Application of lactobacillus fermentum in folic acid production

Abstract

The invention provides an application of lactobacillus fermentum mucus in the aspect of folic acid production. The preservation number of the lactobacillus fermentum mucus TG017 is CCTCC (China Center For Type Culture Collection) NO: M2023513.

Description

Technical Field

[0001] The invention relates to application of fermented mucus lactobacillus in producing folic acid, and belongs to the technical field of microorganisms. Background Art

[0002] Folic acid, also known as vitamin B9 and pteroylglutamic acid, is a water-soluble vitamin composed of pteridine, para-aminobenzoic acid and L-glutamic acid. Folic acid is an essential nutrient for the synthesis of DNA and RNA and is very important for cell division and growth.

[0003] Since folic acid cannot be stored in large quantities in the body, it needs to be taken in regularly through diet or supplements. Some strains also produce folic acid. Yeast and Bacillus are important strains that produce folic acid. In recent years, strains with high folic acid production have become a hot topic of research. Research has focused on isolating folic acid-producing lactic acid bacteria from human colostrum and studying their probiotic potential. For example, Lactobacillus plantarum CKR26 has a high folic acid production.

[0004] Limosilactobacillus fermentum is a probiotic with beneficial functions. It has important value in maintaining and promoting human health, especially in regulating the balance of intestinal microecology, enhancing immunity and preventing diseases. It can strengthen the intestinal barrier by promoting mucus secretion and enhancing the expression of tight junction proteins, thereby protecting intestinal epithelial cells from pathogens. Limosilactobacillus fermentum can secrete antibacterial substances such as organic acids and bacteriocins, which can inhibit or kill pathogens and reduce the colonization of pathogens in the intestine. These beneficial functions make Limosilactobacillus fermentum a hot topic in product research in the food and pharmaceutical fields.

[0005] However, there is no fermentative Lactobacillus mucilaginosus capable of producing high amounts of folic acid in the prior art. Summary of the invention

[0006] The present invention provides an application of fermented mucus lactobacillus in producing folic acid, which can effectively solve the above problems.

[0007] The present invention is achieved in that:

[0008] The invention discloses an application of fermented Lactobacillus mucilaginosus TG017 in producing folic acid. The preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0009] In some embodiments, the fermented Lactobacillus mucilaginosus TG017 is fermented in a culture medium, and folic acid is extracted from the supernatant of the culture medium.

[0010] In some embodiments, the culture medium is MRS medium.

[0011] In some embodiments, the fermentation time of the fermentation is 40-60 hours.

[0012] In some embodiments, the method of extracting folic acid is extraction.

[0013] In some embodiments, the extraction step is: adding a mixture of methyl trioctyl ammonium chloride-isoamyl alcohol and methanol to the supernatant of the fermentation broth and mixing, removing the aqueous phase after centrifugation, then adding NaOH solution and mixing, and then adding methanol and mixing to obtain a folic acid separation product.

[0014] The invention discloses an application of fermented Lactobacillus mucilaginosus TG017 in preparing a probiotic product for supplementing folic acid. The preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0015] The invention discloses an application of fermented Lactobacillus mucilaginosus TG017 in preparing a product containing folic acid. The preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0016] A method for extracting folic acid comprises adding a mixed solution of methyl trioctyl ammonium chloride-isoamyl alcohol and methanol to a primary folic acid product and mixing the mixture evenly, removing the aqueous phase after centrifugation, adding a NaOH solution and mixing the mixture evenly, and then adding methanol and mixing the mixture evenly to obtain a folic acid separation product.

[0017] In some embodiments, in the ammonium chloride-isoamyl alcohol mixture, the molar ratio of methyltrioctylammonium chloride to isoamyl alcohol is 1:3.5-4.5.

[0018] The beneficial effects of the present invention are:

[0019] The fermented mucus Lactobacillus strain TG017 of the present invention has an outstanding high folic acid production capacity. After fermentation and culture in a normal MRS medium, folic acid is extracted using the liquid-liquid method provided by the present invention, and the folic acid yield of the fermentation liquid supernatant after extraction can reach 33.12 μg / mL.

