Lactobacillus reuteri sj-47, exopolysaccharide secreted by the strain, and method for preparing the same

Abstract

The present disclosure provides a Lactobacillus reuteri SJ-47 with a preservation number of CGMCC No. 16416 and an exopolysaccharide secreted by the Lactobacillus reuteri SJ-47 and a preparation method thereof, which comprises: a fermentation culture step: after the Lactobacillus reuteri SJ-47 is inoculated in MRS broth and subjected to fermentation culture treatment, the fermentation broth supernatant is obtained by sterilization and separation; a crude polysaccharide extraction step: the fermentation broth supernatant is concentrated and enriched, and then subjected to first alcohol precipitation, enzymatic deproteinization, second alcohol precipitation, sevage deproteinization, dialysis and freeze-drying treatment to obtain a crude polysaccharide extract. The exopolysaccharide of the Lactobacillus reuteri SJ-47 provided in the present application has high yield and excellent performance, can effectively scavenge DPPH free radicals and hydroxyl free radicals, has good antioxidant capacity, can improve the expression levels of SOD, CAT and GSH, can increase the expression levels of type I collagen and matrix metalloproteinase, and plays a certain regulating role on the cell aging-apoptosis pathway.

Description

Technical Field

[0001] This disclosure belongs to the field of bio-fermentation technology, specifically relating to Lactobacillus reuteri SJ-47, the extracellular polysaccharide secreted by this strain, and its preparation method. Background Technology

[0002] Among all probiotics, lactic acid bacteria are among the best producers of extracellular polysaccharides. Extracellular polysaccharides can act as antioxidants, lower blood cholesterol levels, and also serve as antiviral and anticancer agents. They also possess immunomodulatory activities, such as stimulating the growth of immune cells, inducing nitric oxide production, inducing phagocytosis, and secreting pro-inflammatory and anti-inflammatory cytokines.

[0003] Lactobacillus reuteri is an important member of the Lactobacillus genus in lactic acid bacteria. It enhances human immunity by regulating the host's immune system and gut microbiota, thus playing a probiotic role. However, research on the extracellular polysaccharides of Lactobacillus reuteri is still relatively limited.

[0004] The inventors of this application isolated a novel strain of Lactobacillus reuteri, SJ-47. Studies have shown that the extracellular polysaccharides secreted by Lactobacillus reuteri SJ-47 have excellent antioxidant activity on the skin, specifically manifested in the following ways: 1) effectively scavenging DPPH free radicals and hydroxyl free radicals, exhibiting good antioxidant capacity; 2) increasing the expression levels of SOD, CAT, and GSH; 3) increasing the expression levels of type I collagen and matrix metalloproteinases; and 4) playing a certain regulatory role in the cellular senescence-apoptosis pathway. Summary of the Invention

[0005] A brief overview of this disclosure is given below to provide a basic understanding of certain aspects of it. It should be understood that this overview is not an exhaustive summary of this disclosure. It is not intended to identify key or essential parts of this disclosure, nor is it intended to limit its scope. Its purpose is merely to present certain concepts in a simplified form as a prelude to the more detailed description that follows.

[0006] To solve the above-mentioned technical problems, the technical solution provided in this disclosure is as follows:

[0007] In one aspect, this disclosure provides a Lactobacillus reuteri, specifically Lactobacillus reuteri SJ-47, with accession number CGMCC No. 16416.

[0008] The *Lactobacillus reuteri* SJ-47 disclosed herein was isolated from yogurt by the applicant. The specific procedure was as follows: 5 ml of yogurt sample was briefly shaken for 5 seconds, diluted with physiological saline to a suitable gradient, and spread onto MRS solid medium. The medium was incubated at 37°C for 48 hours. After colonies grew on the plates, smooth, white single colonies were picked with an inoculation loop, Gram-stained, and examined under a microscope. These single colonies were then transferred to MRS solid medium. If the colonies were impure, they were streaked onto MRS medium and incubated continuously until no contaminating bacteria were observed under a microscope. The purified bacterial solution was mixed 1:1 with sterilized 30% glycerol and stored at -80°C. The strain identification sequence number is GenBank: MN480494.

[0009] Secondly, this disclosure provides an extracellular polysaccharide secreted by the aforementioned Lactobacillus reuteri SJ-47.

[0010] Thirdly, this disclosure provides a method for preparing the above-mentioned Lactobacillus reuteri extracellular polysaccharide, the steps of which are as follows:

[0011] Fermentation culture steps: The Lactobacillus reuteri SJ-47 with the above preservation number CGMCC No.16416 was inoculated into MRS broth, fermented, and then sterilized and separated to obtain the supernatant of the fermentation broth;

[0012] Crude polysaccharide extraction steps: The supernatant of the fermentation broth is concentrated and enriched, and then subjected to a first alcohol precipitation, enzymatic hydrolysis to remove protein, a second alcohol precipitation, sevage to remove protein, dialysis, and freeze drying to obtain crude polysaccharide extract.

[0013] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, it further includes: a crude polysaccharide purification step: the crude polysaccharide extract is further purified by passing it through a DEAE-52 fiber resin column to obtain the extracellular polysaccharide; preferably, the DEAE-52 fiber resin column chromatography process uses NaCl solution for elution.

[0014] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, in the fermentation culture step, the Lactobacillus reuteri has been activated and cultured into a bacterial solution before being inoculated into the MRS broth, and the bacterial solution has an OD value of 0.7-1.6 at 600 nm (e.g., 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5).

[0015] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, the pH value of the MRS broth is 5.8-6.5 (e.g., 5.9, 6.0, 6.1, 6.2, 6.3, 6.4).

[0016] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, in the fermentation culture step, the volume ratio (inoculation amount) of the bacterial solution to the MRS broth is 5-30% (e.g., 8%, 10%, 15%, 20%, 25%, 28%).

[0017] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, the fermentation culture step is carried out under the following conditions: fermentation temperature 37-45℃ (e.g., 38℃, 40℃, 42℃, 44℃, etc.), shaking speed 150r / min-180r / min (e.g., 155r / min, 160r / min, 165r / min, 170r / min, 175r / min), and fermentation time 6-16h (e.g., 7h, 8h, 10h, 12h, 14h, 15h, etc.).

[0018] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, in the crude polysaccharide extraction step, the supernatant of the fermentation broth is concentrated to 10-20% of its original volume (e.g., 12%, 14%, 16%, 18%, etc.); during the first alcohol precipitation treatment, the volume ratio of ethanol to the concentrated fermentation broth supernatant is 4:1-10:1 (e.g., 5:1, 6:1, 7:1, 8:1, 9:1, etc.), and the alcohol precipitation is carried out at -4℃ for about 12-24 hours (e.g., 15 hours, 18 hours, 20 hours, 22 hours).

