Bifidobacterium, microbial agent and application of bifidobacterium in production of urolithin A

Abstract

The invention discloses bifidobacterium, a microbial agent and application of the microbial agent in production of urolithin A, and belongs to the technical field of microorganisms. The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on December 26, 2024, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.33209. The bifidobacterium disclosed by the invention can be used for metabolizing ellagic tannin (or ellagic acid) into urolithin A, and the urolithin A has an anti-oxidation effect. The urolithin A is used as an autophagy inducer to induce mitochondrial selective autophagy, so that loss of mitochondrial functions in the aging process is reduced, and the urolithin A can enhance muscle functions by adjusting biosynthesis of mitochondria, so that the anti-aging effect is achieved. Therefore, the bifidobacterium disclosed by the invention can be used in the field of medicine and health as an edible probiotic, and has good development value and huge industrial production potential.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a bifidobacterium, a microbial agent and application thereof in producing urolithin A. Background Art

[0002] Urolithin A (Uro-A) is primarily produced by the metabolism of specific food components by intestinal microbes. For example, foods rich in ellagitannins (or ellagic acid), such as pomegranates, are gradually converted to Uro-A through a series of enzymatic reactions catalyzed by intestinal microbes. In recent years, numerous studies have demonstrated that Uro-A possesses diverse biological activities, including antioxidant, anti-inflammatory, anti-cancer, and anti-aging properties.

[0003] The phenolic hydroxyl group in Uro-A's molecular structure provides hydrogen atoms, reacting with free radicals such as hydroxyl radicals and superoxide anion radicals, transforming them into stable molecules and interrupting free radical chain reactions to achieve antioxidant effects. Uro-A also acts as an autophagy inducer, activating the PINK1 / Parkin ubiquitin-dependent pathway or the BNIP3 receptor to induce selective mitochondrial autophagy, thereby reducing the loss of mitochondrial function during aging. Uro-A can also enhance muscle function by regulating mitochondrial biogenesis, thereby achieving anti-aging effects. Therefore, Uro-A plays a positive role in maintaining human health and has broad application prospects.

[0004] Currently, according to published literature, six strains have been discovered that are capable of converting ellagitannins (or ellagic acid) into Uro-A. These strains are Lactobacillus muciphilus FUA033 (Fang Yaowei, 2024) and Lactobacillus plantarum CCFM1291 (Tang Xin, 2023), which are derived from intestinal contents; Streptococcus thermophilus FUA329 (Fang Yaowei, 2023), which is derived from the breast milk of healthy Chinese women. These three strains are listed in the Chinese Catalogue of Edible Fungi and can be used for food fermentation production; Enterococcus faecium FUA027 (Liu Shu, 2023), Lactococcus gasseri FUA009 (Fang Yaowei, 2022), and Bifidobacterium pseudocatenulatus iniap815 (Gaya P, 2017), which are also derived from intestinal contents but are not listed in the Chinese Catalogue of Edible Fungi and cannot be used for food fermentation production. In summary, only three edible strains with the ability to produce Uro-A have been discovered, and the conversion rate still needs to be improved. Therefore, it is necessary to find a strain with a higher conversion rate that can convert ellagitannin components in the diet to produce Uro-A, which can be used for food fermentation and can be used to produce Uro-A-rich fermentation products in vitro, so as to achieve health benefits through dietary supplementation. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain of Bifidobacterium, a microbial agent and the use thereof in the production of urolithin A, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a strain of Bifidobacterium sp., which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33209.

[0008] The second technical solution of the present invention is a microbial agent, including the bifidobacterium.

[0009] The third technical solution of the present invention is the use of the bifidobacterium or the microbial agent in the production of urolithin A.

[0010] A fourth technical solution of the present invention is a method for producing urolithin A, which utilizes the bifidobacterium to carry out fermentation culture to produce urolithin A.

[0011] A fifth technical solution of the present invention is a fermented product containing urolithin A. The preparation method of the fermented product comprises: inoculating the bifidobacterium into a food containing ellagitannins or ellagic acid, and fermenting the food to obtain the fermented product.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The present invention provides a strain of Bifidobacterium Urolithin A Bifidobacterium and provides a fermented product containing Bifidobacterium Urolithin A Bifidobacterium. The Bifidobacterium Urolithin A Bifidobacterium of the present invention can metabolize ellagitannins (or ellagic acid) into urolithin A. The produced urolithin A can provide hydrogen atoms through the phenolic hydroxyl group in its molecular structure, react with hydroxyl radicals, superoxide anion radicals, etc., thereby interrupting the free radical chain reaction to achieve antioxidant effects. And by acting as an autophagy inducer, activating the PINK1 / Parkin ubiquitin-dependent pathway or the BNIP3 receptor to induce mitochondrial selective autophagy, thereby reducing the loss of mitochondrial function during aging. Urolithin A can also enhance muscle function by regulating mitochondrial biosynthesis, thereby achieving an anti-aging effect. Therefore, the Bifidobacterium Urolithin A Bifidobacterium of the present invention can be used in the field of medicine and health as an edible probiotic, has good development value, and has huge potential for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 The PCR product was detected by agarose gel electrophoresis. M stands for Marker; NTC stands for Negative Control; and 1 stands for Urolithin A Bifidobacterium.