[0020] The liquid-liquid method provided by the invention is used for folic acid extraction, and the extraction rate can reach 59.36%.

[0021] The strain comes from the human intestine and is stable and safe. The strain has a strong resistance to gastrointestinal fluid, and its survival rate in the human body can be guaranteed. Combined with its high folic acid production capacity and its probiotic function as a probiotic, it can better maintain and improve the health of the human intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the growth curve of fermentative Lactobacillus mucilaginosus TG017.

[0024] Figure 2 This is the standard curve for the determination of folic acid content by microbiological method.

[0025] Figure 3 This is the peak diagram of the standard curve concentration gradient folate standard solution determined by high performance liquid chromatography.

[0026] Figure 4 This is the peak diagram of HPLC determination of folic acid standard extract and blank culture medium extract.

[0027] Figure 5 This is the peak diagram of the bacterial supernatant extract measured by HPLC. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0029] The embodiment of the present invention provides an application of fermented Lactobacillus mucilaginosus TG017 in producing folic acid, and the preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0030] The accession number represents the specific identity and origin of the strain, which helps us to track and trace the source. By ensuring the purity and stability of the strain, we can further guarantee the effectiveness of the strain in producing folic acid.

[0031] In some embodiments, the sequence of 16S rDNA of the fermentative Lactobacillus mucilaginosus TG017 is shown as SEQ ID NO: 1.

[0032] SEQ ID NO: 1:

[0033] GTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGGTTTCACATCAGAATTAAAAAAACCGCCTGGCCTCTCTTTTCGCCCCAATAAATCCGGATAACGCTTGGCACCTACGTA

[0034] In some embodiments, a strain of the fermented mucus Lactobacillus TG017 is screened from domestic healthy human feces samples, and the colonies are milky white, opaque, with round protrusions, a smooth surface and neat edges, and the optimal growth temperature is 37°C, the optimal pH is 6 and the anaerobic environment.

[0035] In some embodiments, the fermented Lactobacillus mucilaginosus TG017 is fermented in a culture medium, and folic acid is extracted from the supernatant of the culture medium.

[0036] In some embodiments, the culture medium is MRS medium.

[0037] In some embodiments, the MRS medium formula is: casein digest 10 g / L; beef extract powder 10 g / L; ammonium citrate trihydrate 4 g / L; sodium acetate 5 g / L; magnesium sulfate heptahydrate 0.2 g / L; manganese sulfate tetrahydrate 0.05 g / L; potassium hydrogen phosphate 2 g / L; glucose 20 g / L; Tween-80 1.08 g / L; final pH = 5.7 ± 0.2, sterilized at 115 ° C for 25 min.

[0038] In some embodiments, the seed solution is diluted to an OD of 600 When the concentration reaches 0.8-1.0, inoculate into MRS liquid culture medium at an inoculation rate of 1-5%.

[0039] During the inoculation process, the inoculation amount is controlled to ensure that the strain is evenly distributed in the culture medium, which is beneficial to the subsequent fermentation process.

[0040] In some embodiments, the fermentation time of the fermentation is 40-60 hours.

[0041] During the fermentation time, the fermented Lactobacillus mucilaginosus TG017 can fully grow and metabolize to produce a higher yield of folate primary products. After the culture is completed, we collect metabolites to obtain the desired folate primary products for subsequent folate extraction.

[0042] The embodiment of the present invention aims to explore an efficient and reliable process to meet the demand for high folic acid production by the strain. Through carefully designed culture conditions and steps, it is ensured that the fermented mucus Lactobacillus TG017 can grow stably and produce high folic acid during the fermentation process.

[0043] In some embodiments, the method of extracting folic acid is extraction.

[0044] In some embodiments, the extraction step is: adding a mixture of methyl trioctyl ammonium chloride-isoamyl alcohol and methanol to the supernatant of the fermentation broth and mixing, removing the aqueous phase after centrifugation, then adding NaOH solution and mixing, and then adding methanol and mixing to obtain a folic acid separation product.