[0019] In the above-mentioned method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, the deproteinization process in the crude polysaccharide extraction step includes: reconstituteing the product obtained from the first alcohol precipitation treatment with water to the original sample volume, adding an appropriate amount of papain and mixing well, enzymatically hydrolyzing at room temperature for 2-10 hours (e.g., 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours) and then boiling to inactivate the polysaccharide. Preferably, the papain is a 10 g / L papain solution prepared with phosphate buffer at pH=5, and the papain solution is added at a volume ratio of 2-10% (e.g., 3%, 4%, 5%, 6%, 7%, 8%, 9%, etc.).

[0020] In the above method for preparing Lactobacillus reuteri extracellular polysaccharide, as a preferred embodiment, the crude polysaccharide purification step includes:

[0021] The crude polysaccharide extract was dissolved in 0.1 mol / ml NaCl solution, centrifuged, and insoluble matter was removed. The supernatant was added to a equilibrated DEAE-52 column. After injection, linear elution was first performed with 0.3 mol / ml NaCl solution, followed by gradient elution with 0.1, 0.2, and 0.4 mol / ml NaCl solutions according to the position of the elution peak, until no sugar was detected. After concentration of the eluent, residual salts were removed by dialyzing with flowing distilled water, and the product was then freeze-dried under vacuum to obtain purified Lactobacillus reuteri extracellular polysaccharide.

[0022] The beneficial effects of this disclosure include, but are not limited to:

[0023] 1. The Lactobacillus reuteri SJ-47 disclosed herein has a high yield of secreted extracellular polysaccharides, which promotes cell growth and protects cells after UVA induction. It can be added to cosmetic formulations as an active ingredient to prepare cosmetics including but not limited to masks, serums, sunscreens, lotions, etc.

[0024] 2. This disclosure adopts a fermentation method, using MRS medium as a substrate that has been sterilized and inoculated with Lactobacillus reuteri SJ-47 for fermentation. Fermentation is carried out in a constant temperature and humidity chamber. The fermentation process is simple, easy to operate, and consumes less energy, which can save costs. In addition, the preparation cycle is short, the extraction rate is high, the preparation method is easy to implement, and it can be mass-produced.

[0025] The newly disclosed Lactobacillus reuteri strain was deposited on September 4, 2018, with accession number CGMCCNo.16416, classified and named as Lactobacillus reuteri strain SJ-47. The depository institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0026] Figure 1 The elution curves of the Lactobacillus reuteri extracellular polysaccharide sample isolated and purified in Example 2 are shown;

[0027] Figure 2 The cytotoxic effect of the Lactobacillus reuteri extracellular polysaccharide sample prepared in Example 2 is shown.

[0028] Figure 3 The protective effect of Lactobacillus reuteri extracellular polysaccharide sample prepared in Example 2 after UVA-induced cell damage is shown.

[0029] Figure 4 The total antioxidant capacity (ABTS method) of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 is shown;

[0030] Figure 5 The effect of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 on the intracellular reactive oxygen species (ROS) content is shown.

[0031] Figure 6 The effect of the extracellular polysaccharide of Lactobacillus reuteri prepared in Example 2 on the intracellular malondialdehyde (MDA) content is shown.

[0032] Figure 7 (a) and (b) show the effects of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 on the intracellular superoxide dismutase (SOD) content and the relative expression level of intracellular SOD mRNA, respectively.

[0033] Figure 8 (a) and (b) show the effects of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 on the intracellular catalase (CAT) content and the relative expression level of intracellular CAT mRNA, respectively.

[0034] Figure 9 (a) and (b) show the effects of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 on the intracellular glutathione peroxidase (GSH-Px) content and the relative expression level of intracellular glutathione peroxidase (GSH-Px) mRNA, respectively.

[0035] Figure 10 (a) and (b) show the effects of the Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 on the content of type I collagen (COL-1) in cells and on the relative expression level of COL-1 mRNA in cells, respectively.

[0036] Figure 11 (a) and (b) in Example 2 show the effects of the extracellular polysaccharide of Lactobacillus reuteri prepared on the content of matrix metalloproteinase-1 (MMP-1) in cells and the effect on the relative expression level of MMP-1 mRNA in cells, respectively.

[0037] Figure 12 Figures (a) to (h) show the effects of the extracellular polysaccharide of Lactobacillus reuteri prepared in Example 2 on the relative expression levels of Bax mRNA, Bcl-2 mRNA, Sirt1 mRNA, p16 mRNA, p53 mRNA, AKT mRNA, p21 mRNA, and Foxo mRNA in cells. Detailed Implementation

[0038] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are within the scope of the present invention.

[0039] The technical solutions of this disclosure will be described below with reference to exemplary embodiments.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] The Lactobacillus reuteri SJ-47 used in the following examples was deposited on September 4, 2018, with accession number CGMCC No. 16416.

[0043] Example 1: Isolation, identification, and preservation of Lactobacillus reuteri SJ-47

[0044] 1. Isolation of bacterial strains

[0045] Lactobacillus reuteri SJ-47 was isolated from yogurt. Specifically, 5 ml of yogurt sample was briefly shaken for 5 seconds, diluted with physiological saline to a suitable gradient, and spread onto MRS solid medium. The plates were incubated at 37°C for 48 hours. After colonies grew on the plates, smooth, white single colonies were picked with an inoculation loop, Gram-stained, and examined under a microscope. These single colonies were then transferred to MRS solid medium. If the colonies were impure, they were streaked back into MRS medium and incubated continuously until no contaminating bacteria were observed under a microscope. The purified bacterial solution was mixed 1:1 with sterilized 30% glycerol and stored at -80°C.

[0046] 2. Identification of fungal strains

[0047] The colonies of this strain on MRS agar plates are round, white, with a smooth, moist surface, regular edges, and are opaque. They are Gram-positive.

[0048] Genomic DNA was extracted from SJ-47 following the operating procedures of the bacterial genomic DNA extraction kit (brand: AXYGEN, model: 31516KC4).

[0049] Using the extracted SJ-47 genomic DNA as an amplification template, PCR was performed using universal primers for the fungal ribosomal rDNA region: 27F primer (AGAGTTTGATCMTGGCTCAG) and 1492R primer (TACGGYTACCTTGTTACGACTT). The reaction was performed according to the kit instructions (EX Taq enzyme, model RR001, manufacturer TaKaRa; dNTP, model BK6501A, manufacturer TaKaRa; 10×EX Taq Buffer, model AB5401A, manufacturer TaKaRa). The reaction program was: 94℃ pre-denaturation for 3 min (1 cycle), 94℃ denaturation for 30 sec, 54℃ annealing for 30 sec, and 72℃ extension for 1.5 min (24 cycles).

