Culture medium for improving efficiency of transforming ellagic acid to generate urolithin A by lactobacillus mucus FUA033 and application of culture medium

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a culture medium for improving the efficiency of converting ellagic acid into urolithin A by lactobacillus mucus FUA033 and application of the culture medium. On the basis of a fermentation technology, under the same fermentation condition, the efficiency of converting ellagic acid to generate urolithin A by fermenting lactobacillus mucus FUA033 is improved by 21.13% and reaches 75.21% by optimizing a culture medium. The lactobacillus mucilaginosus FUA033 provides a technical support for preparing urolithin A through industrial green fermentation of the lactobacillus mucilaginosus FUA033 and developing fermented food rich in urolithin A, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to a culture medium for improving the efficiency of fermenting Lactobacillus mucilaginosus FUA033 in converting ellagic acid to produce urolithin A and an application thereof. Background Art

[0002] Ellagic acid is a polyphenolic compound that is widely found in fruits and nuts such as pomegranates, strawberries, walnuts, and chestnuts. It has low water solubility, which makes it difficult to absorb and utilize. Ellagic acid is converted into urolithin by human intestinal microorganisms, especially urolithin A, which has the effects of delaying aging, alleviating muscle decline, anti-inflammatory, anti-tumor, and anti-neurodegenerative diseases, and its nutritional efficacy is significantly improved. However, individual differences in human intestinal flora lead to significant differences in the ability to convert ellagic acid to urolithin A. Only about 40% of people over 40 years old can convert ellagic acid to urolithin A, and it tends to decrease with age. Therefore, in 2018, the U.S. Food and Drug Administration approved urolithin A as a nutritional supplement. Amazon (https: / / www.amazon.com) has developed chemically synthesized urolithin A as a nutritional enhancer product, which is favored by consumers.

[0003] The urolithin A currently on sale is all chemically synthesized. The chemical synthesis of urolithin A is prone to environmental pollution. If a safe strain can be obtained for in vitro fermentation to convert ellagic acid to urolithin A, urolithin A can be prepared green and environmentally friendly by microbial fermentation, or a food rich in ellagic acid can be fermented by the strain, and functional fermented foods rich in urolithin A and probiotics can be prepared, so that people can take in urolithin and probiotics in their daily diet, thereby helping to delay muscle decline and improve overall health. Therefore, probiotics that convert ellagic acid to urolithin A have excellent potential for application. However, there are few recognized safe strains reported for converting ellagic acid to urolithin A, only fermented mucus lactobacillus FUA033, thermophilic streptococcus FUA329, fermented mucus lactobacillus CCFM1286, CCFM1290, CCFM129. Among them, fermented mucus lactobacillus FUA033 has good probiotic properties, high efficiency in converting ellagic acid to urolithin A, and stable fermentation performance, but there is still room for improvement in conversion efficiency. Therefore, it is necessary to further develop a culture medium that improves the efficiency of converting ellagic acid to produce urolithin A by fermenting Lactobacillus mucilaginosus FUA033, so as to provide technical support for the industrial green fermentation preparation of urolithin A using fermenting Lactobacillus mucilaginosus FUA033 and the development of fermented foods rich in urolithin A. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a culture medium and application thereof for improving the efficiency of converting ellagic acid to produce urolithin A by fermenting Lactobacillus mucilaginosus FUA033.

[0005] The technical scheme of the present invention is as follows: a culture medium for efficiently converting ellagic acid to produce urolithin A, the components comprising: 10.0 g / L of trypticase peptone, 10.0 g / L of gelatin peptone, 5.0 g / L of yeast extract powder, 5.0 g / L of sodium chloride, 1.0 g / L of glucose, 1.0 g / L of L-arginine, 1.0 g / L of sodium pyruvate, 0.5 mg / L of vitamin K, 5.0 mg of hemin chloride, 0.16% (V / V) of SL-10 trace element solution, 0.21% (V / V) of Wolfe's vitamin solution, and methyl viologen added to a final concentration of 0.16 mM.