[0045] The embodiment of the present invention extracts and concentrates folic acid in the supernatant of the fermentation broth by liquid-liquid extraction, and the extraction rate is high.

[0046] In some embodiments, the application includes: a method for determining folic acid concentration, comprising:

[0047] The folic acid concentration in the extract was determined using high performance liquid chromatography, wherein the detection wavelength was 280 nm;

[0048] The elution program was: 0.0 to 8.0 minutes, 100% stage A; 8.0 to 15.0 minutes, linear gradient increasing from 0% stage B to 100% stage B; 15.0 to 20.0 minutes, maintaining 100% stage B; 20.0 to 21.0 minutes, linear gradient decreasing from 100% stage B to 0% stage B; 21.0 to 25.0 minutes, maintaining 100% stage A;

[0049] Mobile phase A was a mixture of 20 mmol / L potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 9:1, and the pH value of the mixture was adjusted to 3.0. Mobile phase B was a mixture of acetonitrile and ultrapure water in a volume ratio of 8:2.

[0050] There are many methods for determining folic acid, including microbiological method, high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), fluorescence method and electrochemical analysis. The microbiological method is a traditional determination method that uses the growth of folic acid-dependent microorganisms (such as Lactobacillus rhamnosus) to determine the folic acid content in the sample. The advantage of this method is that the operation is relatively simple, but the disadvantage is that it is time-consuming and the determination of folic acid concentration will be affected by many factors. High performance liquid chromatography (HPLC) can accurately determine the folic acid content in food, including different forms of folic acid derivatives. This method has high sensitivity and accuracy.

[0051] The embodiment of the present invention provides a method for extracting folic acid, comprising adding a mixed solution of methyl trioctyl ammonium chloride-isoamyl alcohol and methanol to a primary folic acid product and mixing the mixture evenly, removing the aqueous phase after centrifugation, adding a NaOH solution and mixing the mixture evenly, and then adding methanol and mixing the mixture evenly to obtain a folic acid separation product.

[0052] The method extracts and concentrates folic acid in the primary folic acid product, improves the extraction method, and has a high extraction rate.

[0053] Deep Eutectic Solvents (DES) are a mixture formed by hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD) through hydrogen bonding. They have the advantages of being environmentally friendly, simple to prepare, low cost, and biodegradable, and have been widely used in extraction and separation technologies.

[0054] DES can be used as an extractant or dispersant during the extraction process. Compared with traditional extraction methods, the extraction method using DES has the advantages of less demand for organic reagents and higher extraction efficiency.

[0055] In some embodiments, in the ammonium chloride-isoamyl alcohol mixture, the molar ratio of methyltrioctylammonium chloride to isoamyl alcohol is 1:3.5-4.5.

[0056] The polarity of DES can be controlled by adjusting the molar ratio of its components, thereby achieving efficient extraction of compounds with different polarities.

[0057] An embodiment of the present invention provides an application of fermented Lactobacillus mucilaginosus TG017 in preparing a probiotic product for supplementing folic acid, and the preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0058] An embodiment of the present invention provides a probiotic product for supplementing folic acid. The probiotic product includes fermented Lactobacillus mucilaginosus TG017. The preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0059] Fermented Lactobacillus mucis TG017 comes from the human strain library and is on the list of edible probiotics, so it has no safety risks for human consumption. It has the advantages of low cost, fast growth rate, large volume, small impact, and strong biological accessibility.

[0060] In some embodiments, the probiotic product is a composition of fermented Lactobacillus mucilaginosus TG017 and other ingredients.

[0061] The ingredients include one or more of prebiotics, fillers, acidulants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, and filter aids.

[0062] The dosage form of the probiotic product can be a solid beverage, a beverage, a compressed candy, a granule, a capsule, a tablet, a pill or an oral liquid.

[0063] The embodiment of the present invention provides an application of fermented Lactobacillus mucilaginosus TG017 in preparing a product containing folic acid, and the preservation number of the fermented Lactobacillus mucilaginosus TG017 is CCTCC NO: M2023513.

