A strain of Lactobacillus reuteri WLRE03 producing ergothioneine and its microbial agent and application

Abstract

The present invention discloses a strain of Lactobacillus reuteri WLRE03 producing ergothioneine and its microbial agent and application, belonging to the field of microbial technology. The strain was deposited in the China Center for Type Culture Collection on December 8, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20232504. (1) The present invention provides a strain of Lactobacillus reuteri WLRE03 with high ergothioneine production, which is isolated from healthy poultry excrement and has the ability to produce ergothioneine; (2) Ergothioneine can alleviate intestinal barrier damage, dysbacteriosis and immune disorders caused by oxidative stress; (3) The ergothioneine production method provided by the present invention has the characteristics of simple operation, easy acquisition, low cost and suitability for industrial production, and can be used as a natural antioxidant in the food industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus reuteri WLRE03 producing ergothioneine, a microbial agent and application thereof. Background Art

[0002] Oxidative stress is a state of imbalance between oxidation and antioxidant effects in the body, and is a negative effect of free radicals produced in the body. Oxidative stress is considered to be an important factor leading to aging and various diseases. The occurrence and development of gastrointestinal diseases are also related to oxidative stress to a certain extent, including gastroesophageal reflux disease, gastroenteritis and colitis. In order to alleviate the adverse effects of oxidative stress, it is necessary to find substances with antioxidant stress properties. Conventional antioxidant stress can be mainly divided into two types: enzymes and non-enzymes. With the development of society and the improvement of people's living standards, the demand for anti-oxidative stress substances is increasing, so finding greener, more effective and safer antioxidant stress substances has become a current research hotspot.

[0003] Ergothioneine (EGT) is a water-soluble, thiol-containing non-protein amino acid that does not self-oxidize at physiological pH and in strong alkaline solutions. As an important active substance, it plays a role in free radical scavenging, maintaining DNA biosynthesis, detoxification, normal cell growth and cellular immunity in the body. It is recognized as a natural antioxidant and has a significant effect on body damage and diseases caused by oxidative stress. The ergothioneine content in edible fungi is high and has natural safety, which is a good resource for preparing ergothioneine, but the ergothioneine content in edible fungi is low, the preparation process is complex and the yield is low.

[0004] Lactobacillus reuteri mainly lives in the gastrointestinal tract of vertebrates such as poultry and humans. It is an internationally recognized excellent probiotic resource that can be used in food and health products, and has the main probiotic effects of lactic acid bacteria. Lactobacillus reuteri is currently reported to be able to produce ergothioneine, but there is no report on Lactobacillus reuteri converting histidine to produce ergothioneine. Therefore, the development of a Lactobacillus reuteri that can specifically convert histidine to produce ergothioneine can enrich the probiotic strain resources and provide more potential value for its industrial production applications. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Lactobacillus reuteri WLRE03 producing ergothioneine and a microbial agent and application thereof, 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 Lactobacillus reuteri (Limosilactobacillusreuteri) WLRE03 that produces ergothioneine. The strain was deposited in the China Center for Type Culture Collection on December 8, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20232504.

[0008] The second technical solution of the present invention is a microbial agent, comprising the Lactobacillus reuteri WLRE03.

[0009] The third technical solution of the present invention is the use of the Lactobacillus reuteri WLRE03 or the microbial agent in the production of ergothioneine.

[0010] A fourth technical solution of the present invention is a method for producing ergothioneine, which utilizes the Lactobacillus reuteri WLRE03 or the microbial agent to produce ergothioneine using histidine as a substrate.

[0011] A fifth technical solution of the present invention is the use of the Lactobacillus reuteri WLRE03 or the microbial agent in the preparation of a drug for alleviating intestinal damage caused by oxidative stress.

[0012] The sixth technical solution of the present invention is a drug for alleviating intestinal damage caused by oxidative stress, comprising the Lactobacillus reuteri WLRE03 or the microbial agent.

