number of biodiversity and a large number of strains, and comparing and screening the Lactobacillus sakei and the strains in the strain bank, the chance of detecting folic acid in the species of extracellular folic acid - producing strains is significantly increased based on the samples. A total of 48 strains were screened before and after this application. Among them, 26 strains showed a positive color reaction in the qualitative reaction. Subsequently, verification and repeated experiments were carried out again, and finally 23 strains were selected for quantitative determination.

[0043] (III) Quantitative screening of strains

[0044] Sample pretreatment of S1:

[0045] Vortex - mix the lactic acid bacteria fermentation broth and centrifuge it at 10000 rpm for 10 min at 4 °C. Take 10 mL of the supernatant, add 40 mL of methanol, dry it with a nitrogen stream, redissolve it with chromatographic - grade methanol, and filter it through a 0.22 - μm filter membrane for standby.

[0046] Preparation of folic acid standard stock solution of S2:

[0047] Weigh 55 - 56 mg (accurate to 0.1 mg) of the standard product, transfer it into a 100 mL volumetric flask with 50 mL of distilled water, add 2 mL of ammonia water. After preparing the solution, calculate the solution volume according to the following formula, and require the folic acid concentration in the stock solution to be 0.50 mg / mL:

[0048]

[0049] m is the mass of the folic acid standard product, in milligrams (mg); c is the purity of the folic acid standard product, in grams per 100 grams (g / 100g).

[0050] Dilute the solution to the scale with water, add water with a pipette to the calculated required volume, mix well, put it into a brown reagent bottle, refrigerate, and the storage period is 4 months. The folic acid standard solution can be diluted 10 times to prepare a folic acid standard product with a mass concentration of 0.05 mg / mL.

[0051] S3 Drawing of the standard curve:

[0052] Dissolve the folic acid standard product in the folic acid buffer solution to prepare standard solutions with concentrations of 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 5 μg / mL, and 10 μg / mL respectively, and measure the folic acid production in the standard solution.

[0053] See Figure 1 , the standard curve: y = 64.367x + 3.09679 R2 = 0.9987; content unit: μg / mL.

[0054] S4 Determination by liquid chromatography:

[0055] Compare the retention times of folic acid between the two to verify whether the strains screened by FACM have the ability to produce folic acid. For liquid chromatography, the stationary phase is a C18 column, the ultraviolet detector (280 nm), and the mobile phase is methanol and water with a volume ratio of 10:90 (containing Na 2 HPO 4 and NaH 2 PO 4 , with a pH of 7.6). The mobile phase is filtered through a 0.45 μm organic membrane and degassed by ultrasonic wave for 15 min. The retention time is 20 min, and the injection volume is 20 μL. The result is expressed as the number of micrograms of folic acid produced per milliliter of the fermentation broth.

[0056] See Figure 2 , the peak value appearing at 5 - 6 min is the characteristic peak of 5 - methyltetrahydrofolic acid. The sample peak time is also around the retention time of 5 - 6 min, and only the 5 - methyltetrahydrofolic acid peak appears, indicating that the separation degree between 5 - methyltetrahydrofolic acid and other substances is good. It shows that the method used in the embodiment of this application can be used for the detection of 5 - methyltetrahydrofolic acid.

[0057] Using the uninoculated medium as a blank control, the folic acid content in the fermentation supernatant of the strain was measured after culturing at 30 °C for 24 h. The obtained results are shown in Figure 3 . The folic acid production of lactic acid bacteria was calculated according to the following formula:

[0058] A = B - A 0 + C

[0059] Where: A is the extracellular folic acid production of lactic acid bacteria; B is the folic acid production in the fermentation broth; A 0 is the folic acid production in the blank group; C is the folic acid loss during the measurement process. The loss was determined by the difference between the folic acid measured after the same sample pretreatment operation was performed on the folic acid standard solution (0.05 mg / mL) and the value measured for the original standard solution.

[0060] (IV) Identification of Lactobacillus plantarum inm30-LP:

[0061] After quantitative and qualitative screening, the dominant strain Lactobacillus plantarum inm30-LP was identified.