[0064] The folic acid yield in the supernatant of the fermented Lactobacillus mucius TG017 culture liquid after extraction can reach 33.12 μg / mL. This strain has a high folic acid yield, and the folic acid produced can be used to prepare products containing folic acid.

[0065] In some embodiments, the product is selected from any one of health products, food or medicine.

[0066] In some embodiments, the dosage form of the product includes injection, suspension, powder, tablet or granule.

[0067] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier;

[0068] In some embodiments, the pharmaceutically acceptable carrier includes any one of a diluent, an excipient, a filler, a binder, a lubricant, and a disintegrant, or a combination of at least two thereof.

[0069] Example 1: Identification of Lactobacillus mucilaginosus TG017.

[0070] A strain of Lactobacillus mucilaginosus TG017 was deposited in the China Center for Type Culture Collection, located at the Wuhan University Collection Center, on April 10, 2022. The deposit number is CCTCC NO: M2023513.

[0071] The single colony of the fermented Lactobacillus mucilaginosus TG017 was streaked and purified on MRS agar plates for 3 rounds.

[0072] The purified single colony was inoculated into MRS liquid culture medium and cultured at 37°C, 200 rpm shaking for 48 hours to obtain bacterial liquid. The bacterial liquid was inoculated at 1% volume on FACM medium containing bromocresol purple, and the colonies in the culture medium plate were repeatedly verified to see whether they could change from dark purple to yellow.

[0073] Example 2: Physiological characteristics of fermented Lactobacillus mucilaginosus TG017.

[0074] 1. Growth curve determination:

[0075] ① Activation of bacterial strains: Take the fermented mucus Lactobacillus TG017 out of the -80°C refrigerator, activate it by streaking on an MRS agar plate using a disposable inoculation loop, and culture it in an anaerobic incubator at 37°C until a single colony grows.

[0076] ② Inoculation liquid: Use a disposable inoculation loop to dip a single colony on the MRS agar plate, inoculate it into MRS liquid culture medium, shake it evenly, and culture it in a shaker at 37°C, 200 rpm for 24 hours.

[0077] ③Add culture medium: dilute the seed solution to OD 600 When the inoculation volume reaches 0.8-1.0, the culture medium is inoculated with 1% of the inoculation volume into MRS liquid culture medium, and 12 groups of culture medium are inoculated. After the inoculation, the culture medium is placed in a shaking incubator at 37°C and 200 rpm for culture, and one group is taken out every 2 hours to measure the OD 600 , until the strain completes logarithmic growth and reaches the plateau phase.

[0078] The growth curve of fermented Lactobacillus mucilaginosus TG017 is as follows Figure 1 shown.

[0079] As can be seen from the figure, 2-8h is the logarithmic growth phase of the strain, and 14h enters the plateau phase.

[0080] 2. Drug sensitivity test

[0081] Dilute the seed solution in 1② to OD 600 When the concentration reaches 0.8-1.0, take 50 μL and spread it evenly on the MRS solid medium. After the bacterial solution is dry, use tweezers to pick up the drug-sensitive tablets and distribute them regularly on the MRS medium. Use tweezers to gently press the drug-sensitive tablets to prevent them from falling off. After anaerobic culture for 24 hours, observe and record the size of the inhibition zone.

[0082] The experimental results are shown in Table 1. Fermented mucus Lactobacillus TG017 was sensitive to 11 drug-sensitive tablets, intermediate to 2 drug-sensitive tablets, and resistant to 4 drug-sensitive tablets.

[0083] Table 1 Drug sensitivity test of fermented mucus Lactobacillus TG017

[0084]

[0085] Example 3: Liquid-liquid extraction of folic acid

[0086] The screened fermented mucus Lactobacillus TG017 was inoculated into MRS liquid culture medium, and cultured at 37° C. and 200 rpm in a shaking incubator for 48 hours to obtain a bacterial liquid.