[0050] The obtained PCR products were sequenced, and the sequencing results showed that the rDNA-16S sequence of SJ-47 is shown below. The sequence was compared online with the rDNA-16S sequences published in the NCBI database, and a BLAST search was performed. The results showed that the nucleic acid sequence of SJ-47 had the highest homology with that of Lactobacillus reuteri, with a similarity of 99%, and its Genebank number is ON629726.1.

[0051] 3. Preservation of microbial strains

[0052] SJ-47 was deposited on September 4, 2018, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 16416. The full name of SJ-47 is Lactobacillus reuteri strain SJ-47, CGMCC No. 16416.

[0053] 4. Microbial strain screening experiment

[0054] Eight colonies selected during the bacterial isolation process in Part 1 of Example 1 above were coded as LB-1 to LB-8. One loopful of each colony was placed in MRS liquid medium and incubated at 37°C for 24 hours. The absorbance at 600 nm was measured. The fermentation broth was then centrifuged at 5000 rpm, and the supernatant was collected. The total sugar content of each fermentation broth was determined using a total sugar content assay kit (Solepro). The final results are expressed as follows:

[0055] C 总糖 =C LB-x / (OD 600 / OD 600min ).

[0056] The fermentation broth was used to extract polysaccharides crudely by alcohol precipitation, and then freeze-dried to obtain lyophilized powder. Then, a crude polysaccharide solution of the same concentration (2 mg / ml) was prepared for DPPH free radical scavenging experiment. The results are shown in Table 1.

[0057] As shown in Table 1, the LB-6 strain has the highest polysaccharide production and DPPH free radical scavenging rate. LB-6 was identified as Lactobacillus reuteri in Example 1 above and named Lactobacillus reuteri SJ-47.

[0058] Table 1 Performance test results of strains LB-1 to LB-8

[0059]

[0060] Example 2: Preparation of extracellular polysaccharides by fermentation of Lactobacillus reuteri SJ-47

[0061] This embodiment uses Lactobacillus reuteri SJ-47 fermentation to prepare extracellular polysaccharides. The specific steps are as follows:

[0062] 1) Fermentation experiment of Lactobacillus reuteri SJ-47

[0063] Prepare MRS broth in advance with a pH of 6.2, pour it into Erlenmeyer flasks, and sterilize them at high temperature in an autoclave. After sterilization, remove the flasks and cool them in a clean bench for later use. Inoculate Lactobacillus reuteri GCMCC No. 16416 into the MRS broth and adjust the OD value at 600 nm to 1.0. Then, inoculate the broth at a rate of 10% (bacterial volume to MRS broth volume ratio) and ferment at 40°C and 150 rpm for 12 hours. After fermentation, sterilize the broth, centrifuge to remove the bacterial cells, and collect the supernatant.

[0064] 2) Extraction of crude extracellular polysaccharides from Lactobacillus reuteri

[0065] The supernatant of the fermentation broth is first concentrated by rotary evaporation to 10% of its original volume in order to reduce the amount of ethanol used.

[0066] The ethanol and concentrated fermentation broth supernatant were mixed at a volume ratio of 4:1, and the sample was precipitated overnight at -4°C for approximately 12 hours. After ethanol precipitation, the sample was reconstituted with water to the original volume, and 4% (v / v) of papain (a 10 g / L papain solution prepared with phosphate buffer at pH 5) was added. The mixture was mixed, and the enzyme was hydrolyzed at room temperature for 2 hours. The enzyme was then boiled for 10 minutes to inactivate it. The precipitate was removed by centrifugation at 5000 rpm for 10 minutes, and the precipitate was collected after ethanol precipitation again.

[0067] The precipitate was reconstituted with water to its original volume to obtain the extract. This extract was then mixed with Sevage's reagent [chloroform: n-butanol = 5:1 (V / V)] at a 5:1 ratio in a separatory funnel. After thorough shaking, the mixture was allowed to stand for 10 minutes. The lower and middle layers were discarded, and the upper layer was collected. This process was repeated three times. The collected polysaccharide solution was further dialyzed. The dialyzed polysaccharide solution was freeze-dried to obtain the crude polysaccharide extract, hereinafter referred to as crude extracellular polysaccharide. The purity of the crude extracellular polysaccharide was 59.3% (by weight).

[0068] 3) Purification of extracellular polysaccharides from Lactobacillus reuteri

[0069] 100g of DEAE resin was soaked in distilled water to allow it to swell fully. After pretreatment with dilute acid and dilute alkali, it was rinsed with distilled water until neutral. The OH-type resin was converted to the Cr-type resin using a high-concentration NaCl solution before being packed into the column. After packing, the column was equilibrated with eluent for 12 hours.

[0070] The crude polysaccharide was dissolved in 0.1 mol / mL NaCl solution, centrifuged, and the insoluble matter was removed. The supernatant was added to a pre-equilibrated DEAE-52 column (2.6 cm × 40 cm). After injection, linear elution was performed initially with 0.3 mol / L NaCl solution, followed by gradient elution with 0.1, 0.2, and 0.4 mol / L NaCl solutions based on the position of the elution peak. Fractional elution was performed using an automated fraction collector, and the sulfuric acid-phenol method was used for detection. An elution curve was plotted with absorbance on the ordinate and elution volume on the abscissa. Elution continued until no sugar was detected. Figure 1 As shown, after concentrating the eluent at 0.1 mol / L, residual salts were removed by dialyzing with flowing distilled water, followed by vacuum freeze-drying to obtain purified Lactobacillus reuteri extracellular polysaccharide.

[0071] Example 3: Performance Test of Extracellular Polysaccharides from Lactobacillus reuteri

[0072] In this embodiment, the purified Lactobacillus reuteri extracellular polysaccharide prepared in Example 2 was subjected to the following series of performance tests.

[0073] 1. Cytotoxicity assay (CCK8 assay)

[0074] Fibroblasts in good logarithmic growth phase were seeded into 96-well plates and cultured overnight at 37°C with 5% CO2. The culture medium was then aspirated, and a blank control group (C), a model group (UVA), and a sample group were set up. Different concentrations of samples were added, with six replicates for each sample. The cell control group received no treatment. Cells were cultured for 24 hours, with three replicates of each concentration sample stimulated with UVA at a dose of 7 J / cm². 2In addition, three parallel studies of samples with different concentrations were not irradiated. Incubation was performed for 12 hours. The culture medium was discarded, and after washing with PBS, 100 μl of serum-free DMEM and 10 μl of CCK8 were added. After incubation for 2-4 hours, the absorbance of each well was measured at 450 nm. Cell viability was calculated using the following formula: Cell viability = (OD value of measurement well - OD value of blank control) / (OD value of cell control group - OD value of blank control) * 100%.

[0075] See the experimental results. Figure 2 , Figure 3 . Figure 2 To investigate the effects of six different concentrations of samples on cell viability, with no sample added as a control group, the study found that as the concentration increased, the samples had no toxic effect on cell survival and promoted cell growth. Figure 3 The study demonstrated the protective effect of UVA-induced samples on cells, showing that after sample treatment followed by UVA induction, cell viability increased with increasing sample concentration.