[0006] A method for converting ellagic acid to produce urolithin A by fermenting Lactobacillus mucilaginosus FUA033, comprising the following steps:

[0007] 1) Activation of bacterial strains: The fermented Lactobacillus mucilaginosus FUA033 stored in a -80°C refrigerator was streaked with three zones on a WAM anaerobic broth solid medium and incubated at 37°C for 48 hours;

[0008] 2) Seed culture: Pick a single colony and inoculate it into WAM liquid medium, and incubate it anaerobically at 37°C for 72 hours as seed liquid;

[0009] 3) Fermentation and conversion of ellagic acid to produce urolithin A: The seed liquid of fermentation Lactobacillus mucilaginosus FUA033 was inoculated into the above culture medium and cultured anaerobically.

[0010] 4) Extraction of urolithin A: centrifuge, extract the fermentation broth with an equal volume of ethyl acetate, acidify with formic acid, separate the upper organic phase and rotary evaporate to obtain the product.

[0011] Preferably, in step 3), the inoculation amount is 2%.

[0012] Preferably, in step 3), the culture temperature under anaerobic conditions is 38° C. and the culture time is 48 h.

[0013] Preferably, in the step 3), the initial pH of the culture under anaerobic conditions is 6.8.

[0014] Preferably, in step 4), the centrifugation conditions are 12000×g and 4° C. for 8 min.

[0015] The invention discloses an application of the culture medium in preparing urolithin A.

[0016] Beneficial effects of the present invention: The culture medium of the present invention significantly improves the efficiency of fermenting Lactobacillus mucilaginosus FUA033 in converting ellagic acid to urolithin A. Under the same fermentation conditions, compared with using WAM liquid culture medium as the fermentation medium, the conversion rate of converting ellagic acid to urolithin A increased by 21.13% to 75.21%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Standard curve for quantitative HPLC determination of urolithin A

[0018] Figure 2 Effects of trace elements on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0019] Figure 3 Effect of methyl viologen on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0020] Figure 4 Effects of vitamins on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0021] Figure 5 Effects of amino acids on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0022] Figure 6 Effect of hydrogen-containing water on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033 DETAILED DESCRIPTION

[0023] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0024] Fermented mucus Lactobacillus FUA033 is disclosed in CN117487707A. The strain has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 13, 2023, with the deposit number CGMCC NO.28447. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the contact number is 010-64807850.

[0025] Ellagic acid standard (content ≥ 95%) and urolithin A standard (content ≥ 97%) were purchased from Sigma-Aldrich, USA; ethyl acetate and anaerobic broth (Wilkins-Chalgren Anaerobe Broth, WAM) were purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; SL-10 trace element solution and Wolfe's vitamin solution were purchased from Beijing Coolaibo Technology Co., Ltd.; compound amino acid injection (14AA-SF) was purchased from Hubei Yibantian Pharmaceutical Co., Ltd.

[0026] Optimization of fermentation components and conditions of strain FUA033 of the present invention.

[0027] Preparation of strain seed solution: The fermentative mucus Lactobacillus FUA033 preserved in glycerol at -80°C was streaked into three zones on WAM solid culture medium, incubated anaerobically at 37°C for 48 h, a single colony was picked and inoculated into WAM liquid culture medium, and incubated anaerobically at 37°C for 72 h as seed solution.

[0028] The strain FUA033 was inoculated with a strain inoculum of 2% to a final concentration of 15 μM EA (ellagic acid) in a volume of 10 ml WAM liquid medium, and liquid paraffin oil was used to seal the oil. The fermentation temperature was 37 ° C for 48 hours. No nutrient elements were added to explore the effects of different amounts of trace element solution, methyl viologen solution, vitamin solution, amino acid solution and hydrogen-containing water on the conversion rate of urolithin A. The single factor levels are as follows: trace elements (0%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%), methyl viologen solution (0mM, 0.05mM, 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM), vitamin solution (0%, 0.05%, 0.1%, 0. 15%, 0.2%, 0.25%, 0.3%, 0.35%), amino acid solution (0%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%) and hydrogen-containing water (0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%). The above concentrations or percentages are the final concentrations or percentages of the substances in the culture medium.

[0029] Extraction of urolithin A: After centrifugation, the fermentation broth was extracted with an equal volume of ethyl acetate. After acidification with 1.5% formic acid, the upper organic phase was separated and rotary evaporated, and 1 mL of organic solvent (acetonitrile: water: formic acid = 80:19.9:0.1, V:V:V) was used for re-dissolution, filtered with a 0.22 μm syringe filter, and placed in a sampling bottle.