[0087] Reagent preparation:

[0088] Preparation of deep eutectic solvent (DES): methyl trioctyl ammonium chloride: isoamyl alcohol = 10 mmol / L: 40 mmol / L, that is, 4.55 mL of methyl trioctyl ammonium chloride and 4.35 mL of isoamyl alcohol were mixed and heated in a 50°C water bath for 20 min.

[0089] 0.5mmol / L NaOH solution: Accurately weigh 0.2g NaOH on a balance and dissolve it in 10mL of deionized water.

[0090] 1 mg / mL folic acid standard solution: Accurately weigh 10 mg of the standard on a balance, add 100 μL of 0.5 mmol / L NaOH solution, and dilute to 10 mL with deionized water.

[0091] Standard curve concentration gradient folic acid standard solution: gradient dilute 1 mg / mL folic acid standard solution to 700 μg / mL, 500 μg / mL, 200 μg / mL, 100 μg / mL, 10 μg / mL, and 1 μg / mL.

[0092] Folic Acid Extract:

[0093] Extraction of folic acid standard: Take 5mL of 100μg / mL folic acid standard solution in a 15mL centrifuge tube, add 100μLDES, 200μL methanol, and vortex mix for 1min. Centrifuge the mixture at 5000rpm for 10min to separate the aqueous phase from the DES phase. Use a syringe to carefully remove the aqueous phase, then add 150μL of 0.5mmol / L NaOH solution, mix thoroughly to dissolve, then add methanol to the 1mL mark, mix thoroughly to dissolve, and obtain the folic acid standard extract, which is stored at 4℃ away from light.

[0094] Extraction of bacterial supernatant: Take 5 mL of bacterial supernatant in a 15 mL centrifuge tube, add 100 μL DES and 200 μL methanol, and vortex mix for 1 min. Centrifuge the mixture at 5000 rpm for 10 min to separate the aqueous phase from the DES phase. Use a syringe to carefully remove the aqueous phase, then add 150 μL 0.5 mmol / L NaOH solution, mix thoroughly to dissolve, then add methanol to the 1 mL mark, mix thoroughly to dissolve, and obtain the bacterial supernatant extract, which is stored at 4°C away from light.

[0095] Blank culture medium extraction: Take 5mL MRS liquid culture medium in a 15mL centrifuge tube, add 100μL DES, 200μL methanol, and vortex mix for 1min. Centrifuge the mixture at 5000rpm for 10min to separate the aqueous phase from the DES phase. Use a syringe to carefully remove the aqueous phase, then add 150μL 0.5mmol / L NaOH solution, mix thoroughly to dissolve, then add methanol to the 1mL mark, mix thoroughly to dissolve, and obtain the blank culture medium extract, which is stored at 4℃ away from light.

[0096] Example 4: Determination of folic acid content by microbiological method

[0097] The folic acid content in the fermentation supernatant of the screened fermented Lactobacillus mucilaginosus TG017 was measured.

[0098] Reagent preparation:

[0099] 20% ethanol solution: Measure 200 mL of anhydrous ethanol and mix with 800 mL of water.

[0100] Sodium hydroxide ethanol solution (0.01 mol / L): Weigh 0.4 g of sodium hydroxide, dissolve it to 1 L with 20% ethanol solution, and mix well.

[0101] Folic acid standard stock solution (20.0 μg / mL): Accurately weigh 20.0 mg of folic acid standard, dissolve it with sodium hydroxide ethanol solution, transfer and dilute to a 1000 mL brown volumetric flask. Store in a refrigerator at 2°C-4°C away from light.

[0102] Folic acid standard intermediate solution (0.200 μg / mL): Accurately pipette 1.00 mL of folic acid standard stock solution into a 100 mL brown volumetric flask, dilute to volume with sodium hydroxide ethanol solution, mix well and store in a refrigerator at 2°C to 4°C away from light.

[0103] Folic acid standard working solution (0.200ng / mL): Accurately pipette 1.00mL of folic acid standard intermediate solution into 1000mL, dilute with water to the mark in a volumetric flask, mix well and use immediately.

[0104] Bacterial culture medium for folic acid determination: 40 μL of LGG (standard strain of Lactobacillus rhamnosus) bacterial solution (OD600=0.8-1.0) was added to 10 mL of culture medium and mixed for later use.