[0076] 2. Total antioxidant capacity assay (ABTS method)

[0077] 1) Preparation of cell lysis supernatant:

[0078] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 × 10⁶ cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and blank control, model, and sample groups were set up. Different concentrations of samples were added (serum-free DMEM culture medium was added to the model group and blank control). Cells were cultured for 24 hours and then stimulated with UVA at a dose of 7 J / cm². 2 The control group was not irradiated and incubated for 12 hours. The cells were then removed, placed on ice, and washed twice with PBS. Cells were scraped off with a cell scraper and collected into centrifuge tubes. The cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded to obtain cell pellet. 200 μl of lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatant was collected to obtain the cell lysis supernatant.

[0079] 2) Preparation of ABTS working solution:

[0080] First, prepare ABTS working stock solution by mixing 400 μl of ABTS with 400 μl of oxidant solution. After storing at room temperature in the dark for 14 hours, dilute with PBS 50 times to obtain ABTS working solution.

[0081] 3) Preparation for standard curve determination:

[0082] Dilute the standards with PBS to make 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM solutions of 10 mM Trolox.

[0083] 4) Determination of total antioxidant capacity:

[0084] Add 200 μl of ABTS working solution to each well of the 96-well plate, and 10 μl of PBS solution to each blank control well. Add 10 μl of Trolox standard solutions of various concentrations to each standard curve well. Add 10 μl of each sample to each sample well and mix gently. After incubating at room temperature for 4 minutes, measure the absorbance (A) at a wavelength of 734 nm. 734 The total antioxidant capacity of the samples was calculated based on the standard curve. The obtained standard curve was: y = -0.4357x + 0.5565, R² = 0.9921.

[0085] See the experimental results. Figure 4 The model group (UVA) was a cell group that was irradiated with UVA without any sample added, and the blank group (C) was a cell group that was not irradiated and without any sample added. UVA irradiation reduces the total antioxidant capacity of cells. After statistical difference calculation, the sample group was compared with the model group and the blank group. It was found that the sample concentrations of 250 μg / ml and 500 μg / ml can significantly improve the total antioxidant capacity of fibroblasts.

[0086] 3. Reactive Oxygen Species Detection (ROS)

[0087] Fibroblasts in the logarithmic growth phase were seeded into each well of a six-well plate with 2 ml of fibroblast suspension diluted in complete DMEM medium. A blank control group, a model group, and a sample group were set up. The cell count per well was controlled at 5 × 10⁶. 5 Cells were cultured overnight at 37°C in a 5% CO2 incubator. The culture medium was discarded, and different concentrations of samples were added and incubated for 24 hours. Cells were stimulated with UVA at a UV radiation dose of 7 J / cm². 2 The control group was not irradiated.

[0088] For the blank control group, model group, and sample group, after removing the samples, the cells were washed twice with PBS, digested with 0.5 ml of 0.25% trypsin, centrifuged to collect the cell pellet, and then diluted with 1 ml of serum-free culture medium at a 1:1000 ratio. The cells were incubated at 37°C for 20 minutes, inverting every 3-5 minutes. The cells were washed three times with serum-free cell culture medium to thoroughly remove any uninfiltrated DCFH-DA. After centrifugation at 5000 rpm for 5 minutes, the supernatant was discarded to obtain the cell pellet. PBS was added to prepare a cell suspension, bringing the cell count to 1 × 10⁶ cells / day. 6 / ml. Spotting was performed on a plate with three replicates for each sample, and measurements were taken using a fluorescence microplate reader at an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0089] See the experimental results. Figure 5The model group (UVA) was a cell group that was irradiated with UVA without any additional sample, and the blank group (C) was a cell group that was neither irradiated nor had any additional sample added. UVA irradiation significantly increased the reactive oxygen species content of fibroblasts. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml had a significant effect on reducing the reactive oxygen species content.

[0090] 4. Lipid oxidation detection

[0091] When animal or plant cells experience oxidative stress, lipid oxidation occurs. Some fatty acids are oxidized and gradually decompose into a series of complex compounds, including malondialdehyde (MDA). The level of lipid oxidation can be detected by measuring the level of MDA, which can serve as one of the indicators for assessing the severity of cellular stress.

[0092] This experiment used the Beyotime lipid oxidation (MDA) detection kit for detection.

[0093] 1) Sample preparation:

[0094] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 x 10⁶ cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded. A blank control group, a model group, and a sample group were set up, with different concentrations of sample added (serum-free DMEM culture medium was added to the model group and the blank control group). Cells were cultured for 24 h, then stimulated with UVA at a dose of 7 J / cm². The blank group was not irradiated. Cells were then incubated for 12 h. Cells were removed, placed on ice, and washed twice with PBS. Cells were scraped off with a cell scraper and collected in centrifuge tubes. The cells were centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and 200 μl of lysis buffer was added to lyse the cells. The cells were centrifuged at 12000 rpm at 4°C for 5 min, and the supernatant was collected to obtain the cell lysis supernatant.

[0095] 2) Preparation of the reagent kit:

[0096] a. Preparation of thiobarbituric acid (TBA) stock solution: Weigh an appropriate amount of TBA and prepare a TBA stock solution with a concentration of 0.37% using TBA preparation solution.

[0097] b. Preparation of MDA detection working solution: Based on the number of samples to be tested (including controls), refer to Table 2 below to prepare an appropriate amount of fresh MDA detection working solution before testing.

[0098] Table 2

[0099] Number of tests 1 time 10 times 20 times 50 times TBA diluent 150μl 1500μl 3000μl 7500μl TBA storage solution 50μl 500μl 1000μl 2500μl antioxidants 3μl 30μl 60μl 150μl

[0100] c. Dilution of standards: Take an appropriate amount of standard and dilute it with distilled water to 1, 2, 5, 10, 20, and 50 μM for subsequent preparation of standard curves.

[0101] 3) Sample determination:

[0102] a. Add 0.1 ml of PBS solution as a blank control to a centrifuge tube or other suitable container, add 0.1 ml of the above-mentioned standards at different concentrations to prepare a standard curve, and add 0.1 ml of sample for measurement; then add 0.2 ml of MDA detection working solution. Refer to Table 3 below to set up the detection reaction system:

[0103] Table 3

[0104] Blank control Standard products sample Homogenate, lysis buffer or PBS 0.1ml － － Standard products － 0.1ml － Sample to be tested － － 0.1ml MDA detection working solution 0.2ml 0.2ml 0.2ml

[0105] b. After mixing, heat in a 100°C boiling water bath for 15 minutes. Take care to avoid boiling over and splashing during heating. If using a boiling water bath, use centrifuge tubes with a lockable lid or screw cap, or seal the centrifuge tube opening with parafilm and prick a small hole with a needle.

