A strain of Lactobacillus plantarum ZJUIDS16 with high folic acid production and its application

Abstract

The invention provides a plant lactobacillus strain with high folic acid production (Lactobacillus plantarum) ) ZJUIDS16 and its application. The strain preservation number is: CGMCC NO.26162. The strain has a high ability to metabolize and produce folic acid, can tolerate the gastrointestinal environment, the culture fluid has no antibiotic resistance, inhibits harmful pathogens in the intestine, and has a strong antioxidant capacity. Therefore, the plant lactobacillus ZJUIDS16 of the present invention can be used in the preparation of functional foods, health products and pet foods. The foods include: fermented fruit and vegetable juices, fermented dairy products, bacterial powders, etc. Lactic acid bacteria bacteriocins can also be fermented to prepare food preservatives, which are used in food processing and storage. The plant lactobacillus ZJUIDS16 provided by the present invention can be widely used in the development of probiotics and other related products.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms, and in particular relates to a plant lactobacillus ZJUIDS16 with high folic acid production and application thereof, in particular to a plant lactobacillus ZJUIDS16 producing 5-methyltetrahydrofolate and application thereof. Background Art

[0002] Folic acid is a water-soluble B vitamin composed of purine, para-aminobenzoic acid and polyglutamic acid tail. It is widely distributed and commonly found in animals, plants and traditional fermented foods. Folic acid has important physiological functions. When it is deficient, it will have an adverse effect on the development of the nervous system of fetuses and infants. Studies have found that folic acid also has the functions of anti-tumor, prevention and treatment of cardiovascular and cerebrovascular diseases and intestinal diseases. However, higher animals such as humans cannot synthesize the folic acid they need due to the lack of corresponding folic acid synthesis genes. These folic acids can only be taken in from the outside through diet or absorption of folic acid produced by intestinal flora. In addition, chemically synthesized folic acid has the disadvantages of high absorption rate but potential side effects, while natural folic acid has high safety, good absorption effect and no toxic side effects when taken into the human body. Lactic acid bacteria are widely used in the food industry and healthcare industry. At the same time, studies have confirmed that lactic acid bacteria in intestinal microorganisms can metabolize and produce vitamins such as folic acid, which is one of the main sources of vitamins for the host. Therefore, lactic acid bacteria are the best choice for biosynthesis of folic acid.

[0003] Therefore, screening lactic acid bacteria with high folic acid production capacity has high application value. At the same time, it was found that the strain has obvious advantages over other lactic acid bacteria in terms of acid and bile salt resistance, is suitable for growth in the gastrointestinal environment and has the ability to proliferate, has no antibiotic resistance, does not release hemolytic toxins, has antibacterial activity and strong antioxidant capacity, and is applied to fermented dairy products, and has a significant advantage in determining folic acid content. Therefore, the invented plant lactobacillus can provide a basis for the development of probiotic products. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Lactobacillus plantarum ZJUIDS16 with high folic acid production, which is a strain of Lactobacillus plantarum ZJUIDS16 that produces 5-methyltetrahydrofolate. The present invention provides a strain of Lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS16 that produces 5-methyltetrahydrofolate. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on November 21, 2022, and the classification name is: Lactobacillus plantarum, the deposit number is: CGMCC NO.26162, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0005] Furthermore, the complete 16SrDNA sequence of the Lactobacillus plantarum ZJUIDS16 is shown in SEQ ID No.1.

[0006] Furthermore, Lactobacillus plantarum ZJUIDS16 has obvious advantages over other lactic acid bacteria in terms of acid and bile resistance, is suitable for the gastrointestinal environment and has the ability to proliferate; the culture medium of Lactobacillus plantarum ZJUIDS16 (Lactobacillus plantarum) has no antibiotic resistance, indicating that the strain does not carry drug-resistant genes; Lactobacillus plantarum ZJUIDS16 (Lactobacillus plantarum) has antibacterial activity.

[0007] Another object of the present invention is to provide the use of Lactobacillus plantarum ZJUIDS16 in the preparation of functional foods, health products and pet foods.

[0008] The functional foods include fermented fruit and vegetable juices, fermented dairy products, bacterial powder and other foods.

[0009] The health care product comprises: a bacterial agent or bacterial powder with high folic acid production.

[0010] The pet food includes: pet milk powder and pet probiotic powder.

[0011] Another object of the present invention is to provide the use of Lactobacillus plantarum ZJUIDS16 in the preparation of food preservatives.

[0012] Furthermore, the lactic acid bacteria bacteriocin prepared by fermentation of Lactobacillus plantarum can be used as a preservative for the preservation of foods such as meat products, dairy products, and alcoholic beverages, or combined with other preservative technologies and applied as a barrier technology in food processing and storage.