[0105] In a 15mL centrifuge tube, add 0.00mL, 0.25mL, 0.50mL, 1.00mL, 1.50mL, 2.00mL, 2.50mL, 3.00mL, 4.00mL, and 5.00mL of folic acid standard working solution, respectively, and make up to 5mL with water, which is equivalent to a folic acid content of 0.00ng, 0.05ng, 0.10ng, 0.20ng, 0.30ng, 0.40ng, 0.50ng, 0.60ng, 0.80ng and 1.00ng in the centrifuge tube; mix well to obtain standard assay solutions of each concentration gradient.

[0106] Add 0.5 mL of bacterial supernatant to a 50 mL centrifuge tube, add 20 mL of sodium hydroxide ethanol solution (0.01 mol / L), and ultrasonicate for 1 h. Transfer to a 100 mL volumetric flask and dilute to 100 mL with ultrapure water.

[0107] Under aseptic operation conditions, add 5 mL of standard assay solution of each concentration gradient and 5 mL of TG017 bacterial supernatant diluted after ultrasonic treatment to a 15 mL centrifuge tube with 5 mL of folic acid assay bacterial culture medium, and mix well. Incubate at 37°C, 200 rpm shaker for 24 hours. After the culture is completed, take 200 μL and measure the absorbance at 540 nm.

[0108] In this embodiment, the diluted bacterial liquid supernatant is an unextracted bacterial liquid supernatant.

[0109] Three sets of repeated experiments were performed for each concentration gradient of the standard solution. When drawing the standard curve, the average value of each standard point was used for calculation. With folic acid content as the horizontal axis, the average absorbance value of each standard point was OD 540 Draw the standard curve with y as the vertical axis.

[0110] The standard curve for determining folic acid content by microbiological method is as follows Figure 2 The formula is y = 0.1252x + 0.0052, R 2 =0.995.

[0111] The calculation formula for determining folic acid content by microbiological method is:

[0112] Where, C is the concentration of folic acid in the sample (ng / mL); C 1 is the folic acid content (ng) calculated from the standard curve; V is the volume of the sample when preparing the assay solution (mL).

[0113] The concentration of the supernatant of the bacterial solution diluted 1000 times was 3.49 μg / mL, and the concentration of the supernatant of the bacterial solution diluted 5000 times was 2.60 μg / mL.

[0114] Because the measured dilution multiples are too many and the folic acid concentration far exceeds the standard curve, the measured data is not accurate. Other methods are needed to determine the folic acid content in the fermentation supernatant of fermented Lactobacillus mucilaginosus TG017.

[0115] Example 5: Determination of folic acid production by high performance liquid chromatography (HPLC)

[0116] Reagent preparation:

[0117] 20mmol / L potassium dihydrogen phosphate solution: Accurately weigh 2.7218g of potassium dihydrogen phosphate on a balance, dilute to 1L with ultrapure water, and adjust the pH value to 3.0.

[0118] Mobile phase preparation:

[0119] A: 20mmol / L potassium dihydrogen phosphate solution: acetonitrile = 9:1 (V:V). pH = 3

[0120] B: acetonitrile: ultrapure water = 8:2 (V:V).

[0121] Elution program: 0.0 to 8.0 minutes, 100% step A; 8.0 to 15.0 minutes, linear gradient increasing from 0% step B to 100% step B; 15.0 to 20.0 minutes, maintaining 100% step B; 20.0 to 21.0 minutes, linear gradient decreasing from 100% step B to 0% step B; 21.0 to 25.0 minutes, maintaining 100% step A.

[0122] HPLC conditions: The HPLC instrument was Waters, the chromatographic column was ACQUITY UPLC-C18 4.6×250 mm, the flow rate was 1 mL / min, the column temperature was 27°C, the injection volume was 10 μL, and the measurement wavelength was 280 nm.

[0123] HPLC was used to measure the folic acid standard solutions with different standard curve concentration gradients and a standard curve was drawn.