[0062] Table 2 Folic acid production of common folic acid-producing strains

[0063] Strain Folic acid yield (μg / mL) Lactobacillus plantarum 0.397 Lactobacillus fermentum 0.084 Lactobacillus acidophilus + Lactococcus lactis 0.017-0.1 Lactobacillus sakei 0.2397 Lactococcus lactis 0.814

[0064] Combined with Table 2 and Figure 3 It can be seen that the folic acid production ability of Lactobacillus plantarum inm30-LP of the present application is significantly higher than that of other common lactobacilli.

[0065] See Figure 4 , after culturing Lactobacillus plantarum inm30-LP on MRS solid medium for 48 h, the surface edges are all smooth, the shape is regular and round, and it is milky white.

[0066] See Figure 5 , smear of Lactobacillus plantarum inm30-LP colonies: Gram staining is positive, does not produce spores, and is rod-shaped.

[0067] See Figure 6 , the genomic DNA of the target strain was extracted using the Ezup column bacterial genomic DNA extraction kit. The extracted genomic DNA of lactic acid bacteria was used as a template for PCR amplification. The PCR experiment of 16S rDNA was carried out using the universal bacterial primers 27F and 1492R. After the PCR reaction amplification was completed, the PCR product was taken for agarose gel detection and photography, and the amplified fragment length was about 1500 bp.

[0068] The PCR product was sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. The obtained 16S rDNA sequence is shown in the sequence listing. BLAST sequence alignment was performed on the NCBI website, and the results showed that the sequence had a homology of more than 99% with the 16S rDNA sequence for the identification of Lactobacillus plantarum.

[0069] Optimization of the Fermentation Method of Lactobacillus plantarum inm30-LP in Example 2

[0070] Select the strain culture time, initial fermentation pH value, fermentation time, fermentation temperature, inoculum size, and pABA as the main factors affecting folic acid production, and conduct single-factor experiments.

[0071] The experimental process is as follows:

[0072] Inoculate Lactobacillus plantarum in MRS liquid medium, culture at 30 °C for 36 - 48 h, then transfer to MRS broth medium at an inoculum size of 1 - 3% (v / v), add 0 - 25 mg / L of p-aminobenzoic acid, with an initial pH of 4 - 6, a fermentation temperature of 25 - 37 °C, and a fermentation time of 20 - 36 h to obtain the fermentation broth.

[0073] (1) Influence of Strain Culture Time

[0074] Culture the strain for 36 h, 40 h, and 48 h respectively at 30 °C, inoculate the strain at an inoculum size of 1%, ferment for 24 h at 30 °C, with an initial pH of 6.0, and do not add p-aminobenzoic acid. After completion, analyze the folic acid production according to the method in Example 1.

[0075] Table 3 Influence of Strain Culture Time on Folic Acid Production

[0076] Cultivation time (h) Folic acid yield (μg / mL) 36h 2.02±0.50 40h 2.25±0.07 48h 1.75±0.67

[0077] (2) Influence of pABA Addition Amount

[0078] Add pABA to the liquid medium in amounts of 8 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, and 25 mg / L respectively. Culture the strain for 36 h at 30 °C, inoculate the strain at an inoculum size of 3%, ferment for 24 h at 30 °C, with an initial pH of 6.0. After completion, analyze the folic acid production according to the method in Example 1. The results of different groups have significant differences, and 20 mg / L is the optimal addition amount.

[0079] Table 4 Influence of pABA Addition Amount on Folic Acid Production

[0080] pABA (mg / L) Folic acid yield (μg / mL) 8 10.62±3.94c 10 14.03 ± 1.05bc 15 17.37±1.90b 20 25.33±1.42a 25 14.4 ± 0.96bc

[0081] (3) Influence of Inoculum Size

[0082] Inoculate the strain with inoculation amounts of 1%, 2%, and 3% respectively, culture the strain for 36 h at a culture temperature of 30°C, ferment for 24 h at a fermentation temperature of 30°C, with an initial pH of 6.0, and without adding p-aminobenzoic acid. Detect the folic acid yield according to the method in Example 1. The results show that there are significant differences in inoculating the strain with different inoculation amounts.