[0013] The present invention provides a plant lactobacillus that produces 5-methyltetrahydrofolate in view of the lack of lactic acid bacteria with high folate production capacity and convenient and accurate determination methods and culture conditions. The present invention improves its yield by optimizing the process of culture and fermentation conditions through a single factor method, and compares and verifies with a control strain to investigate the effect of plant lactobacillus on folate production. Studies have shown that the strain has a higher metabolic folate production capacity, can tolerate the gastrointestinal environment, has no antibiotic resistance in the culture fluid, inhibits harmful pathogens in the intestine, and has a strong antioxidant capacity. Therefore, the plant lactobacillus ZJUIDS16 of the present invention can be widely used in the development of probiotics and other related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1This is a colony morphology diagram of Lactobacillus plantarum ZJUIDS16 of the present invention.

[0015] Figure 2 The Gram-stained bacterial morphology of Lactobacillus plantarum ZJUIDS16 of the present invention is shown in FIG.

[0016] Figure 3 This is an electrophoresis identification diagram of 16SrDNA of Lactobacillus plantarum ZJUIDS16 of the present invention.

[0017] Figure 4 This is the phylogenetic tree of Lactobacillus plantarum ZJUIDS16.

[0018] Figure 5 This is the standard curve of folic acid.

[0019] Figure 6 This is the result of the self-agglutination rate determination of Lactobacillus plantarum ZJUIDS16.

[0020] Figure 7 The hemolysis type result is detected on the blood agar plate. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0022] Example 1 Isolation and identification of Lactobacillus plantarum ZJUIDS16

[0023] 1 Isolation of Lactobacillus plantarum ZJUIDS16

[0024] 1.1 Sample source

[0025] The strain used in the present invention is derived from Northeast pickled vegetable samples, and a total of 50 samples were collected.

[0026] 1.2 Isolation and purification of strains

[0027] Take about 5g of sample and collect it in a sterile tube, and immediately send it to the laboratory for strain isolation and purification. When isolating, take 1g of sample and put it into 9mL of MRS liquid culture medium, vortex mix it and enrich it at 37℃ for 48h; then take 1mL of culture medium in the clean bench, dilute it tenfold with sterile saline, and select 10 -5 , 10 -6 , 10 -7Three dilution gradients, take 100% of the bacterial suspension for each dilution gradient, apply it on MRS solid medium, and culture it at 37℃ for 48h. After the culture, select a plate with 30 to 300 single colonies from the MRS solid medium, pick a typical colony, and separate it by streaking on the MRS agar plate several times until the colony morphology on the entire plate is consistent, pick a single colony and expand it in MRS liquid medium. The obtained strain is frozen and stored in a -80℃ refrigerator in MRS liquid medium containing 40% (w / v) glycerol.

[0028] 2. Identification of Lactobacillus plantarum ZJUIDS16

[0029] 2.1 Colony characteristics

[0030] After Lactobacillus plantarum ZJUIDS16 was cultured in MRS solid medium for 48 hours, the surface edges were smooth, the shape was regular and round, and the color was milky white. Figure 1 .

[0031] 2.2 Morphology under microscope

[0032] Colony smear of Lactobacillus plantarum ZJUIDS16: Gram staining is positive, does not produce spores, rod-shaped, see Figure 2 .

[0033] 2.3 16S rDNA identification

[0034] The target strain genomic DNA was extracted using the Ezup column bacterial genomic DNA extraction kit. The extracted lactic acid bacteria genomic DNA was used as a template for PCR amplification. The bacterial universal primers 27F and 1492R were used to perform PCR experiments on 16SrDNA. After the PCR reaction was completed, the PCR product was taken for agarose gel detection and photography. The length of the amplified fragment was about 1500bp, as shown in Figure 1. Figure 3 The PCR products were sent to Beijing Liuhe BGI Technology Co., Ltd. for sequencing. After comparing the homology, the phylogenetic tree results were as follows: Figure 4 As shown, a BLAST sequence comparison was performed on the NCBI website, and the results showed that the sequence had a homology of more than 99% with the identified 16S rDNA sequence of Lactobacillus plantarum.

[0035] Based on the sequence alignment results of strain ZJUIDS16 and the physiological and biochemical results, it was determined that the screened lactic acid bacteria ZJUIDS16 was Lactobacillus plantarum ZJUIDS16.

[0036] Example 2 Determination of folic acid production of Lactobacillus plantarum ZJUIDS16

[0037] 1 Quantitative screening of high folate-producing strains and determination of folate content

[0038] 1.1 Sample pretreatment

[0039] The lactic acid bacteria fermentation broth was shaken and mixed, and then centrifuged (10000rpm, 10min), 10 ml of the supernatant was taken, and deproteinized with methanol (the volume ratio of the sample to be tested and methanol during methanol deproteinization was 1:4), dried with a nitrogen stream, re-dissolved with methanol (chromatographic grade), filtered with a 0.22μm filter membrane, and set aside.

[0040] 1.2 Preparation of folic acid standard stock solution

[0041] Weigh 55-56 mg (accurate to 0.1 mg) of standard, transfer to a 100 mL volumetric flask with 50 mL of distilled water, add 2 mL of ammonia water, and after the solution is prepared, calculate the volume of the solution according to the following formula. The folic acid concentration in the stock solution is required to be 0.50 mg / mL:

[0042] m is the mass of the folic acid standard in milligrams (mg); c is the purity of the folic acid standard in g / 100g. Dilute the solution to the mark with water, add water to the required volume with a pipette, mix thoroughly, put into a brown reagent bottle and refrigerate. The shelf life is 4 months. The folic acid standard solution can be diluted 10 times to prepare the folic acid standard to a mass concentration of 0.05 mg / mL.