[0124] The concentration gradient of the standard curve was 1 μg / mL, 10 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 700 μg / mL, and 1 mg / mL.

[0125] The peak diagram of the standard curve concentration gradient determination is as follows Figure 3 As shown in Figure 2. The retention time of folic acid is 5 minutes.

[0126] The peak areas of the standard samples with concentrations of 1 μg / mL, 10 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 700 μg / mL, and 1 mg / mL are 21500, 252740, 2724606, 5372863, 13669238, 20124356, and 27978503, and the formula is y=2.82×10 4 x-9.84×10 4 , standard curve R 2 =0.999, which is consistent with the linear relationship of the standard curve.

[0127] The folic acid standard extract, bacterial supernatant extract and blank culture medium extract after extraction in Example 4 were measured by HPLC.

[0128] It should be pointed out that the corresponding folic acid content of the folic acid standard extract before extraction is 500 μg.

[0129] The results of the test are as follows Figure 4 and 5 shown.

[0130] Figure 4The peak area of ​​the extracted folic acid standard solution was 8064764, and the peak area of ​​the unextracted 500μg / mL folic acid standard solution was 13585713, and the calculated extraction rate was 59.36%. The peak area of ​​the blank MRS liquid culture medium after extraction was 381.

[0131] Figure 5 In the figure, the peak area corresponding to the supernatant of the extracted bacterial solution is 4520485.

[0132] Substituting the standard curve and extraction rate into the calculation, it can be obtained that the folic acid yield in the supernatant of the fermented Lactobacillus mucosa TG017 culture liquid after extraction is 33.12 μg / mL, indicating that the strain has a high folic acid yield.

[0133] In summary, the present invention extracts folic acid from the metabolites of fermented Lactobacillus mucilaginosus TG017 to obtain a folic acid separation product with a high extraction rate, and determines its folic acid content by high performance liquid chromatography, and it is found that the strain has a high folic acid yield.

[0134] The strain's high folate production capacity, combined with its stability and safety from human sources, its lack of toxicity, and its strong resistance to gastrointestinal fluids, can provide more options for subsequent drug dosage forms.

[0135] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of fermented Lactobacillus mucilaginosus TG017 in producing folic acid, characterized in that: The deposit number of the fermented mucus lactobacillus TG017 is CCTCC NO: M2023513.

2. The use according to claim 1, characterized in that: The fermented Lactobacillus mucilaginosus TG017 is fermented in a culture medium, and folic acid is extracted from the supernatant of the culture medium.

3. The use according to claim 2, characterized in that: The culture medium is MRS culture medium.

4. The use according to claim 2, characterized in that: The fermentation time of the fermentation is 40-60h.

5. The use according to claim 2, characterized in that: The method for extracting folic acid is extraction.

6. The use according to claim 5, characterized in that: The extraction step is: adding a mixed solution of methyl trioctyl ammonium chloride-isoamyl alcohol and methanol to the supernatant of the fermentation liquid and mixing, removing the aqueous phase after centrifugation, then adding NaOH solution and mixing, and then adding methanol and mixing to obtain a folic acid separation product.

7. An application of fermented Lactobacillus mucilaginosus TG017 in preparing a probiotic product for supplementing folic acid, characterized in that: The deposit number of the fermented mucus lactobacillus TG017 is CCTCC NO: M2023513.

8. An application of fermented Lactobacillus mucilaginosus TG017 in preparing a product containing folic acid, characterized in that: The deposit number of the fermented mucus lactobacillus TG017 is CCTCC NO: M2023513.

9. A method for extracting folic acid, characterized in that: A mixed solution of methyltrioctylammonium chloride-isoamyl alcohol and methanol are added to the primary folic acid product and mixed evenly. The aqueous phase is removed after centrifugation, and then a NaOH solution is added and mixed evenly. Then, methanol is added and mixed evenly to obtain a folic acid separation product.

10. The method according to claim 9, characterized in that In the ammonium chloride-isoamyl alcohol mixed solution, the molar ratio of methyl trioctyl ammonium chloride to isoamyl alcohol is 1:3.5-4.5.

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