[0083] Table 5 Effects of inoculation amount on folic acid yield

[0084] Inoculation amount / % Folic acid yield (μg / mL) 1 2.03±1.19 2 2.47±0.04 3 3.41±0.43

[0085] (IV) Effects of initial pH value of fermentation

[0086] Set the pH to 4, 4.5, 5, 5.5, and 6 respectively, culture the strain for 48 h at a culture temperature of 30°C, inoculate the strain with an inoculation amount of 3%, ferment for 24 h at a fermentation temperature of 30°C, without adding p-aminobenzoic acid, and analyze the folic acid yield according to the method in Example 1.

[0087] Table 6 Effects of initial pH value of fermentation on folic acid yield

[0088] pH Folic acid yield (μg / mL) 4 2.15±0.22b 4.5 3.08 ± 0.22ab 5 4.09±0.95a 5.5 3.37 ± 0.92ab 6 3.11 ± 0.58ab

[0089] (V) Effects of fermentation time

[0090] Culture the strain for 36 h at a culture temperature of 30°C, inoculate the strain with an inoculation amount of 3%, ferment at a fermentation temperature of 30°C, with an initial pH of 6.0, and the addition amount of p-aminobenzoic acid is 15 mg / L. Ferment for 20 h, 24 h, and 36 h respectively, and analyze the folic acid yield according to the method in Example 1.

[0091] Table 7 Effects of fermentation time on folic acid yield

[0092] Fermentation time (h) Folic acid yield (μg / mL) 20 14.03±1.05 24 17.37±1.90 36 17.97±8.48

[0093] (VI) Effects of fermentation temperature

[0094] Culture the strain for 36 h at a culture temperature of 30°C, inoculate the strain with an inoculation amount of 3%, ferment for 24 h, with an initial pH of 5.0, and the addition amount of p-aminobenzoic acid is 10 mg / L. Set the fermentation temperatures to 25°C, 30°C, and 37°C respectively. After fermentation, analyze the folic acid yield according to the method in Example 1.

[0095] Table 8 Effects of fermentation temperature on folic acid yield

[0096] Temperature (°C) Folic acid yield (μg / mL) 25 13.74±1.96 30 14.42±3.43 37 17.24±4.22

[0097] (VII) Effects of control strain and co-fermentation

[0098] Set the experimental group: Lactobacillus plantarum inm30-LP;

[0099] Set control group 1: Lactobacillus sakei LZ217;

[0100] Set control group 2: Lactobacillus plantarum inm30-LP + Lactobacillus sakei LZ217.

[0101] The process conditions are all as follows: The strain is cultured for 40 h, the culture temperature is 30 °C, the strain is inoculated at an inoculation amount of 3%, fermented for 24 h, the fermentation temperature is 30 °C, the initial pH is 5.0, and the addition amount of p-aminobenzoic acid is 15 mg / L. After the fermentation is completed, the folic acid yield is analyzed according to the method in Example 1.

[0102] Combined with Figure 7 、 Figure 8 , according to the data statistical analysis, it can be seen that the difference between the two strains is significant (P < 0.05), and there is no significant difference from the fermentation of the synergistic strain. Therefore, Lactobacillus plantarum inm30-LP of the present application has a high ability to metabolize and produce folic acid.

[0103] Performance detection of Lactobacillus plantarum inm30-LP

[0104] I. Acid tolerance experiment:

[0105] Pick a single colony of Lactobacillus plantarum inm30-LP and expand it in MRS liquid medium for 18 h. Inoculate the expanded bacterial suspension into MRS broth medium at a volume of 1% (v / v). After culturing at 37 °C for 18 h, the concentration of the bacterial liquid reaches 10 8 CFU / mL;

[0106] Then, the culture solution is centrifuged at 5000 r / min for 5 min at 4 °C to collect the bacterial cells. After washing twice with phosphate buffer (PBS), the bacterial cells are suspended in MRS liquid medium with a pH of 3.0 and cultured at 37 °C for 3 h to obtain a test sample.