[0013] Based on the above technical solution, the present invention has the following technical effects:

[0014] (1) The present invention provides a strain of Lactobacillus reuteri WLRE03 with high ergothioneine production, which is isolated from the excrement of healthy poultry and has the ability to produce ergothioneine; (2) Ergothioneine can alleviate intestinal barrier damage, dysbacteriosis and immune disorders caused by oxidative stress; (3) The ergothioneine production method provided by the present invention has the characteristics of simple operation, easy acquisition, low cost and suitability for industrial production, and can be used as a natural antioxidant in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0016] Figure 1 This is the HPLC test result of ergothioneine standard;

[0017] Figure 2is a standard curve for the quantification of ergothioneine in the fermentation broth;

[0018] Figure 3 This is the HPLC test result of ergothioneine in the supernatant after histidine fermentation by Lactobacillus reuteri WLRE03;

[0019] Figure 4 It is the quantitative situation of Lactobacillus reuteri WLRE03 metabolizing histidine to produce ergothioneine;

[0020] Figure 5 The ability of ergothioneine produced by Lactobacillus reuteri WLRE03 to scavenge DPPH free radicals;

[0021] Figure 6 The ability of ergothioneine produced by Lactobacillus reuteri WLRE03 to scavenge hydroxyl radicals;

[0022] Figure 7 Ergothioneine produced by Lactobacillus reuteri WLRE03 improves LPS-induced oxidative damage in colon tissue;

[0023] Figure 8 Ergothioneine produced by Lactobacillus reuteri WLRE03 alleviated the trend of decreased number of colonic goblet cells induced by LPS. DETAILED DESCRIPTION

[0024] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0027] 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 description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0030] An embodiment of the present invention provides a strain of Lactobacillus reuteri (Limosilactobacillusreuteri) WLRE03 that produces ergothioneine. The strain was deposited in the China Center for Type Culture Collection on December 8, 2023, with a deposit address of Wuhan University, Wuhan, China, and a deposit number of CCTCC NO: M 20232504.

[0031] The embodiment of the present invention further provides a microbial agent, comprising the Lactobacillus reuteri WLRE03.

[0032] The embodiment of the present invention also provides the use of the Lactobacillus reuteri WLRE03 or the microbial agent in the production of ergothioneine.

[0033] The embodiment of the present invention also provides a method for producing ergothioneine, which uses the Lactobacillus reuteri WLRE03 or the microbial agent and histidine as a substrate to produce ergothioneine.

[0034] In some specific embodiments, the concentration of Lactobacillus reuteri should be 1×10 9 CFU.

[0035] In some specific embodiments, the concentration of histidine is 100 mmol / L.

[0036] The embodiment of the present invention also provides the use of the Lactobacillus reuteri WLRE03 or the microbial agent in the preparation of a drug for alleviating intestinal damage caused by oxidative stress.

[0037] The embodiment of the present invention further provides a drug for alleviating oxidative stress intestinal damage, comprising the Lactobacillus reuteri WLRE03 or the microbial agent.

[0038] Furthermore, the drug also includes histidine and pharmaceutically acceptable excipients.

[0039] Example 1

[0040] Screening and identification of ergothioneine-producing Lactobacillus reuteri

[0041] Fresh healthy poultry (chicken, duck and pigeon) feces samples were collected from Jiangda South Road Farmers' Market in Qingshan Lake District, Nanchang, Jiangxi Province. Lactobacillus reuteri selective culture medium was used to screen single colonies with rod-shaped morphology. Gene fragments were amplified using 16S rRNA universal primers. The products were identified by 1.2% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for nucleic acid sequence determination. The base sequences in the determination results were entered into the NCBI database for comparison, and the strain species was preliminarily determined. The strain was further compared with the bacterial genera in the database and a phylogenetic tree was constructed. The specific species was determined to be Lactobacillus reuteri, named WLRE03.

[0042] The Lactobacillus reuteri WLRE03 was deposited in the China Center for Type Culture Collection on December 8, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20232504.

[0043] Example 2

[0044] Qualitative Analysis of Ergothioneine Produced by Lactobacillus reuteri

[0045] HPLC was used to qualitatively analyze ergothioneine in Lactobacillus reuteri culture fluid, and the specific method was as follows:

[0046] (1) Sample pretreatment: Filter the supernatant of Lactobacillus reuteri through a 0.22 μm aqueous membrane, take 200 μL of the filtered supernatant into a sampling bottle, and store at 4°C for later use.

[0047] (2) Reagents: Mobile phase: methanol: water = 1:99, use 20% acetic acid to adjust the mobile phase pH to 5. Mobile phase preparation: 1% methanol solution: prepare 150 mL, take 147 mL of double distilled water, add 1.5 mL of methanol (chromatographic grade), adjust the pH to 5, and make up to 150 mL; 20% acetic acid preparation: prepare 10 mL, take 2 mL of acetic acid (chromatographic grade) and add it to 8 mL of double distilled water, mix well, transfer to a 50 mL centrifuge tube, seal with a sealing film, and place at 4°C for use.