[0106] c. Cool to room temperature in a water bath, then centrifuge at 1000g for 10 minutes at room temperature. Add 200 μL of supernatant to a 96-well plate, and then measure the absorbance at 532 nm using a microplate reader.

[0107] d. Calculation of MDA content: The molar concentration of MDA in the sample can be directly calculated from the standard curve. After calculating the MDA content in the sample solution, the MDA content in the initial sample can be expressed by the protein content per unit weight, for example, μmol / mg protein.

[0108] See the experimental results. Figure 6 The model group (UVA) was a cell group that was irradiated with UVA without any sample added, and the blank group (C) was a cell group that was not irradiated and without any sample added. UVA irradiation significantly increased the MDA content of fibroblasts. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml all had a highly significant effect on reducing MDA content.

[0109] 5. SOD activity detection

[0110] This experiment used the Beyotime Total SOD Activity Kit (NBT method) for detection.

[0111] 1) Preparation of cell samples:

[0112] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 × 10⁶ cells per well. 5Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and blank control, model, and sample groups were set up. Different concentrations of samples were added (serum-free DMEM culture medium was added to the model group and blank control). Cells were cultured for 24 hours and then stimulated with UVA at a dose of 7 J / cm². 2 The control group was not irradiated and incubated for 12 hours. The cells were then removed, placed on ice, and washed twice with PBS. Cells were scraped off with a cell scraper and collected into centrifuge tubes. The cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded to obtain cell pellet. 200 μl of lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatant was collected to obtain the cell lysis supernatant.

[0113] 2) Preparation of the reagent kit:

[0114] a. Preparation of NBT / enzyme working solution: Prepare an appropriate volume of NBT / enzyme working solution (160 μl per reaction). Mix 158 μl of SOD detection buffer, 1 μl of NBT, and 1 μl of enzyme solution thoroughly to prepare 160 μl of NBT / enzyme working solution. Prepare an appropriate amount of NBT / enzyme working solution according to the number of samples to be tested (including standards). Store the prepared NBT / enzyme working solution at 4°C or on ice. It can be used on the same day, but it is recommended to prepare and use it immediately.

[0115] b. Preparation of the reaction initiation working solution: Dissolve and mix the reaction initiation solution (40X) from the kit. Dilute with 39 μl of SOD detection buffer per 1 μl of reaction initiation solution (40X), and mix well. This is the reaction initiation working solution. Prepare an appropriate amount of reaction initiation working solution according to the number of samples to be tested (including standards). Store the prepared reaction initiation working solution at 4°C or on ice. It can be used on the same day, but it is recommended to prepare and use it immediately.

[0116] 3) Sample determination:

[0117] a. Refer to the table below to set up the sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order shown in Table 4. After adding the reaction start-up working solution, mix thoroughly.

[0118] Table 4

[0119]

[0120]

[0121] b. Incubate at 37℃ for 30 minutes. Note: There was no significant difference in SOD activity detected after incubation of 25 to 35 minutes. To ensure consistency of test results, incubate for 30 minutes.

[0122] c. Measure absorbance at 560 nm.

[0123] See the experimental results. Figure 7 In (a) and (b), the model group consists of cells irradiated with UVA without any added sample, while the blank group consists of cells that were neither irradiated nor added with any sample. UVA irradiation reduces cellular SOD activity. Statistical comparison between the sample group and the model group showed that a sample concentration of 500 μg / ml increased SOD activity. UVA irradiation also reduces the relative expression level of cellular SOD mRNA. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml all significantly increased the relative expression level of cellular SOD mRNA.

[0124] 6. Catalase detection

[0125] This experiment used the Beyotime catalase detection kit for detection.

[0126] 1) Preparation of the reagent kit:

[0127] a. Prepare a 250 mM hydrogen peroxide solution. The hydrogen peroxide concentration provided in this kit is approximately 1 M. Since hydrogen peroxide is not very stable, the actual concentration must be determined before use. Dilute the approximately 1 M hydrogen peroxide solution 100 times with the catalase detection buffer provided in this kit to a concentration of approximately 10 mM. Measure the absorbance value A at a wavelength of 240 nm. 240 .

[0128] Concentration calculation formula: c = A / (ε × b). Where: c is the sample concentration (unit: mol / L or M); A is the absorbance; ε is the wavelength-dependent molar extinction coefficient (unit: L × mol). -1 ×cm -1 Or M -1 ×cm -1 The molar extinction coefficient of hydrogen peroxide is 43.6 M. -1 cm -1 b = optical path length (in cm).

[0129] Therefore: Hydrogen peroxide concentration (M) = A 240 / (43.6×b); that is: hydrogen peroxide concentration (mM) = 22.94×A 240 / b, thus calculating the actual concentration of hydrogen peroxide provided by this kit.

[0130] Then, prepare a 250mM hydrogen peroxide solution based on the actual hydrogen peroxide concentration.

[0131] b. Prepare a 5mM hydrogen peroxide solution. Prepare a 5mM hydrogen peroxide solution based on the actual hydrogen peroxide concentration obtained from the measurement.

[0132] c. Prepare the chromogenic working solution. Dissolve the chromogenic substrate on ice, aliquot appropriately, and avoid repeated freeze-thaw cycles as much as possible. Keep other reagents on ice for later use. Take an appropriate amount of peroxidase and dilute it with the chromogenic substrate at a ratio of 1:1000 to prepare the chromogenic working solution. For example, take 5 μl of peroxidase, add 5 ml of chromogenic substrate, and mix well to obtain 5 ml of chromogenic working solution.

[0133] 2) Sample preparation:

[0134] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 × 10⁶ cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and blank control, model, and sample groups were set up. Different concentrations of samples were added (serum-free DMEM culture medium was added to the model group and blank control). Cells were cultured for 24 hours and then stimulated with UVA at a dose of 7 J / cm². 2 The control group was not irradiated and incubated for 12 hours. After incubation, the cells were removed and placed on ice, washed twice with PBS. Cells were scraped off with a cell scraper and collected in centrifuge tubes. The tubes were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was collected. 200 μl of lysis buffer was added to lyse the cells, and the mixture was centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatant was collected to obtain the cell lysis supernatant. The sample was diluted with the catalase detection buffer provided in this kit. At least an equal volume of catalase detection buffer was added to dilute the lysed sample.

[0135] 3) Determination of standard curve:

[0136] a. Transfer 0, 12.5, 25, 50, or 75 μL of prepared 5 mM hydrogen peroxide solution to 1.5 mL or 0.5 mL plastic centrifuge tubes. Add catalase detection buffer to each tube until the final volume is 100 μL. Mix well. The hydrogen peroxide solution concentrations at this point are 0, 0.625, 1.25, 2.5, and 3.75 mM, respectively. Higher concentrations of hydrogen peroxide standard solution can be prepared if needed.