[0107] The pour plate method is used to count the viable bacteria in the test samples at 0 h and 3 h. The poured plates are cultured at 37 °C for 48 h to determine their survival rates. The survival rate calculation formula is as follows:

[0108]

[0109] In the above formula, N 0 is the number of viable bacteria in the test sample at 0 h (CFU / mL); N t is the number of viable bacteria in the test sample at 3 h (CFU / mL).

[0110] II. Bile salt tolerance experiment:

[0111] Pick a single colony of Lactobacillus plantarum inm30-LP and expand it in MRS liquid medium for 18 h. Then inoculate the expanded bacterial suspension into MRS broth medium at a volume fraction of 1% (v / v). After culturing at 37 °C for 18 h, the concentration of the bacterial suspension reaches 10 8 CFU / mL. Then vortex and mix well, and inoculate it into MRS broth medium containing 0.3% (m / v) bile salts at a volume fraction of 2% (v / v). Culture at 37 °C for 3 h to obtain the test sample.

[0112] Use MRS broth medium without bile salts as the control sample. The pour plate method is used to count the viable bacteria in the test sample. The poured plates are cultured at 37 °C for 48 h. The bile salt tolerance of the strain is expressed by the following formula:

[0113]

[0114] In the above formula, N 0 is the number of viable bacteria in the control sample (CFU / mL); N t is the number of viable bacteria in the test sample (CFU / mL).

[0115] Table 9 Acid and bile salt tolerance results of Lactobacillus plantarum inm30-LP

[0116] Strain Acid tolerance survival rate / % Bile salt tolerance rate / % Lactobacillus plantarum inm30-LP 76.89 31.87

[0117] The pH of gastric juice is generally around 2-3. Only lactic acid bacteria with acid tolerance can enter the gastrointestinal tract to play a role. As shown in Table 9, Lactobacillus plantarum inm30-LP has good acid tolerance and can successfully pass through the acidic environment in the stomach to reach the small intestine.

[0118] The bile salt content in the human small intestine is about 0.3%. Only lactic acid bacteria with bile salt resistance can maintain their viability and play the role of lactic acid bacteria in the small intestine. As shown in Table 9, the bile salt tolerance rate of Lactobacillus plantarum inm30-LP is 31.87%, indicating that it has strong bile salt tolerance characteristics.

[0119] III. Antibiotic sensitivity of Lactobacillus plantarum inm30-LP

[0120] The Kirby-Bauer disk diffusion method is used to measure the antibiotic sensitivity of lactic acid bacteria strains, referring to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI).

[0121] First, prepare MRS solid medium in an Erlenmeyer flask. After sterilizing the medium, keep it in a 55 °C water bath for heat preservation. After cooling, take it out and place it in a sterilized laminar flow hood. Then, the bacterial suspension of Lactobacillus plantarum inm30-LP (10 8The CFU / mL) was inoculated into an Erlenmeyer flask at a volume of 1% (v / v), shaken until evenly mixed, and then poured into a sterile Petri dish to make an LB plate of 15 mL / dish. After the MRS plate solidified, two antibiotic discs were gently attached to each plate with forceps. Finally, the plates with the attached antibiotic discs were placed at 37 °C and cultured for 24 h. After the culture was completed, the diameter of the inhibition zone was measured with a vernier caliper and recorded.

[0122] Table 10 Results of the sensitivity of Lactobacillus plantarum inm30-LP to antibiotics

[0123] Drug name Abbreviation Tablet content Inhibition zone diameter (mm) Erythromycin ERM 15.00 30.81±0.80 Ciprofloxacin CFX 5.00 8.82±0.80 Lincomycin LIN 2.00 12.43±2.40 Tetracycline TET 30.00 10.11±0.10 Ceftriaxone CTR 30.00 29.85±9.80 Cefazolin CZ 30.00 15.75±5.70 Ampicillin AM 10.00 18.67±8.60 Penicillin PEN 10.00 32.19±2.10 Gentamicin GEM 10.00 19.14±9.10

[0124] With the widespread use of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria has become increasingly serious. Long-term intake of drug-resistant lactic acid bacteria will bring great difficulties to clinical treatment. As shown in Table 10, according to the drug sensitivity test standard of CLSI, the sensitivity of the strain to 9 common antibiotics was evaluated. Lactobacillus plantarum inm30-LP was sensitive to 3 antibiotics (erythromycin, penicillin, ceftriaxone), and it was most sensitive to penicillin, with an inhibition zone of 32.19 mm. It can be seen that Lactobacillus plantarum inm30-LP is sensitive to common antibiotics and will not harm human health.