[0043] 1.3 Liquid chromatography determination

[0044] The HPLC method for rescreening of folic acid-producing bacteria was used to analyze the fermentation broth sample and the 0.50 mg / mL folic acid standard sample after pretreatment. The operation steps are as follows. The retention time of folic acid between the two samples was compared to verify that the strain screened by FACM has the ability to produce folic acid. The liquid phase was selected as a C18 column with a UV detector (280 nm); the mobile phase was methanol: water (containing Na 2 HPO 4 and NaH 2 PO 4 , pH 7.6)=10:90, mobile phase was filtered through 0.456) organic membrane and then ultrasonically degassed for 15min; retention time 20min; injection volume 20μL, the results were expressed in micrograms of folic acid produced per milliliter of fermentation broth. The culture medium without inoculation was used as a blank control, and the folic acid content in the fermentation supernatant after the strain was cultured at 30℃ for 24h was determined. The results were expressed in the mass of folic acid per milliliter of fermentation broth. The folic acid production of lactic acid bacteria was calculated as follows:

[0045] A=B-A 0 +C

[0046] Where: A is the extracellular folate production of lactic acid bacteria; B is the folate production in the fermentation broth; A0 is the folate production in the blank group; C is the folate loss during the determination process. The loss is calculated by adding a folate standard solution (0.05 mg / mL) and performing the same sample pretreatment operation as above, and the difference between the folate measured by the original standard solution.

[0047] 1.4 Drawing of standard curve

[0048] The folic acid standard was dissolved in folic acid buffer and prepared into standard solutions with concentrations of 1μg / mL, 1.5μg / mL, 2μg / mL, 5μg / mL, and 10μg / mL, respectively, and the folic acid production in the standard solution was determined. See the standard curve diagram for details. Figure 5 , the formula is as follows: y = 64.367x + 3.09679R 2 =0.9987; content unit: μg / mL.

[0049] The comparison of folic acid production of different strains is shown in Table 1. The present invention screened out lactic acid bacteria with better folic acid production ability by measuring the folic acid production of 50 strains through liquid chromatography.

[0050] Table 1 Comparison of folic acid production of different strains

[0051]

[0052] Example 3 Confirmation of acid resistance and bile resistance of Lactobacillus plantarum ZJUIDS16

[0053] 1. Acid resistance test

[0054] A single colony of Lactobacillus plantarum ZJUIDS16 was selected and expanded in MRS liquid medium for 18 h. The expanded bacterial suspension was inoculated into MRS broth medium at 1% (v / v). After 18 h of incubation at 37 °C, the concentration of the bacterial suspension reached 10 8 CFU / mi. The culture solution was centrifuged at 5000r / min for 5min at 4℃ to collect the bacteria, washed twice with phosphate buffer (PBS), and then suspended in MRS liquid culture medium with pH=3.0 and cultured at 37 for 3h. MRS without pH adjustment was used as a control, and the live bacteria in the 0h and 3h samples were counted by pouring plate method. The poured plates were cultured at 37 for 48h, and their survival rate was determined. The survival rate calculation formula is as follows:

[0055] In the above formula, N 0 N is the number of viable bacteria of the test strain at 0h (CFU / mL); t It is the number of viable bacteria (CFU / mL) of the test strain at 3 hours.

[0056] 2. Bile salt tolerance test

[0057] The activated and enlarged Lactobacillus plantarum ZJUIDS16 bacterial suspension was inoculated into MRS broth at a volume of 1% (v / v). After culturing at 37°C for 18 h, the concentration of the bacterial suspension reached 10 8 CFU / mi. Vortex mix, inoculate 2% into MRS broth containing 0.3% (m / v) ox bile salts (control is MRS broth without ox bile salts), and culture at 37°C for 3h. Then count the number of viable bacteria in the sample by pouring plate method. The poured plate is cultured at 37°C for 48h. The bile salt tolerance of the strain is expressed by the following formula:

[0058]

[0059] Table 2 Acid and bile resistance of Lactobacillus plantarum ZJUIDS16

[0060] strain pH = 3.0, 3h, survival rate in MRS 0.3% bile salt, 3h, survival rate in MRS Lactobacillus plantarum ZJUIDS16 68.31％ 41.60％

[0061] As shown in Table 2, the pH of gastric juice is generally around 2 to 3, and only lactic acid bacteria with acid tolerance can enter the gastrointestinal tract to play a role. As shown in the table, the test results show that the acid tolerance rate of ZJUIDS16 is 68.31%, and it can smoothly pass through the acidic environment in the stomach to reach the small intestine. The bile salt content in the human small intestine is about 0.30%, and lactic acid bacteria with bile salt resistance can maintain vitality in the small intestine and play the role of lactic acid bacteria. The results show that the bile salt tolerance rate of Lactobacillus plantarum ZJUIDS16 is 41.60%, which is significantly better than the control strain and has a strong bile salt resistance. Probiotics must be able to tolerate a series of adverse environments such as gastric acid and bile in the gastrointestinal tract and survive to play their probiotic effects. The plant lactobacillus ZJUIDS16 provided by the present invention can grow and proliferate under the condition of pH=3.0, and it can smoothly pass through the acidic environment in the stomach to reach the small intestine.