[0137] b. Take 4 μL of each solution and add it to one well of a 96-well plate. Add 200 μL of the colorimetric working solution. A was measured after incubation for at least 15 minutes. 520 However, the incubation time should not exceed 45 minutes.

[0138] 4) Sample determination:

[0139] a. Referring to Table 5, take x μL (0-40 μL) of sample into a 1.5 ml plastic centrifuge tube, add catalase detection buffer to a volume of 40 μL (i.e., add 40-x μL of catalase detection buffer), and mix well. Then add 10 μL of 250 mM hydrogen peroxide solution, mix quickly with a pipette, and react at 25°C for 1-5 minutes.

[0140] Table 5

[0141] Blank control sample Sample volume 0μl xμl Catalase detection buffer 40μl 40-xμl 250mM hydrogen peroxide solution 10μl 10μl

[0142] b. Add 450 μL of catalase reaction termination solution, and mix by inverting or Vortexing to terminate the reaction. Steps c and d below must be completed within 15 minutes of terminating the reaction.

[0143] c. Add 40 μL of catalase detection buffer to a clean plastic centrifuge tube, then add 10 μL of the above reaction system that has been terminated and mixed, and mix well.

[0144] d. Take 10 μL from the 50 μL system in the previous step and add it to one well of a 96-well plate. Add 200 μL of the colorimetric working solution.

[0145] e. Incubate at 25°C for at least 15 minutes, then measure A. 520 However, the incubation time should not exceed 45 minutes.

[0146] See the experimental results. Figure 8 In Figures (a) and (b), the model group consists of cells irradiated with UVA without any added sample, while the control group consists of cells that were neither irradiated nor treated with any added sample. UVA irradiation reduces CAT cell activity. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml significantly increased CAT cell activity. UVA irradiation also reduces the relative expression level of CAT mRNA in cells. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml significantly increased the relative expression level of CAT mRNA in cells. 7. Glutathione peroxidase detection

[0147] This experiment used the Beyotime Total Glutathione Peroxidase Detection Kit for detection.

[0148] 1) Sample preparation

[0149] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 × 10⁶ cells per well. 5Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and blank control, model, and sample groups were set up. Different concentrations of samples were added (serum-free DMEM culture medium was added to the model group and blank control). Cells were cultured for 24 hours and then stimulated with UVA at a dose of 7 J / cm². 2 The control group was not irradiated and incubated for 12 hours. The cells were then removed, placed on ice, and washed twice with PBS. Cells were scraped off with a cell scraper and collected into centrifuge tubes. The cells were centrifuged at 5000 rpm for 5 minutes, and the supernatant was discarded to obtain cell pellet. 200 μl of lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000 rpm at 4°C for 5 minutes. The supernatant was collected to obtain the cell lysis supernatant.

[0150] 2) Preparation of the reagent kit:

[0151] a. Preparation of 62.5 mM NADPH solution. Add 220 μL of Milli-Q grade pure water to the 11.5 mg NADPH provided in this kit, dissolve and mix well to obtain the 62.5 mM NADPH solution.

[0152] b. Preparation of 75mM GSH solution. Add 433 μL of Milli-Q grade pure water to the 10 mg GSH provided in this kit, dissolve and mix well to obtain the 75mM GSH solution.

[0153] c. Preparation of GPx detection working solution. Based on the number of samples to be tested (including controls), prepare an appropriate amount of GPx detection working solution, requiring 40 μL of GPx detection working solution per test. The prepared GPx detection working solution should only be used on the same day and should be stored on an ice bath if possible.

[0154] d. Preparation of 30 mM peroxide reagent solution. Take 21.5 μL of peroxide reagent (Cum-OOH) and add 5 mL of Milli-Q grade pure water, mix well, and the 30 mM peroxide reagent solution is prepared. It should be stored on an ice bath if possible.

[0155] e. All reagents must be incubated in a water bath at 25°C before use.

[0156] 3) Sample determination

[0157] a. Using a 96-well plate, add the detection buffer, the sample to be tested, and the GPx detection working solution in sequence, mix well, add 40 μL of GPx detection working solution, and incubate at room temperature for 15 minutes to consume the GSSG in the sample and eliminate interference with subsequent detection.

[0158] b. Add 10 μL of 30 mM peroxide reagent solution to each well and mix well.

[0159] c. Immediately use an enzyme-linked immunosorbent assay (ELISA) reader to measure A.340 At this point, the reading is recorded as 0 minutes. Measurement A is taken continuously for 5 minutes or automatically every minute. 340 Data was recorded continuously for 5 minutes, yielding 6 data points. See the experimental results below. Figure 9 In (a) and (b), the model group consisted of cells irradiated with UVA without any sample, while the blank group consisted of cells that were neither irradiated nor treated with any sample. UVA irradiation significantly downregulated the activity of glutathione peroxidase (p<0.001), while cells treated with the sample and then stimulated with UVA showed a significant protective effect, especially the 500 μg / mL extracellular polysaccharide (p<0.001). UVA irradiation reduced the relative expression level of cellular GSH-px mRNA. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml all significantly increased the relative expression level of cellular GSH-px mRNA.

[0160] 8. COL-1 detection

[0161] 1) Experimental preparation

[0162] a. Dilution of Standards: Adjust the volume of the lyophilized standard powder to 150 μl (160 ng / ml) with standard diluent, then mix for 30 seconds. Take five clean EP tubes, add 150 μl of standard diluent to each tube, and label them 80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, and 5 ng / ml, respectively. Add 150 μl of the stock solution to the tube labeled 80 ng / ml, mix well, and repeat the process with the next tube until the last tube. Add standard diluent directly to the zero well.

[0163] b. Dilution of biotin antigen: Centrifuge the concentrated biotin antigen at 6000-10000 rpm for 30 seconds, then take 1 ml of biotin antigen dilution solution into the concentrated biotin antigen, mix for 15 seconds until completely dissolved, then pour all the liquid into the dilution solution bottle and mix well to obtain the working solution of biotin antigen.

[0164] c. Dilution of avidin-HRP: Centrifuge the concentrated biotin antigen at 6000-10000 rpm for 30 seconds, transfer all the concentrated avidin-HRP to the dilution bottle, mix well and the resulting solution is the avidin-HRP working solution.

[0165] d. Washing solution dilution: Pour 25 times the concentrated solution into a 500ml volumetric flask, and dilute to 500ml with distilled water to obtain the working solution.

[0166] 2) Detailed experimental procedures

[0167] a. Do not add sample to the blank well; only add colorimetric reagents A and B and stop solution for zeroing.

[0168] b. Standard wells: Add 50 μl of diluted standard to each well, and add 50 μl of standard / sample diluent to each zero well, followed by 50 μl of biotin antigen working solution.