[0016] Figure 2 This is a colony diagram of Bifidobacterium UrolithinABifidobacterium.

[0017] Figure 3 This is the ultra-high performance liquid chromatogram of Uro-A. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0024] An embodiment of the present invention provides a strain of Bifidobacterium, which was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33209.

[0025] The embodiment of the present invention also provides a microbial agent, including the bifidobacterium.

[0026] The embodiments of the present invention also provide the use of the bifidobacterium or the microbial agent in producing urolithin A.

[0027] The embodiment of the present invention also provides a method for producing urolithin A, which produces urolithin A by fermentation culture using the bifidobacterium.

[0028] In some specific embodiments, the fermentation culture conditions include: using ellagitannins or ellagic acid as a fermentation substrate.

[0029] An embodiment of the present invention further provides a fermented product containing urolithin A. The preparation method of the fermented product comprises: inoculating the bifidobacterium into a food containing ellagitannins or ellagic acid, and fermenting the food to obtain the fermented product.

[0030] In some specific embodiments, the food containing ellagitannins or ellagic acid includes pomegranate, persimmon, mulberry, raspberry, blackberry, walnut, pistachio and walnut.

[0031] In some specific embodiments, the fermentation preparation conditions include: fermentation temperature of 30-37° C., 200-400 rpm, and fermentation time of 24-72 h.

[0032] The strains obtained by screening of the present invention were sequenced and analyzed, and the 16s DNA was as follows. Figure 1As shown, the obtained sequence was compared in NCBI-Blast, and the results showed that the bacterial species was Bifidobacterium of the genus Bifidobacterium, and was named "Urolithin A Bifidobacterium".

[0033] The colonies of the bifidobacteria on the MRS solid medium can form round, smooth, raised, neatly edged colonies with a color of cream to white and reflective. The size is usually (0.5-1.3) μm × (1.5-8.0) μm. Figure 2 shown.

[0034] In one embodiment of the present invention, the activated Urolithin A Bifidobacterium seed solution is inoculated into a culture medium containing ellagic acid at an inoculum amount of 5% to 8% (v / v), and fermented at 36° C. under anoxic conditions and 400 rpm shaking for 24 hours.

[0035] In one embodiment of the present invention, the Uro-A detection method is to take a certain amount of fermentation broth, add 2 times the volume of acetonitrile for extraction, and repeat 3 times, evaporate to dryness by rotary evaporator to a yellow-brown oily liquid, dissolve in dimethyl sulfoxide, and finally filter through a 0.22 μm filter membrane and detect by HPLC.

[0036] In one embodiment of the present invention, the activated UrolithinABifidobacterium seed solution is inoculated into the ellagic acid-rich substance reaction system at an inoculation rate of 5% to 8% (v / v), and fermented at 36°C under anoxic conditions and 400 rpm shaking for 24 hours.

[0037] In one embodiment of the present invention, the substances rich in ellagic acid include but are not limited to berries and nuts.

[0038] In one embodiment of the present invention, the berries include but are not limited to pomegranate, persimmon, mulberry, raspberry, and the like.

[0039] In one embodiment of the present invention, the nuts include but are not limited to walnuts, cashews, walnuts, pistachios, etc.

[0040] In one embodiment of the present invention, the substance rich in ellagic acid is pomegranate juice, and the specific processing method is as follows:

[0041] The pH of pomegranate juice squeezed from peeled pomegranates is first adjusted to 5.8-6.2, and a nitrogen source required for bacterial growth is then provided. The juice is then pasteurized (85°C, 20 minutes), immediately placed in a 4°C environment for rapid cooling to rupture the bacterial cells, and stored at 4°C for later use. The nitrogen source comprises: 10 g / L tryptone, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate, and 2 g / L diammonium citrate.

[0042] The pomegranate-derived EA (ellagic acid) involved in the embodiments of the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of ≥98%. In addition, the pomegranates involved in each embodiment were picked from a pomegranate orchard in Yicheng District, Zaozhuang City.