[0062] Example 4 Confirmation of the self-agglutination ability of Lactobacillus plantarum ZJUIDS16

[0063] Wash the lactic acid bacteria precipitate twice with high-temperature sterilized PBS liquid culture medium, resuspend it to make the absorbance of OD610 be 0.5, and obtain lactic acid bacteria suspension; take an appropriate amount of bacterial liquid and put it into a sterilized clean test tube, incubate it at 37℃, and measure the OD at different time points within 24 hours. 610 The absorbance value.

[0064] Among them A 0 = absorbance value at 0h, A t = absorbance value of th.

[0065] The results are as follows Figure 6As shown. Compared with the control strain GQ1701, ZJUIDS16 has better self-agglutination performance. The higher the self-agglutination rate, the higher the self-agglutination rate of the strain. From the data, we can see that the self-agglutination rate of all strains increases with time within 24 hours, and the self-agglutination rate of ZJUIDS14 reaches about 80% at 24 hours.

[0066] The self-agglutination rate of the strain is related to its adhesion in the intestine. The higher the agglutination rate, the higher the adhesion in the gastrointestinal tract. When the self-agglutination rate of the strain is high enough, the bacteria can form a biological protective film when they aggregate to a certain number, thereby protecting the gastrointestinal mucosa. In addition, the agglutination of the strain can keep the bacteria alive, improve the upper digestive tract of the human body against pathogens, and form steric hindrance to prevent pathogens from colonizing in the intestine.

[0067] Example 5 Verification of the Antioxidant Capacity of Lactobacillus plantarum ZJUIDS16

[0068] 5 Determination of antioxidant capacity of Lactobacillus plantarum ZJUIDS16

[0069] 5.1 Sample preparation

[0070] The strains stored in the glycerol tube were streaked on MRS solid medium and incubated at 37°C for 48 hours. A single colony was picked with an inoculation loop and inoculated into sterile MRS liquid medium. The culture was incubated at 37°C for 18-24 hours to obtain a culture solution. The culture solution was adjusted with distilled water to a lactic acid bacteria concentration of 10 10 CFU / mL, centrifuge at 4°C, 8000×g for 20 min, collect the supernatant as the fermentation supernatant. Resuspend and wash the bacterial pellet after centrifugation with 0.02M PBS buffer (pH=7.4), centrifuge at 4°C, 8000×g for 20 min, repeat 3 times. Resuspend the washed bacterial cells in PBS buffer and adjust the bacterial concentration to 10 10 CFU / mL, and the bacterial suspension was obtained.

[0071] 5.2 Determination of total antioxidant capacity

[0072] Total antioxidant capacity (FRAP method) requires adding 150 μL TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, mixed at V:V:V=10:1:1, prepared before use) and 20 μL of sample to the ELISA plate, shaking and mixing, reacting at 37°C for 10 minutes, and measuring the absorbance of the solution at 593nm. Substitute the measured absorbance of the sample into the ferrous sulfate standard curve, and the antioxidant capacity of the sample is expressed as ferrous sulfate equivalent (μmol FeSO 4 Each sample was repeated 3 times and the average value was calculated.

[0073] Ferrous sulfate standard curve: Prepare ferrous sulfate solutions of different mass concentrations (0μM, 50μM, 100μM, 200μM, 400μM, 600μM, 800μM), mix ferrous sulfate solutions of different molar concentrations, 10mM TPTZ buffer, and 0.3M acetate buffer at V:V:V=1:1:10, take 170μL of the mixture and add it to the ELISA plate, react at 37℃ for 10min, and measure the absorbance of the solution at 593nm. Draw the standard curve with the mass concentration of ferrous sulfate as the horizontal axis and the absorbance as the vertical axis.

[0074] 5.3 Determination of reducing capacity

[0075] Take 1mL of sample in a centrifuge tube, add 0.2M, pH 6.6 PBS solution and 1% (w / v) potassium ferricyanide solution 1mL each, and mix well. Place in a 50℃ water bath for 20min, and cool in an ice bath. Then add 1mL of 10% (w / v) trichloroacetic acid, centrifuge at 6000×g for 5min, take 1mL of supernatant, add 1mL of 0.1% (w / v) ferric chloride and 1mL of distilled water, mix well, let it stand for 10min, and measure the absorbance of the sample at 700nm. Use PBS buffer or MRS liquid culture medium instead of the sample as the blank group. Make 3 parallels for each sample and calculate the average value.