[0048] (3) Instrument parameters: Column: C18 column; Flow rate: 1 mL / min; Column temperature: 30°C; Injection volume: 2 μL; Detection wavelength: 254 nm; Retention time: 4 min; Detector: UV absorption detector.

[0049] (4) Qualitative analysis: The ergothioneine standard has an absorption peak at a characteristic wavelength of 254 nm. According to its retention time and response area ( Figure 1; 3.778min and 228997mAU*s respectively), to determine whether there are response peaks and retention times similar to those of the standard in the metabolites of Lactobacillus reuteri WLRE03, and then determine whether the metabolites contain ergothioneine.

[0050] Example 3

[0051] Quantitative Analysis of Ergothioneine Production by Lactobacillus reuteri by Spectrophotometry

[0052] (1) Preparation of standard curve: Take ergothioneine standard and prepare standard dilutions with concentrations of 40, 20, 10, 5, and 2.5 μM respectively with double distilled water. Then, take 200 μL of each dilution and mix it evenly with 140 μL of 2'2-dithiodipyridine. After incubation at 25°C for 5 min, take 200 μL of the mixture and add it at OD 343 According to the measurement results, a standard curve is drawn with concentration as the horizontal axis and absorbance as the vertical axis.

[0053] (2) Sample determination: Experimental group: 5 mL of Lactobacillus reuteri was inoculated into MRS medium at a 1% inoculum volume and cultured anaerobically at 37°C for 16 h. After 16 h, centrifuge at 6000 r / min for 10 min, take 200 μL of the supernatant and put it into a 1.5 mL centrifuge tube, add 140 μL of 2'2-dithiodipyridine, mix well, and store at 25°C for 5 min. Take 200 μL of the reaction solution after the reaction, add it to the ELISA plate, and measure its OD value at 343 nm. Blank group: replace the fermentation supernatant with 200 μL of double distilled water.

[0054] Calculation result: OD (实际) =OD (实验) -OD (空白) , and finally calculate the corresponding value according to the standard curve.

[0055] Figure 2 The standard curve drawn by different concentrations of ergothioneine standards is, y = 8845.2x-61124, R 2 =0.9951, where y represents the peak area and x represents the EGT concentration (mg / L). Figure 3 The HPLC analysis results of fermentation metabolites of Lactobacillus reuteri WLRE03 using 100 mM histidine as the culture medium substrate show a characteristic absorption peak at a retention time of 3.774 min. Figure 4 The figure shows the difference in ergothioneine concentration produced by Lactobacillus reuteri WLRE03 in the presence or absence of 100 mM histidine. Histidine can increase the ergothioneine produced by Lactobacillus reuteri WLRE03 to 45 mg / L, which is about 4 times that in the absence of histidine.

[0056] Example 4

[0057] In vitro antioxidant assay of ergothioneine

[0058] 1. DPPH free radical scavenging ability

[0059] Weigh an appropriate amount of DPPH and dissolve it in 95% ethanol to make the final concentration of DPPH 0.2 mM. The DPPH solution should be prepared and used immediately, and stored in the dark.

[0060] Blank group (A0): Take 125 μL of 95% ethanol and 1 mL of 0.2 mM DPPH, and react at room temperature in the dark for 30 minutes. After the reaction, take 200 μL of the reaction solution to the ELISA plate and measure its absorbance at 517 nm.

[0061] Experimental group: Lactobacillus reuteri cultured for 16 hours in MRS was centrifuged at 6,000r / min for 5 minutes, the supernatant was discarded, the bacteria were washed once with PBS, and then the bacteria were resuspended with an equal volume of 100mM histidine. After anaerobic culture at 37°C for 4 hours, centrifuged under the same centrifugation conditions. After centrifugation, 125μL of the supernatant was taken and 1ml of 0.2mM DPPH was added. The reaction was carried out at room temperature in the dark for 30 minutes. 200μL of the reaction solution was taken into the ELISA plate and the absorbance was measured at 517nm.

[0062] Control group: centrifuge Lactobacillus reuteri cultured for 16 hours at 6,000r / min for 5 minutes, discard the supernatant, wash the bacteria once with PBS, and resuspend the bacteria with an equal volume of double distilled water. Incubate anaerobically at 37°C for 4 hours, and centrifuge under the same centrifugation conditions after 4 hours. Take 125μL of the supernatant after centrifugation and add 1ml of DPPH with a concentration of 0.2mM, and react at room temperature in the dark for 30 minutes. Take 200μL of the reaction solution to the ELISA plate and measure the absorbance at 517nm.