[0125] IV. Pathogen inhibitory ability of Lactobacillus plantarum inm30-LP

[0126] The antibacterial activity of lactic acid bacteria was determined by the internationally common agar diffusion method.

[0127] First, the four indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes) stored in frozen state were activated 2-3 times on LB solid medium, and then the well-activated single colonies were picked and inoculated into LB medium and cultured at 37 °C for 18 h. Then, the bacterial cells were collected by centrifugation, resuspended in physiological saline to make the concentration reach 10 8 CFU / mL. Then, the indicator bacteria suspension was added to the sterilized LB solid medium cooled to 55 °C at a volume of 1% (v / v), mixed evenly and poured into a Petri dish, 15 mL / dish. After condensation, the previously placed sterile Oxford cup was removed.

[0128] After the activated Lactobacillus plantarum inm30-LP was cultured in MRS medium for 18 h, it was centrifuged at 8000 rpm for 5 min at 4 °C to collect the supernatant, and the cell precipitate was discarded. Then, the supernatant was added to the cup wells (200 μL / well) and cultured at 37 °C. After 24 h, the diameter of the inhibition zone was measured. The uninoculated MRS medium with a pH of 6.2 was used as a blank control. The strains with obvious inhibition zones around the small holes were selected, and the diameter of the inhibition zone was measured, with each measurement repeated three times.

[0129] Table 11 Results of the inhibitory ability of Lactobacillus plantarum inm30-LP against pathogenic bacteria

[0130]

[0131] a, b: P-value < 0.05

[0132] Note: Different lowercase letters indicate significant differences in the size of the inhibition zone of the bacterial strains (P < 0.05).

[0133] Staphylococcus aureus is the most common pathogenic bacterium in human purulent infections. Some Escherichia coli can cause severe diarrhea and septicemia, and some Salmonella species can also cause human food poisoning. Bacteriocins, organic acids, hydrogen peroxide and other antibacterial products produced by the metabolism of lactic acid bacteria can inhibit the growth of these pathogenic bacteria alone or in combination.

[0134] As shown in Table 11, the strain has a certain inhibitory effect on 4 pathogenic bacteria. The strain has a significant inhibitory effect on Staphylococcus aureus and Escherichia coli (P < 0.05). Among them, the supernatant has the best inhibitory effect on Listeria monocytogenes, and the inhibition zone reaches 20.1 mm. Therefore, the metabolites produced during the growth of Lactobacillus plantarum inm30-LP all have certain antibacterial activities, play an important role in maintaining the intestinal microecological balance, and have a health promotion effect.

[0135] The Lactobacillus plantarum inm30-LP of the present application can be widely used in the development of probiotic and other related products, and five application examples are used for specific application descriptions.

[0136] Application Example 1 Preparation of bacteriocin:

[0137] (1) Preparation of the fermentation supernatant of Lactobacillus plantarum inm30-LP producing 5-methyltetrahydrofolic acid. Inoculate Lactobacillus plantarum inm30-LP into 100 mL of MRS liquid medium with an inoculation loop, place it in an incubator at 37 °C for 18 h, and then transfer it to 250 mL of sterilized MRS liquid medium at an inoculation amount of 2% (v / v). Then place it in a bacterial incubator at 37 °C for 18 h to obtain a fermentation broth.

[0138] The obtained fermentation broth is centrifuged at 10000 r / min for 20 min to collect the bacterial cells. Then the bacterial cells are suspended in distilled water and centrifuged at 10000 r / min for 20 min to collect the supernatant. The pH value is adjusted to 6.0 with NaOH, and then filtered through a 0.45 μm filter membrane to obtain a sterile fermentation supernatant, which is stored in a 4 °C refrigerator for later use.