[0076] Reducing ability (%) = [(A s -A b ) / A b ]*100%Wherein: A s - Absorbance of sample group; A b -Blank group absorbance

[0077] 5.4 DPPH free radical scavenging ability determination

[0078] Prepare different concentration gradients of V C Solution (0-30μg / ml). Add 100μL of the sample to be tested (or V C 100 μL 0.2 m M DPPH ethanol solution (prepared with anhydrous ethanol, stored at 4°C away from light, prepared and used immediately), shake well and keep away from light for 30 minutes at room temperature, and measure the absorbance of the solution at 517 nm; use 100 μL anhydrous ethanol to replace 100 μL DPPH ethanol solution as the blank group; use 100 μL PBS buffer (or MRS liquid culture medium) to replace 100 μL of the sample to be tested as the control group, and use 100 μL PBS buffer (or MRS liquid culture medium) and anhydrous ethanol mixed solution as the blank zero. Repeat 3 times for each sample and calculate the average value. DPPH free radical scavenging ability (%) = [1-(A s -A b ) / Ac ]*100%Wherein: A s - Absorbance of sample group; A b -Blank group absorbance; A c - Absorbance of control group.

[0079] Table 3 In vitro antioxidant activity of Lactobacillus plantarum ZJUIDS16

[0080]

[0081] Example 6 Confirmation of the hydrophobicity of Lactobacillus plantarum ZJUIDS16

[0082] Wash the lactic acid bacteria precipitate twice with high temperature sterilized PBS liquid medium and resuspend to OD 610 The absorbance is about 0.5, and the lactic acid bacteria suspension is obtained; 2 ml of the lactic acid bacteria suspension is mixed with 2 ml of xylene, and the mixture is thoroughly shaken in a 37°C water bath for 5 min, and the OD of the aqueous phase is measured after 0 h and 2 h respectively. 610 Absorbance value.

[0083] Among them A 0 = absorbance value at 0h, A t = absorbance value of th.

[0084] Table 4 Surface hydrophobicity of different strains (%)

[0085] Bacteria Hydrophobicity ZJUIDS16 56.74％

[0086] The hydrophobicity of ZJUIDS16 was measured to be 56.74%, significantly higher than that of the control strain, indicating that the strain has a strong adhesion ability and can adhere to the human intestine and improve the health of the intestinal flora.

[0087] Example 7 Confirmation of the anti-hemolysis ability of Lactobacillus plantarum ZJUIDS16

[0088] This test uses the blood agar plate culture method to detect the hemolytic type of Lactobacillus plantarum ZJUIDS16, the method is as follows:

[0089] (1) ZJUIDS16 was activated and cultured in liquid MRS medium at 37°C;

[0090] (2) Use a sterile inoculating loop to pick up the activated third generation culture medium and streak it on a blood agar plate (purchased from Beijing Luqiao Technology Co., Ltd.);

[0091] (3) Incubate the plate at 37°C for 48 h;

[0092] (4) Observe the color change of the blood agar plate to see if there is a hemolysis zone. There are three types of hemolysis and their judgment methods: α (type A) hemolysis: hemolysin is produced, but it is incomplete hemolysis. There is a 1-2 mm narrow, translucent, grass-green hemolysis zone around the colonies on the blood agar plate. β (type B) hemolysis: complete hemolysis. There is a 2-4 mm wide, clearly defined, colorless and transparent hemolysis zone around the colonies on the blood agar plate. β (type C) hemolysis: hemolysin is not produced. There is no hemolysis zone around the colonies on the blood agar plate. The experimental results are as follows: Figure 7 As shown, Lactobacillus plantarum ZJUIDS16 grew well in the blood agar plate, and there was no obvious change in the color of the culture medium near the colonies, so it was judged that Lactobacillus plantarum had no hemolysis phenomenon.

[0093] Example 8 Confirmation of antibiotic sensitivity of Lactobacillus plantarum ZJUIDS16

[0094] The antibiotic sensitivity of lactic acid strains was measured by paper diffusion method. This research method refers to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI). MRS solid culture medium was prepared using a wide-mouth conical flask. After the culture medium was sterilized, it was placed in a 55°C water bath for insulation. After the temperature dropped, it was taken out and placed in a sterilized clean bench. The lactic acid bacteria suspension (108CFU / mL) was inoculated into the conical flask at 1% and shaken until mixed. After the lactic acid bacteria suspension was mixed with the MRS solid culture medium, it was poured into a sterile plate to make a 15mL / plate LB plate. After the MRS plate solidified, two antibiotic paper discs were evenly applied to each plate with tweezers. The plate with the antibiotic paper discs was placed at 37°C and cultured for 24 hours. After the culture was completed, the diameter of the inhibition zone was measured and recorded with a vernier caliper.

[0095] The diameters of the inhibition zones of Lactobacillus plantarum ZJUIDS16 to antibiotics are shown in Table 5. According to the CLSI drug sensitivity test standard, the sensitivity of the strain to 9 common antibiotics was evaluated, and the results are shown in Table 5.

[0096] Table 5 Diameter of inhibition zone of sensitivity of Lactobacillus plantarum ZJUIDS16 to antibiotics

[0097]

[0098] Lactobacillus plantarum ZJUIDS16 was sensitive to three antibiotics (erythromycin, penicillin, and ceftriaxone), and was most sensitive to penicillin, with an inhibition zone of 32.19 mm. The experimental results showed that Lactobacillus plantarum ZJUIDS16 was sensitive to common antibiotics.