[0030] Determination of urolithin A: The samples before and after fermentation of L.fermentum FUA033 were analyzed by high performance liquid chromatography to measure the content of urolithin A. Chromatographically pure acetonitrile was used as phase A and 1% formic acid in water as the mobile phase, and separation was performed on an Agilent ZORBAX SB-C18 column by gradient elution. The injection volume was 5 μL, the flow rate was 1 mL / min, the column temperature was 35 °C, and the detection wavelength was set to 305 nm for chromatographic analysis. The gradient elution program was: 0-15 min, 0-20% acetonitrile; 15-20 min, 20-70% acetonitrile; 20-21 min, 70-95% acetonitrile; 21-24 min, 95-100% acetonitrile; 24-25 min, 100-20% acetonitrile. With different concentration gradients of urolithin A as the horizontal coordinate and the peak area determined by HPLC as the vertical coordinate, a standard curve was drawn to obtain the curve fitting regression equation.

[0031] Calculation of conversion rate of ellagic acid into urolithin A

[0032]

[0033] Where: η is the conversion rate, C Uro-A is the concentration of urolithin A, C EA is the initial concentration of ellagic acid.

[0034] 1. Quantitative determination of urolithin A by high performance liquid chromatography

[0035] The standard curve was drawn with different concentration gradients of urolithin A as the horizontal axis and the peak area determined by HPLC as the vertical axis. The results are shown in Figure 1 As shown, the curve fitting regression equation y = 5.92632x + 2.13943, R 2 =0.99967.

[0036] 2. Effects of trace elements on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0037] When the trace element addition amount was 0.15%, strain FUA033 had the best effect in converting ellagic acid to urolithin A. The results are shown in the attached Figure 2 As shown, it reaches 63.76%. Trace elements are in a state of deficiency and cannot meet the needs of enzymatic reactions, resulting in limited activity of related enzymes and low conversion rates. However, if the concentration of trace elements is too high, some metal ions may competitively bind to the catalytic center of the enzyme, hinder substrate binding, and reduce the efficiency of the enzymatic reaction.

[0038] 3. Effect of methyl viologen on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0039] As attached Figure 3As shown, with the increase of methyl viologen concentration, the conversion rate of urolithin A gradually increases. When the methyl viologen concentration is 0.15mM, the conversion rate of urolithin A is the highest, reaching 63.34%. The present invention finds that low concentration of methyl viologen can enhance the efficiency of electron transfer, promote enzyme-catalyzed reduction reaction, and improve the conversion rate of urolithin A. However, too high methyl viologen concentration has an inhibitory effect on the strain, resulting in a decrease in metabolic efficiency. This may be related to the strong oxidizing property of methyl viologen, and excessive oxidants interfere with the normal metabolic process of cells.

[0040] 4. Effect of vitamins on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0041] As attached Figure 4 As shown, when the vitamin addition amount is 0.20%, the strain FUA033 has the best effect of converting ellagic acid to urolithin A, reaching 61.47%. The present invention finds that appropriate vitamin supplementation not only accelerates fermentation, but also maintains strain vitality by regulating the antioxidant mechanism in the cell, thereby improving fermentation stability. At lower concentrations, the addition of vitamins can supplement the required cofactors and activate the catalytic activity of the enzyme, thereby improving the conversion efficiency and promoting the conversion of ellagic acid to urolithin A. However, excessive vitamins may interfere with the normal metabolism of cells.

[0042] 5. Effect of amino acids on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0043] As attached Figure 5 As shown in the figure, when the addition amount was 0.1%, the conversion rate of ellagic acid to urolithin A by fermentation mucus lactobacillus FUA033 was the highest, reaching 59.83%, and the optimal addition amount of amino acid solution was determined to be 0.1%. Adding a small amount of composite amino acids can provide a nitrogen source for FUA033, thereby improving the conversion efficiency of ellagic acid, while excessive amino acids require lactic acid bacteria to consume more energy for metabolism, which may lead to uneven distribution of its metabolic resources and affect the synthesis of urolithin A.