[0169] c. Sample wells: Add 50 μl of sample, then add 50 μl of biotin antigen working solution. Gently shake, cover with sealing film, and incubate at 37°C for 30 min.

[0170] d. Dilute the 25x concentrated washing solution with 25x distilled water and set aside.

[0171] e. First wash: Carefully peel off the sealing film, discard the liquid, spin dry, add 200μl of washing solution to each well, let stand for 30s and then discard, repeat this 5 times, and drain.

[0172] f. Add 50 μl of avidin-HRP to the standard wells and sample wells, shake gently, cover with the sealing film, and incubate at 37°C for 30 min.

[0173] g. Second wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry.

[0174] h. Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop at 37°C in the dark for 10 minutes.

[0175] i. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow).

[0176] j. Measurement: Zero the sample using the blank well and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. The measurement should be performed within 10 minutes after the stop solution is applied.

[0177] 1. Calculation: Calculate the standard curve and regression equation based on the concentration and OD value, use specialized software for calculation, and select the logistic curve (four parameters) as the fitting model.

[0178] See the experimental results. Figure 10In (a) and (b), the model group consists of cells irradiated with UVA without any added sample, while the blank group consists of cells that were neither irradiated nor added with any sample. UVA irradiation reduces the content of type I collagen in cells. Statistical comparison between the sample group and the model group showed that sample concentrations of 250 μg / ml and 500 μg / ml significantly increased the content of type I collagen in cells. UVA irradiation also reduces the relative expression level of COL-1 mRNA in cells. Statistical comparison between the sample group and the model group showed that sample concentrations of 250 μg / ml and 500 μg / ml significantly increased the relative expression level of COL-1 mRNA in cells.

[0179] 9. MMP-1 detection

[0180] Fibroblasts in good logarithmic growth phase were counted and seeded into 6-well culture plates, with the cell count controlled at 5 × 10⁶ cells per well. 5 Cells were cultured overnight at 37°C and 5% CO2. The culture medium was discarded, and blank control, model, and sample groups were set up. Different concentrations of samples were added (serum-free DMEM culture medium was added to the model group and blank control). Cells were cultured for 24 hours and then stimulated with UVA at a dose of 7 J / cm². 2 The control group was not irradiated and incubated for 12 hours. The cell supernatant was collected using sterile 1.5 mL centrifuge tubes, centrifuged at 1000 g for about 15 minutes at 2–8 °C, and the supernatant was carefully collected.

[0181] The detection was performed using the Cusabio human matrix metalloproteinase (MMP-1) enzyme-linked immunosorbent assay kit.

[0182] The regression equation obtained from the standard curve is as follows: Logistic model: A=a / (1+b×exp(-cρ)), where a=10.078 228 341; b=373.792 111 796; c=7.781627 571 1.

[0183] See the experimental results. Figure 11In (a) and (b), the model group consists of cells irradiated with UVA without any added sample, while the blank group consists of cells that were neither irradiated nor added with any sample. UVA irradiation increases the content of MMP-1 in cells. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml all significantly reduced the content of MMP-1 in cells. UVA irradiation increases the relative expression level of MMP-1 mRNA in cells. Statistical comparison between the sample group and the model group showed that sample concentrations of 100 μg / ml, 250 μg / ml, and 500 μg / ml all significantly reduced the relative expression level of MMP-1 mRNA in cells.

[0184] 10. Apoptosis-senescence detection

[0185] UVA irradiation induces the upregulation of apoptosis-related genes, leading to cell death and functional exhaustion. To understand the effect of the Lactobacillus reuteri extracellular polysaccharide in this application on the apoptosis pathway, the following experiments were also conducted.

[0186] 1) Total RNA extraction from cells: When cells reached 80% confluence, 1 mL of Trizol was added to lyse the cells under ice-cold conditions; 0.2 mL of chloroform was added, and the mixture was shaken by hand for 2 min; the mixture was centrifuged at 8000 r / min for 15 min at 4 °C; the upper aqueous phase was collected, and an equal volume of isopropanol (approximately 700 μL) was added, shaken, and allowed to stand for 15 min; the mixture was centrifuged at 12000 r / min for 10 min, the supernatant was discarded, 1 mL of 70% ethanol was added to the precipitate for washing, and the mixture was centrifuged at 8000 r / min for 10 min; the ethanol washing was repeated once; the mixture was dried at room temperature for 15 min; 40 μL of DEPC-treated ddH2O was added, and the mixture was dissolved for 10 min; the mixture was stored at -80 °C.

[0187] 2) cDNA First-Strand Synthesis: The first strand of cDNA was synthesized using the TINAGEN FastQuant RT Kit (With gDNase) (Genomic De-generated). Preparation of the mixing system: 8 μL total RNA plus 2 μL 5×gDNA Buffer. After thorough mixing, briefly centrifuge and incubate at 42°C for 4 min. Then place on ice. Prepare the mixing system according to the system in Table 6. Thoroughly mix the reverse transcription reaction system with the reaction solution from the gDNA removal step and incubate at 42°C for 15 min. Then incubate at 95°C for 3 min and place on ice. Add 1 mL ddH2O to the obtained cDNA, dilute and mix well, and store at -20°C.

[0188] Table 6

[0189] Component volume Total RNA / mRNA 2μl Anchored Oligo(dT)18Primer(0.5ug / ul) 1μl 2xES Reaction Mix 10μl Transcript RT / RI Enzyme Mix 1μl gDNA Remover 1μl RNase-free water 5μl Total volume 20μl

[0190] 3) Primer and probe design and synthesis: Primers were designed using PrimerExpress software based on the gene sequences published in NCBI, with the housekeeping gene GADPH as the internal reference gene, and specific primer sequences for a total of 8 genes were developed.

[0191] 4) Real-time PCR: The procedure was performed according to the SuperReal quantitative PCR kit instructions. The total reaction volume was 20 μL, including 1 μL cDNA template, 10 μL 10×SuperReal PreMix Plus buffer, 0.3 μL each of positive and negative primers, 0.25 μL hot-start enzyme, and ddH2O to a final volume of 20 μL. Cycling parameters: 95℃ pre-denaturation for 2 min, followed by 40 cycles of 95℃ for 15 s, 65℃ for 15 s, and 72℃ for 30 s. Fluorescence data were collected at 72℃. The reaction was performed on an ABI 7300 quantitative PCR instrument. Experimental results are shown below. Figure 12 The study showed the effect of extracellular polysaccharides from Lactobacillus reuteri on the relative expression levels of Bax mRNA, Bcl-2 mRNA, Sirt1 mRNA, p16 mRNA, p53 mRNA, AKT mRNA, p21 mRNA, and Foxo mRNA in cells. The model group consisted of cells irradiated with UVA without any additional samples, while the control group consisted of cells that were neither irradiated nor treated with any additional samples.