[0099] With the widespread use of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is becoming more and more serious. Long-term intake of drug-resistant lactic acid bacteria will bring great difficulties to clinical treatment. The Lactobacillus plantarum ZJUIDS16 provided by the present invention is sensitive to common antibiotics and will not cause harm to human health.

[0100] Example 9 Confirmation of the pathogen inhibition ability of Lactobacillus plantarum ZJUIDS16

[0101] The antibacterial activity of lactic acid bacteria was determined by the internationally accepted agar diffusion method. The four frozen indicator strains (Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes) were activated 2 to 3 times on LB solid medium. The activated single colonies were picked out and placed in LB medium and cultured at 37 for 18 hours. The bacterial cells were collected by centrifugation and resuspended in physiological saline to a concentration of 10 8 CFU / mL. Add the indicator bacteria suspension at 1% (v / v) to the sterilized LB solid culture medium cooled to about 55°C, mix well and pour into the culture dish (15mL / dish), and remove the sterile Oxford cup placed in advance after condensation. After the activated Lactobacillus plantarum ZJUIDS16 is expanded and cultured in MRS culture medium for 18 hours, the supernatant is collected after centrifugation (8000rpm, 5min, 4m), and the bacterial precipitate is discarded. The metabolic supernatant of Lactobacillus plantarum ZJUIDS16 is added to the cup holes (200 holes (holes), and the uninoculated MRS culture medium (pH=6.2) is used as a blank control. Culture at 37°C and measure the diameter of the inhibition zone after 24 hours. Select the strains with obvious inhibition zones around the small holes, measure the diameter of the inhibition zones, and repeat three times for each.

[0102] Table 6 Diameter data of sensitive zones of Lactobacillus plantarum ZJUIDS16 to 9 common antibiotics

[0103]

[0104] As shown in Table 6, the strain had a certain inhibitory effect on the four pathogens. The strain had a significant inhibitory effect on Staphylococcus aureus and Escherichia coli (P<0.05), and the supernatant had the best inhibitory effect on Listeria monocytogenes, with an inhibition zone of 20.1 mm. Therefore, the metabolites produced during the growth of Lactobacillus plantarum ZJUIDS16 have certain antibacterial activity and can improve human intestinal health.

[0105] Staphylococcus aureus is the most common pathogen in human suppurative infection, some Escherichia coli can cause severe diarrhea and sepsis, and some Salmonella species can also cause food poisoning in humans. Antibacterial products such as bacteriocins, organic acids, and hydrogen peroxide produced by lactic acid bacteria metabolism can inhibit the growth of these pathogens alone or together. The metabolites of plant lactobacillus ZJUIDS16 provided by the present invention have a certain antagonistic effect on these four pathogens, which plays an important role in maintaining the balance of intestinal microecology and has a health-promoting effect.

[0106] Example 10 Preparation of functional fermented fruit and vegetable juice using Lactobacillus plantarum ZJUIDS16

[0107] 1. Processing process of fermented fruit and vegetable juice:

[0108] Raw materials → cleaning → flash evaporation → beating → mixing → homogenization → sterilization → cooling → inoculation → closed fermentation → post-ripening → filling → refrigeration

[0109] 2. Operation points

[0110] (1) Wash and cut into pieces: Wash, peel and cut the pumpkin and dragon fruit into small pieces;

[0111] (2) Flash evaporation: The enzyme is inactivated by flash evaporation for 0.5-1 min at 121°C and the exhaust is rapid;

[0112] (3) Pulping: According to the ratio of pumpkin to water (mass ratio) = 1:1, gradually put the pumpkin and water into a colloid mill and grind them, performing coarse grinding and fine grinding once each. Pulp the dragon fruit with a pulper until the pulp is uniform and free of lumps;

[0113] (4) Mixing and homogenization: Pumpkin juice 15% (v / v), dragon fruit juice 30% (v / v), and then use sucrose to adjust the soluble solid content to 10 。 Brix, add 0.2% (w / v) stabilizer CMC-Na and mix evenly, adopt two-stage homogenization method, first low pressure (15MPa), then high pressure (25MPa), so that the diameter of melon pulp particles is 2-3μm;

[0114] (5) Sterilization and cooling: The prepared composite fruit and vegetable juice is kept at 100°C for 10 min and then cooled to about 40°C;

[0115] (6) Inoculation and fermentation: Under sterile conditions, activated Lactobacillus plantarum ZJUIDS16 was inoculated, and the initial bacterial count was controlled at 1×10 7 CFU / mL. Fermented at 37℃ for 24h;

[0116] (7) Post-ripening: After fermentation, place in a 4°C refrigerator for 3 h;

[0117] (8) Filling and refrigeration: After ripening, fill into 250 mL sterilized glass bottles and send to cold storage for refrigeration.