[0044] 6. Effect of hydrogen-containing water on the conversion of ellagic acid to urolithin A by fermentative Lactobacillus mucilaginosus FUA033

[0045] As attached Figure 6 As shown in the figure, when the amount of hydrogen-containing water added was 1.5%, the conversion rate of strain FUA033 to urolithin A reached 58.71%. The appropriate amount of hydrogen-containing water added can provide reducing hydrogen for strain FUA033 to convert ellagic acid to urolithin A, which can promote the reaction and increase the conversion rate. However, the solubility of hydrogen in the culture medium is limited, resulting in its promotion effect being limited.

[0046] 7. Effects of different conditions on the conversion of ellagic acid to urolithin A by fermentation of Lactobacillus mucilaginosus FUA033

[0047] According to the results of the single factor experiment, considering that amino acids and hydrogen-containing water had no significant effect on the production of urolithin A by fermenting Lactobacillus mucilaginosus FUA033, the addition amount of trace element solution, vitamin solution and methyl viologen solution, which had a significant effect on the conversion of ellagic acid to urolithin A by strain FUA033, were used as factors to study the effect of different ratios of each factor on the conversion rate of urolithin A.

[0048] Table 1 Different ratios to study the conversion rate of urolithin A

[0049]

[0050] The optimum fermentation medium is: trypticase peptone 10.0g / L, gelatin peptone 10.0g / L, yeast extract powder 5.0g / L, sodium chloride 5.0g / L, glucose 1.0g / L, L-arginine 1.0g / L, sodium pyruvate 1.0g / L, vitamin K 0.5mg / L, hemin chloride 5.0mg, trace element solution added 0.16% (V / V), vitamin solution added 0.21% (V / V), methyl viologen added to a final concentration of 0.16mM, pH 7.1±0.2 (25°C). In the optimum fermentation medium, 37°C, anaerobic culture for 48h, the maximum conversion rate is 76.19%±0.42%.

[0051] Comparative example: CN117487707A A fermented mucus lactobacillus FUA033 and its application

[0052] Patented culture conditions of bacteria:

[0053] Fermentation medium: 10.0 tryptic peptone, 5.0 sodium chloride, 10.0 peptone, 1.0 L-arginine, 1.0 glucose, 5.0 yeast extract powder, 1.0 sodium pyruvate, 0.005 hemin, 10.0005 vitamin K, pH 7.1±0.1 (25°C). The seed liquid was inoculated at a 2% inoculum into the culture medium containing ellagic acid substrate and cultured in an anaerobic workstation at 37°C for 72 hours. The strain conversion rate was 55% at this time.

Claims

1. A culture medium for efficiently converting ellagic acid to urolithin A, comprising: Tryptic peptone 10.0 g / L, gelatin peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 5.0 g / L, glucose 1.0 g / L, L-arginine 1.0 g / L, sodium pyruvate 1.0 g / L, vitamin K 0.5 mg / L, hemin 5.0 mg, SL-10 trace element solution added 0.16% (V / V), Wolfe's vitamin solution added 0.21% (V / V), methyl viologen added to a final concentration of 0.16 mM.

2. A method for converting ellagic acid to produce urolithin A by fermenting Lactobacillus mucilaginosus FUA033, comprising the following steps: 1) Activation of bacterial strains: The fermented Lactobacillus mucilaginosus FUA033 stored in a -80°C refrigerator was streaked with three zones on a WAM anaerobic broth solid medium and incubated at 37°C for 48 hours; 2) Seed culture: Pick a single colony and inoculate it into WAM liquid medium, and incubate it anaerobically at 37°C for 72 hours as seed liquid; 3) Fermentation and conversion of ellagic acid to produce urolithin A: inoculating the seed liquid of fermentation Lactobacillus mucilaginosus FUA033 into the culture medium of claim 1 and culturing anaerobically; 4) Extraction of urolithin A: centrifuge, extract the fermentation broth with an equal volume of ethyl acetate, acidify with formic acid, separate the upper organic phase and rotary evaporate to obtain the product.

3. The method according to claim 2, characterized in that In the step 3), the inoculation amount is 2%.

4. The method according to claim 2, characterized in that: In the step 3), the culture temperature under the anaerobic condition is 38° C. and the culture time is 48 hours.

5. The method according to claim 2, characterized in that: In the step 3), the initial pH of the culture under anaerobic conditions is 6.

8.

6. The method according to claim 2, characterized in that In the step 4), the centrifugation conditions are 12000×g and 4° C. for 8 min.

7. Use of the culture medium according to claim 1 in the preparation of urolithin A.

Images