[0043] The culture medium involved in the embodiments of the present invention is as follows:

[0044] MRS solid medium: tryptone 10.0 g / L, beef extract 10.0 g / L, glucose 20.0 g / L, sodium acetate 2.0 g / L, yeast powder 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, magnesium sulfate 0.1 g / L, Tween 80 1 mL / L, agar 15.0 g / L, pH adjusted to 5.8 ± 0.2 (20°C).

[0045] MRS liquid medium: tryptone 10.0 g / L, beef extract 10.0 g / L, glucose 20.0 g / L, sodium acetate 2.0 g / L, yeast powder 5.0 g / L, diammonium hydrogen citrate 2.0 g / L, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, magnesium sulfate 0.1 g / L, Tween 80 1 mL / L, pH adjusted to 5.8 ± 0.2 (20°C).

[0046] Ellagic acid MRS liquid medium: 10.0 g / L tryptone, 10.0 g / L beef extract, 20.0 g / L glucose, 2.0 g / L sodium acetate, 5.0 g / L yeast extract, 2.0 g / L diammonium hydrogen citrate, 2.0 g / L potassium dihydrogen phosphate, 0.05 g / L manganese sulfate, 0.1 g / L magnesium sulfate, and 1 mL / L Tween 80. Prepare 500 ml of MRS liquid medium and add 1 g of ellagic acid. Adjust the pH to 5.8 ± 0.2 (20°C).

[0047] The detection methods involved in the embodiments of the present invention are as follows:

[0048] The content of Uro-A was determined by HPLC:

[0049] HPLC detection: Agilent Technologies 1290 ultra-high performance liquid chromatograph was used, liquid column: Elite Supersil ODS2 (250×4.6 mm, 5 μm); mobile phase: 0.1% formic acid in water (phase A), acetonitrile (phase B); spectral scanning was performed to determine the maximum absorption wavelength at 305 nm; elution conditions: flow rate 1.0 ml / min, gradient elution. Uro-A peak time: 17.27 min (such as Figure 3 HPLC Uro-A determination).

[0050] Example 1

[0051] Screening of Uro-A-producing strains

[0052] (1) Strain screening

[0053] The fruit juice probiotic strains used in this embodiment are composite bacteria (Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, and Bifidobacterium) provided by Shandong Zhongke Bioengineering Co., Ltd.

[0054] Fresh, ripe pomegranates were peeled and squeezed for juice. The pomegranate juice was placed in a fermentation container, inoculated with probiotic strains of the juice, and fermented at 37°C for 30 days to obtain a pomegranate juice fermentation product. The fermentation product was subjected to microbial separation and purification, and isolated and cultured using the plate streak method to obtain a single colony. This was then fermented in vitro in MRS liquid medium (containing ellagic acid). The fermentation broth was extracted with acetonitrile and then rotary evaporated to a yellow-brown oily liquid. The liquid was re-dissolved in dimethyl sulfoxide and tested by HPLC, thereby screening a strain capable of converting ellagic acid to produce Uro-A, ultimately obtaining a target strain.

[0055] (2) Identification of bacterial species

[0056] The 16S rDNA of the Uro-A producing strain obtained by screening was amplified and sequenced (the nucleotide sequence of the amplified 16S rDNA is shown in SEQ ID NO.3). The obtained sequence was compared with the nucleic acid sequence in NCBI-Blast, which showed that the strain was a Bifidobacterium, and was named Urolithin A Bifidobacterium.

[0057] The primers used for 16S rDNA amplification are as follows:

[0058] 27F (forward SEQ ID NO. 1): 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0059] 1492R (reverse SEQ ID NO. 2): 5'-GGTTACCTTGTTACGACTT-3'.

[0060] The 16S rDNA amplification program was as follows: 94°C for 5 min; 30 cycles (90°C for 30 s; 55°C for 30 s; 72°C for 2 min); 72°C for 10 min; and 12°C for 2 min.

[0061] PCR products were detected by agarose gel electrophoresis. Figure 1 shown.

[0062]

[0063] (3) Observation of colony characteristics

[0064] A single colony of Bifidobacterium UrolithinABifidobacterium was picked and inoculated into MRS solid medium, cultured at 36℃ for 24h, and the colony characteristics of the strain on MRS solid medium were observed ( Figure 2 ). It was observed that the colonies of the bifidobacterium on the MRS solid culture medium were white, round and convex, with a diameter of about 3 mm.

[0065] The Bifidobacterium provided by the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on December 26, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33209.