[0118] Example 11 Preparation of bacterial powder using a 5-methyltetrahydrofolate-producing Lactobacillus plantarum ZJUIDS16

[0119] Use an inoculation loop to inoculate Lactobacillus plantarum ZJUIDS16 into 50mL MRS liquid culture medium, and place it in a 37°C incubator for 18 hours. Then transfer it to a sterilized 250mL MRS liquid culture medium with a 5% inoculation amount. Place it in a bacterial incubator at 37°C and culture it for 24 hours. Centrifuge the resulting fermentation liquid, then discard the supernatant to collect the bacterial precipitate, rinse the bacterial body twice with sterile phosphate buffer (PBS), each time for 10 minutes, and the centrifugal speed is 5000r / min. The bacterial precipitate of Lactobacillus plantarum ZJUIDS16 can be obtained.

[0120] Weigh 4.1g of trehalose, 2.8g of sodium glutamate, and 8g of sucrose respectively and dissolve them in 10mL of 40℃ distilled water, filter and sterilize them with a 0.22μm microporous filter membrane, then add them into 90mL of sterile water for later use. Dissolve 15g of skim milk powder in distilled water to obtain 100mL of 15% skim milk powder solution, sterilize at 110℃ for later use.

[0121] The prepared Lactobacillus plantarum ZJUIDS16 bacterial precipitate was fully mixed with the protective agent solution at a ratio of 1:5 to obtain a bacterial suspension. 5 mL of the bacterial suspension was divided into 10 mL sterile stoppered glass bottles, pre-frozen at -70°C, taken out after 2 hours and freeze-dried in a vacuum freeze dryer. The freeze-drying conditions were vacuum degree 5 Pa, partition heating temperature 20°C, cold trap temperature -55°C, and freeze-dried for 30 hours before breaking to obtain Lactobacillus plantarum ZJUIDS16 bacterial powder.

[0122] Example 12 Preparation of solid beverage using a 5-methyltetrahydrofolate-producing Lactobacillus plantarum ZJUIDS16

[0123] 3 parts of whole milk powder, 1-2 parts of tea theanine, 4-5 parts of gamma-aminobutyric acid, 0.20 parts of vitamin C, 2-5 parts of compound probiotics, 16.02-36.02 parts of inulin, 5.01-15.01 parts of oligofructose, 7.01-17.01 parts of erythritol, as well as sweeteners, thickeners and flavors are added; the compound probiotics include a mixture of thermophilic streptococcus, Bifidobacterium lactis Bla08, Lactobacillus acidophilus LA85, Lactobacillus plantarum BL21, Lactobacillus plantarum Lp90 and Lactobacillus plantarum ZJUIDS16, each group of substances synergistically enhances the effect, is prepared according to the above proportion, mixed evenly and packaged to obtain the finished product.

[0124] Example 13 Preparation of fruit and vegetable juice fermented by using a 5-methyltetrahydrofolate-producing Lactobacillus plantarum ZJUIDS16

[0125] Select fresh pumpkin and pitaya as raw materials, wash, peel (pumpkin removes pulp), and cut into small pieces. Use flash evaporation to inactivate enzymes, 0.5-1min, 121℃ treatment, and exhaust quickly. According to the ratio of pumpkin: water (weight ratio) = 1:1, gradually put pumpkin and water into the colloid mill for grinding, and grind them coarsely and finely once each. Beat the pitaya with a beater until the pulp is uniform and free of lumps. Blending and homogenization: According to 15% pumpkin juice and 30% pitaya juice, adjust the soluble solid content to 10°Brix with sucrose, add 0.2% stabilizer CMC and mix evenly, use two-stage homogenization method, first low pressure (15MPa), then high pressure (25MPa), so that the diameter of the melon pulp particles is 2-3μm. The prepared composite fruit and vegetable juice is kept at 100℃ for 10min and cooled to about 40℃. Under sterile conditions, inoculate activated Lactobacillus plantarum ZJUIDS16, and control the initial bacterial count at 107 CFU / mL. Ferment at 37°C for 24 hours. After fermentation, place in a 4°C refrigerator for 3 hours. After ripening, fill into 250mL sterilized glass bottles and send to cold storage.

[0126] Example 14 Preparation of a 5-methyltetrahydrofolate-producing Lactobacillus plantarum ZJUIDS16 starter

[0127] The original strain of Lactobacillus plantarum ZJUIDS16 was inoculated into 11% skim milk (sterilized at 115℃ for 20min), cultured at 37℃ for 18-24h until curdled, and activated for two generations continuously as the mother starter. The mother starter was inoculated into 11% skim milk at an inoculum of 3%-5% and sterilized for 20min, cultured at 37℃ for 18-24h until curdled, at which time the number of viable bacteria could reach 10 9 -10 10 CFU / mL, and the Lactobacillus plantarum ZJUIDS16 fermentation agent was obtained.

[0128] Example 15 Preparation of functional fermented milk by fermentation with Lactobacillus plantarum ZJUIDS16

[0129] The pretreated milk was heated to about 60°C, 6% sucrose was added, and after being fully dissolved, homogenized under a pressure of 20MPa. Then, after heat treatment at 95°C for 5 minutes, the milk was cooled to 38°C. The plant lactobacillus ZJUIDS16 starter prepared according to Example 10 was added at a 5% inoculum amount, and the milk was fermented at 37°C for 14-18 hours until the milk curdled. After cooling, the milk was refrigerated at 4°C to obtain the functional fermented milk of plant lactobacillus ZJUIDS16.