[0139] (2) Ammonium sulfate precipitation

[0140] To the sterile fermentation supernatant of Lactobacillus plantarum inm30-LP, while stirring with a magnetic stirrer, slowly add the pre-weighed solid ammonium sulfate powder until the final ammonium sulfate saturation is 60 wt%. Then place it at 4°C until sufficient precipitation occurs. Next, centrifuge at 4°C for 20 min at a speed of 10,000 r / min to separate the precipitate. Then dissolve the precipitate with 50 mmol / L phosphate buffer (PBS) with a pH of 6.0 to obtain the crude bacteriocin solution after ammonium sulfate treatment. Finally, store it in a 4°C refrigerator for later use.

[0141] (III) G-25 Sephadex gel chromatography

[0142] After passing the crude bacteriocin solution obtained after ammonium sulfate treatment through a 0.22 μm filter membrane, slowly add it to the G-25 Sephadex gel chromatography and elute with ultrapure water at a flow rate of 1.0 mL / min. At the same time, detect and collect the protein fractions at 280 nm (i.e., collect the active fractions with antibacterial activity), and use BaCl 2 Detect whether there is salt residue in the collected protein fractions. The protein fraction that does not produce a precipitate with BaCl 2 The protein fraction that produces a precipitate is the purified bacteriocin solution, and collect it.

[0143] (IV) Freeze concentration

[0144] After pre-freezing the purified bacteriocin solution in a -80°C ultra-low temperature refrigerator for at least 2 h, quickly take it out and put it into a vacuum freeze dryer for vacuum treatment. Maintain the vacuum degree at about 0.1 P. After concentrating to 1 / 5 of the original volume, take out the bacteriocin under reduced pressure and put it into a 4°C refrigerator to slowly dissolve, obtaining the bacteriocin of Lactobacillus plantarum inm30-LP.

[0145] Application Example 2 Preparation of functional fermented fruit and vegetable juice:

[0146] (1) Cleaning and cutting: First, wash, peel, and cut the pumpkin and pitaya into small pieces.

[0147] (2) Flash evaporation: Use the method of flash evaporation to inactivate enzymes, treat at 121°C for 0.5 - 1 min, and quickly exhaust the air.

[0148] (3) Pulping: According to the mass ratio of pumpkin:water = 1:1, gradually put the pumpkin and water into a colloid mill for grinding, and perform coarse grinding and fine grinding once each to obtain pumpkin juice; at the same time, use a blender to pulp the pitaya until the pulp is uniform and without lumps to obtain pitaya juice.

[0149] (4) Blending and homogenization: Mix according to 15% (v / v) pumpkin juice and 30% (v / v) pitaya juice, then adjust the soluble solids content to 10 °Bx with sucrose, add 0.2% (w / v) stabilizer CMC-Na and mix evenly. Use a two-stage homogenization method, first at low pressure (15 MPa), then at high pressure (25 MPa), to make the diameter of the melon flesh particles 2 - 3 μm, obtaining a compound fruit and vegetable juice.

[0150] (5) Sterilization and cooling: Keep the prepared compound fruit and vegetable juice at 100 °C for 10 min, and then cool it to about 40 °C.

[0151] (6) Inoculation and fermentation: Under sterile conditions, inoculate the activated Lactobacillus plantarum inm30-LP into the cooled compound fruit and vegetable juice, and control the initial bacterial count at 1×10 7 CFU / mL, and ferment at a constant temperature of 37 °C for 24 h.

[0152] (7) Ripening: After fermentation, place it in a 4 °C refrigerator for 3 h.

[0153] (8) Filling and refrigeration: After ripening, fill it into 250 mL sterilized glass bottles and send them to the cold storage for refrigeration.

[0154] Application Example 3 Preparation of starter culture:

[0155] Inoculate the original strain of Lactobacillus plantarum inm30-LP into 11 wt% skim milk (sterilized at 115 °C for 20 min), and culture it at 37 °C for 18 - 24 h until coagulation. Activate it continuously for two generations as the mother starter culture.