[0066] Example 2

[0067] Uro-A was prepared by using Bifidobacterium UrolithinABifidobacterium and Lactobacillus plantarum (purchased from Taiwan Asia Core Biotechnology Co., Ltd.) in MRS solid medium (containing ellagic acid).

[0068] (1) Activation of Bifidobacterium UrolithinABifidobacterium and Lactobacillus plantarum

[0069] Use an inoculating loop to streak a strip of both bacterial cultures onto MRS solid medium and incubate inverted at 36°C for 24 hours. Transfer a single colony to MRS liquid medium and incubate at 36°C for 12 hours. After mixing, inoculate each culture at a 5% (v / v) inoculum into fresh MRS liquid medium. Repeat this activation process three times to obtain the activated bacterial culture.

[0070] (2) Preparation of MRS liquid culture medium containing ellagic acid

[0071] Add 10.0 g / L tryptone, 10.0 g / L beef extract, 20.0 g / L glucose, 2.0 g / L sodium acetate, 5.0 g / L yeast powder, 2.0 g / L diammonium hydrogen citrate, 2.0 g / L potassium dihydrogen phosphate, 0.05 g / L manganese sulfate, 0.1 g / L magnesium sulfate, and 1 mL / L of Tween 80 to prepare 500 ml of MRS liquid culture medium, add 1 g of ellagic acid, and adjust the pH to 5.8 ± 0.2 (20°C).

[0072] (3) Fermentation of Bifidobacterium UrolithinABifidobacterium and Lactobacillus plantarum

[0073] The activated bacterial liquid obtained in step (1) was inoculated into the treated liquid culture medium obtained in step (2) at an inoculum rate of 5% (v / v), and cultured at 36° C. and 400 rpm for 24 h, and the fermentation liquid was extracted.

[0074] (4) Detection of Uro-A content

[0075] 20 mL of fermentation broth was dissolved in 40 mL of acetonitrile and extracted three times. The extract was evaporated to a yellow-brown oil using a rotary evaporator. 5 mL of chromatographic-grade dimethyl sulfoxide was added and sonicated for 10 minutes (to promote dissolution). After passing through a 0.22 μm organic filter, the extract was transferred to a sample vial and assayed for Uro-A content. The results are shown in Table 1.

[0076] Table 1: Urolithin A Bifidobacterium and Lactobacillus plantarum metabolism Uro-A production (unit: uM)

[0077]

[0078] The study found that in a culture medium with ellagic acid as a substrate, after 24 hours, the bacterial liquid was taken out and measured by ultra-high performance liquid chromatography (HPLC) technology, which confirmed that Bifidobacterium UrolithinABifidobacterium has the ability to convert ellagic acid (EA) into Uro-A (see Figure 3 Under the same experimental conditions, the conversion efficiency of Bifidobacterium was higher than that of Lactobacillus plantarum, nearly twice that of Lactobacillus plantarum, and the Uro-A production was more significant.

[0079] Example 3

[0080] A fermented product containing Uro-A

[0081] The activated Bifidobacterium UrolithinABifidobacterium bacterial liquid is inoculated into the pomegranate juice fermentation culture medium at an inoculum amount of 5% (v / v), and the culture is shaken at 400 rpm at 36.5°C for 24 to 72 hours to obtain a fermentation product rich in probiotics and Uro-A.

[0082] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the implementation methods of the present invention. A person skilled in the art will be able to make other variations or modifications based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A strain of Bifidobacterium, characterized in that, This strain was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on December 26, 2024. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33209.

2. Microbial inoculant, characterized in that, Including the Bifidobacterium as described in claim 1.

3. Use of the Bifidobacterium as described in claim 1 or the microbial agent as described in claim 2 in the production of urolithin A.

4. A method for producing urolithin A, characterized in that, Fermentation culture is carried out using the Bifidobacterium as described in claim 1 to produce urolithin A.

5. The method according to claim 4, characterized in that, The conditions for the fermentation culture include using ellagitannin or ellagic acid as the fermentation substrate.

6. A fermented product containing urolithin A, characterized in that, The preparation method of the fermentation product includes inoculating the Bifidobacterium as described in claim 1 into a food containing ellagitannin or ellagic acid and carrying out fermentation preparation, which is the fermentation product.

7. The fermented product according to claim 6, wherein The foods containing ellagitannin or ellagic acid include: pomegranate, persimmon, mulberry, raspberry, walnut, pistachio, and walnut.

8. The fermented product according to claim 6, wherein The conditions for the fermentation preparation include: the fermentation temperature is 30 - 37°C, 200 - 400 rpm, and the fermentation time is 24 - 72 h.

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