[0063] DPPH clearance = [1-{(A1-A2) / A0}] x 100%;

[0064] Among them, A1 125 μL reaction mixture + 1 mL DPPH; A2 125 μL reaction mixture + 1 mL double distilled water; A0 125 μL 95% ethanol + 1 mL DPPH.

[0065] 2. Hydroxyl free radical scavenging ability

[0066] Preparation of culture supernatant: centrifuge Lactobacillus reuteri cultured with MRS for 16 hours at 6,000r / min for 5 minutes, discard the supernatant, wash the cells once with PBS, and then resuspend the cells with an equal volume of 100mM histidine / double distilled water. After anaerobic culture at 37°C for 4 hours, centrifuge under the same centrifugation conditions and collect the supernatant for use.

[0067] Experimental group: Take 100μL of 2.5mmol / L o-phenanthroline solution, add 100μL of 0.02mol / L PBS and 100μL of distilled water, mix well. Then add 100μL of 2.5mmol / LFeSO4 solution and 100μL of 20mmol / LH2O2 solution, mix well, and incubate at 37℃ for 1h. Take 200μL of the mixture and add it to the ELISA plate, and measure its absorbance at a wavelength of 536nm.

[0068] Control group: A s The culture supernatant was replaced with an equal volume of 100 mM histidine / double distilled water.

[0069] OH removal rate % = {(A s -A p ) / (A b -A p )}x 100;

[0070] Among them, A p A is the absorbance of the mixed solution (reaction mixture); b A is the absorbance of the mixed solution when 100 μL of distilled water replaces 100 μL of H2O2 solution; s The absorbance of the mixed solution when 100 μL of culture supernatant replaced 100 μL of distilled water.

[0071] Figure 5 and Figure 6 This indicates that the fermentation broth of Lactobacillus reuteri WLRE03 has a strong ability to scavenge DPPH free radicals and OH free radicals, indicating that it has a strong antioxidant capacity.

[0072] Example 5

[0073] 1. Ergothioneine relieves oxidative stress intestinal damage

[0074] The model group was the mice that were intraperitoneally injected with lipopolysaccharide (LPS) for 4 days to induce intestinal oxidative damage in C57BL / 6 male mice; the experimental group was the mice that were continuously gavaged with Lactobacillus reuteri WLRE03 and histidine (100mM) in free drinking water for 28 days and intraperitoneally injected with LPS for 4 days before the end of gavage, i.e., the Lactobacillus reuteri WLRE03 and histidine relief group; and the control group was the mice that were intraperitoneally injected with normal saline. 2 The colon tissues of left and right mice were fixed in 10% tissue fixative and stored at 4°C. Tissue sections were made and stained with H&E to observe the pathological changes of the colon tissues of mice.

[0075] The status of the colon after staining is as follows Figure 7As shown in the figure, after 4 consecutive intraperitoneal injections of lipopolysaccharide (LPS), the villus structure of the model group became significantly shorter and looser (yellow arrow), the submucosal part of the colon was obviously edematous (black arrow), the crypt base showed obvious inflammatory infiltration (red arrow), and crypt abscess (blue arrow). In the remission group, it can be observed that the above pathological conditions have been significantly improved and can basically return to the same level as the control group.

[0076] 2. Analysis of changes in the number of goblet cells in mouse colon

[0077] Goblet cells are an important component of the intestinal mucosal barrier and play an important role in maintaining intestinal homeostasis. In order to observe the degree of damage to the intestinal barrier of mice by oxidative stress, AB-PAS staining was performed on the above sections. Figure 8 This indicates that ergothioneine produced by Lactobacillus reuteri WLRE03 and histidine fermentation can effectively alleviate the trend of decreased goblet cell number in the model group (purple part), thereby further improving the intestinal barrier.

[0078] Obviously, the above embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A production method of ergothioneine, characterized in that, Lactobacillus reuteri WLRE03 or a microbial agent containing Lactobacillus reuteri WLRE03 is used to produce ergothioneine using histidine as a substrate; The Lactobacillus reuteri WLRE03 was deposited in the China Center for Type Culture Collection on December 8, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20232504.

2. A drug for alleviating oxidative stress intestinal damage, characterized in that: The invention comprises the Lactobacillus reuteri WLRE03, histidine and pharmaceutically acceptable auxiliary materials.

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