[0156] Inoculate the mother starter culture into 11 wt% skim milk (sterilized at 115 °C for 20 min) at an inoculation amount of 3 - 5% (v / v), and culture it at 37 °C for 18 - 24 h until coagulation. At this time, the viable bacteria count can reach 10 9 -10 10 CFU / mL, thus obtaining the Lactobacillus plantarum inm30-LP starter culture.

[0157] Application Example 4 Preparation of functional fermented milk:

[0158] Heat the pretreated cow milk to about 60 °C, add 6 wt% sucrose, fully dissolve it, homogenize it under a pressure of 20 MPa, then heat-treat it at 95 °C for 5 min, cool it to 38 °C, and then inoculate the Lactobacillus plantarum inm30-LP starter culture prepared in Application Example 3 at an inoculation amount of 5% (v / v), ferment it at 37 °C for 14 - 18 h until coagulation, and refrigerate it at 4 °C after cooling, thus obtaining the functional fermented milk of Lactobacillus plantarum inm30-LP.

[0159] Application Example 5 Preparation of bacterial powder:

[0160] Step 1, inoculating Lactobacillus plantarum inm30-LP into 50 mL of MRS liquid culture medium with an inoculating loop, placing it in a 37° C. incubator and culturing it for 18 h, then transferring it to a sterilized 250 mL of MRS liquid culture medium with a 5% (v / v) inoculation amount, placing it in a bacterial incubator at 37° C. and culturing it for 24 h to obtain a fermentation liquid;

[0161] Step 2, the obtained fermentation broth is centrifuged at 5000r / min, the supernatant is discarded, the bacterial precipitate is collected, and the bacterial cells are rinsed twice with sterile phosphate buffer (PBS), each time for 10min, to obtain Lactobacillus plantarum inm30-LP bacterial precipitate;

[0162] Step 3, 4.1 g of trehalose, 2.8 g of sodium glutamate, and 8 g of sucrose were weighed and dissolved in 10 mL of 40°C distilled water, filtered and sterilized with a 0.22 μm microporous filter membrane, and then added to 90 mL of sterile water to obtain a first solution for later use; 15 g of skim milk powder was dissolved in distilled water to obtain 100 mL of a 15 wt% skim milk powder solution, which was sterilized at 110°C for later use;

[0163] The first solution and the skimmed milk powder solution are uniformly mixed in a volume ratio of 1:5 to obtain a protective agent solution;

[0164] Step 4, according to a volume ratio of 1:5, the above-mentioned Lactobacillus plantarum inm30-LP bacterial precipitation and the protective agent solution are thoroughly mixed to obtain a bacterial suspension, and then 5 mL of the bacterial suspension is divided into 10 mL sterile stoppered glass bottles, and pre-frozen at -70°C. After 2 hours, take out and place in a vacuum freeze dryer for freeze drying. The freeze drying conditions are a vacuum degree of 5 Pa, a partition heating temperature of 20°C, and a cold trap temperature of -55°C. After freeze drying for 30 hours, the Lactobacillus plantarum inm30-LP bacterial powder is obtained by crushing.

[0165] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A Lactobacillus plantarum inm30-LP that produces 5-methyltetrahydrofolic acid, characterized in that: The preservation date is December 26, 2022, the preservation unit is the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 26278.

2. A fermentation method, characterized in that: Inoculate the Lactobacillus plantarum described in claim 1 into MRS liquid medium, culture for 36 - 48 h, and then transfer it to MRS broth medium at an inoculation amount of 1 - 3% (v / v). 8 - 25 mg / L of p-aminobenzoic acid is also added to the MRS broth medium. The initial pH is 4 - 6, the fermentation temperature is 25 - 37 °C, and the fermentation time is 20 - 36 h to obtain a fermentation broth.

3. A fermentation method according to claim 2, characterized in that: The addition amount of p-aminobenzoic acid is 20 mg / L.

4. A fermentation method according to claim 2, characterized in that: The inoculation amount is 3%.

5. A fermentation method according to claim 2, characterized in that: The initial fermentation pH is 5.

6. Use of the Lactobacillus plantarum described in claim 1 in the preparation of probiotic products.

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