[0130] 1. Preparation of Fermented Milk for Determination of Folic Acid Content

[0131] The folic acid content was determined using the method 1.3 in Example 2.

[0132] 2. Preparation and flavor evaluation of fermented milk

[0133] The fermented milk was prepared by strain fermentation as the experimental material. The samples were stored in a 4℃ refrigerator in the laboratory after preparation, and all indicators were measured within the shelf life.

[0134] 3. Sensory evaluation

[0135] 3.1 Screening of evaluators: In view of the persistence of sensory experiments, the recruitment and screening of evaluators are mainly targeted at students majoring in food. According to the method described in GB / T 16291.1-2012, the basic taste recognition ability, observation threshold, and recognition threshold of the evaluators are measured respectively, and finally 6 candidates with sensitive sensory organs are determined, with a male-female ratio of 1:1.

[0136] 3.2 Yogurt sensory descriptors: After sensory training, the final sensory evaluation words for yogurt smell are sweet, sour, milky, creamy, and taste; the sensory evaluation words for taste are sweet, sour, milky, and creamy. Define the above descriptors and determine the reference sample. Select a transparent food-grade plastic cup with a lid, weigh 10g of yogurt sample and put it into 2 tasting cups, and conduct sensory evaluation of smell and taste respectively. The number of yogurt samples evaluated in each test is controlled within 5, and the sample number uses a 3-digit random number. The evaluator rinses his mouth with mineral water after tasting each sample, and repeats each sample twice. Sensory scoring standard (0-9): 0 (none); 1-3 (weak); 4-6 (medium intensity); 7-9 (strong).

[0137] Table 7 Sensory evaluation of strains with different folate production

[0138] Bacteria ZJUIDS16 LZ217 Sweetness <![CDATA[4.3±0.23 a ]]> <![CDATA[5.1±0.14 a ]]> sour taste <![CDATA[4.4±0.13 b ]]> <![CDATA[4.1±0.39 c ]]> Milky flavor <![CDATA[4.4±0.62 a ]]> <![CDATA[3.4±0.13 a ]]> Sour and fatty taste <![CDATA[6.2±0.31 a ]]> <![CDATA[6.1±0.75 a ]]> Steaming flavor <![CDATA[2.2±0.3 a ]]> <![CDATA[4.1±0.3 a ]]> Odor intensity <![CDATA[4.5±0.45 a ]]> <![CDATA[2.2±0.53 a ]]> Preference <![CDATA[6.2±0.23 a ]]> <![CDATA[4.9±0.63 b ]]>

[0139] The results of the odor sensory analysis of the prepared yogurt and commercially available yogurt are shown in Table 3. The odor sensory analysis data show the flavor differences between different commercially available yogurts. The yogurt flavor index score is 3.7-5.6, the cooking flavor score is 1.7-4.2, and the preference score is 2.8-6.7. The milk flavor in the yogurt was measured to be of medium intensity, the milk flavor was weak, and there was also a weak cooking flavor. According to the evaluator's odor sensory preference, sample ZJUIDS16 has a high preference; LZ217 has a moderate preference.

[0140] Example 16 Comparative Example of Yield and Functional Properties of High Folic Acid Producing Strains

[0141] In order to demonstrate the advantages of the present invention in terms of folic acid production and other probiotic functions, the folic acid production capacity and probiotic functions of the strain of the present invention and other disclosed strains were compared. The comparison results are shown in Table 8.

[0142] Table 8 Comparative examples of extracellular folate content of lactic acid bacteria compared with literature disclosure examples

[0143]

[0144]

[0145] As shown in Table 8, Lactobacillus plantarum has a higher folate production than other strains in the published literature. Lactobacillus plantarum has a synthetic gene sequence for high folate production. Therefore, the subsequent experiments using Lactobacillus plantarum have a good theoretical basis and experimental significance, and it has the superiority of strain types in improving the folate production of lactic acid bacteria.

[0146] Since the existing technology mainly adopts common bacterial strains to ferment and produce fermented dairy products, there is a tendency of product homogeneity, focusing on product functions, and lacking products rich in high-quality nutrients. Currently, there are no fermented dairy products made with high-folate-producing bacterial strains, and there are no fermented dairy products with high folic acid content. Therefore, in comparison with commercially available products, the present invention has better originality and innovation.

[0147] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum that produces high levels of folate ( Lactobacillus plantarum )ZJUIDS16, characterized in that, The strain is classified and named as Lactobacillus plantarum, and was deposited in the General Microbiology Center of China Culture Collection Administration on November 21, 2022, with the deposit number: CGMCC NO. 26162.

2. The plant lactobacillus ZJUIDS16 according to claim 1, characterized in that The complete sequence of its 16S rDNA is shown in SEQ ID No.

1.

3. Application of Lactobacillus plantarum ZJUIDS16 according to claim 1 in preparing food and health products.

4. The use according to claim 3, characterized in that: The food includes: fermented fruit and vegetable juice, fermented dairy products, and bacterial powder.

5. The use according to claim 3, characterized in that: The health care product comprises: a bacterial agent or bacterial powder with high folic acid production.

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