[0192] from Figure 12 As shown in (a), UVA irradiation increases the relative expression level of Bax mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly reduced the relative expression level of Bax mRNA in cells.

[0193] from Figure 12 As shown in (b), UVA irradiation reduces the relative expression level of Bcl-2 mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly increased the relative expression level of Bcl-2 mRNA in cells.

[0194] from Figure 12 As shown in (c), UVA irradiation reduces the relative expression level of Sirt1 mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly increased the relative expression level of Sirt1 mRNA in cells.

[0195] from Figure 12As shown in (d), UVA irradiation increases the relative expression level of P16 mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly reduced the relative expression level of P16 mRNA in cells.

[0196] from Figure 12 As can be seen from (e), UVA irradiation increases the relative expression level of P53 mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly reduced the relative expression level of P53 mRNA in cells.

[0197] from Figure 12 As shown in (f), UVA irradiation reduces the relative expression level of AKT mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly increased the relative expression level of AKT mRNA in cells.

[0198] from Figure 12 As can be seen from (g), UVA irradiation reduces the relative expression level of P21 mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly increased the relative expression level of P21 mRNA in cells.

[0199] from Figure 12 As can be seen from (h), UVA irradiation increases the relative expression level of Foxo mRNA in cells. Statistical comparison between the sample group and the model group revealed that a sample concentration of 500 μg / ml significantly reduced the relative expression level of Foxo mRNA in cells.

[0200] Finally, it should be noted that in this disclosure, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0201] Although this disclosure has been described above through specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this disclosure within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this disclosure. sequence list <110> Beijing Technology and Business University <120> Lactobacillus reuteri SJ-47, the extracellular polysaccharide secreted by this strain and its preparation method <130> PD210560CN0195 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1493 <212> DNA <213> Lactobacillus reuteri <400> 1 gtgtgcctaa tacatgcaag tcgtacgcac tggcccaact aattgatggt gcttgctgaa 60 ttgacgatgg atcaccagtg agtggcggac gggtgagtaa cacgtaggta acctgccccg 120 gagcggggaa taacatttgg aaacagatgc taataccgca taacaacaaa agccgcatgg 180 tttttctgga aagatggctt tggctatcac tctggggatgg acctgcggtg catttagcta 240 gttggtaagg taacggctta cccaaggcga tgatgcatag ccgagttgag agactgatcg 300 gccacaagga actgagacac ggtccataac ttctacggga ggcagcagta gggaatcttc 360 cacaatgggc gcaagctgat ggagcaacac cgcgttatta agaaagggtt tcggccgctt 420 aaactctgtt gttggagaag aacgtgcgtt agagtaactg ttacgcagtg acggtatcca 480 accagaaagt cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540 tatccggatt tattgggcgt aaagcgagcg caggcggttg cttaggtctg atgtggaaac 600 tcggcttaac cgaagaagtg catcggaaac cgggcgactt gagtgcagaa gaggacagtg 660 gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720 gctgtctggt ctgcaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780 accctggtag tccatgccgt aaacgatgag tgctaggtgt tggagggttt cggcccttca 840 gtgcctgttc taacgcatta atgcactccg cctggggagt acgaccgcaa ggttgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtgaagcatg tggtttaatt cgaagctacg 960 cgaagaacct taccaggtct tgacatcttg cgctaacctt agagataagg cgttcccttc 1020 ggggacgtta atgacaggtg gtgcatggtc gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttgtta ctagttgcca gcattaagtt ggggactcta 1140 gtgagactgc cggtgacaaa ccgggagaag gtggggacga cgtcagatca tcatgcccct 1200 tatgaccctg ggctacacac gtgctacaat ggacggtaca acgagtcgca aactcgcgag 1260 agtaagctaa tctcttaaag ccgttctcac ttcggactgt aggctgcaac tcgcctacac 1320 gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccatgggagt ttgtaacgcc caaagttcgg tggcctaacc 1440 1493

Claims

1. A type of Lactobacillus reuteri, characterized in that, The Lactobacillus reuteri mentioned is Lactobacillus reuteri ( Lactobacillus reuteri SJ-47, with accession number CGMCC No.16416.

2. A method for preparing Lactobacillus reuteri extracellular polysaccharide, characterized in that, include: Fermentation culture steps: Lactobacillus reuteri SJ-47 with preservation number CGMCC No. 16416 was inoculated into MRS broth, fermented, and then sterilized to obtain the supernatant of the fermentation broth; Crude polysaccharide extraction steps: The supernatant of the fermentation broth is concentrated and enriched, and then subjected to a first alcohol precipitation, enzymatic hydrolysis to remove protein, a second alcohol precipitation, sevage to remove protein, dialysis, and freeze drying to obtain crude polysaccharide extract.

3. The preparation method according to claim 2, characterized in that, In the fermentation and culture step, the Lactobacillus reuteri has been activated and cultured into a bacterial solution before being inoculated into the MRS broth, and the bacterial solution has an OD value of 0.7-1.6 at 600 nm.

4. The preparation method according to claim 3, characterized in that, During the fermentation and culture step, the pH value of the MRS broth is 5.8-6.

5.

5. The preparation method according to claim 4, characterized in that, In the fermentation culture step, the volume ratio of the bacterial solution to the MRS broth is 5-30%.

6. The preparation method according to any one of claims 2-5, characterized in that, In the fermentation culture step, the fermentation culture conditions are as follows: fermentation temperature 37-45℃, shaking speed 150 r / min-180 r / min, fermentation time 6-16h.

7. The preparation method according to any one of claims 2-5, characterized in that, In the crude polysaccharide extraction step, the fermentation broth supernatant is concentrated to 10-20% of its original volume; during the first alcohol precipitation treatment, the volume ratio of ethanol to the concentrated fermentation broth supernatant is 4:1-10:1, and the alcohol precipitation is carried out at -4℃ for 12-24 hours.

8. The preparation method according to claim 7, characterized in that, In the crude polysaccharide extraction step, the protein removal process includes: reconstitute the product obtained from the first alcohol precipitation treatment with water to the original sample volume, add an appropriate amount of papain and mix well, enzymatically hydrolyze at room temperature for 2-10 hours, and then boil to deactivate.

9. The preparation method according to claim 8, characterized in that, In the crude polysaccharide extraction step, the papain is a 10 g / L papain solution prepared with a phosphate buffer solution at pH=5, and the papain solution is added at a volume ratio of 2-10%.

10. The preparation method according to any one of claims 2-5 and 8-9, characterized in that, Also includes: crude polysaccharide purification Steps: The crude polysaccharide extract was further purified by passing it through a DEAE-52 fiber resin column; NaCl solution was used for elution during the further purification process on the DEAE-52 fiber resin column.

11. The preparation method according to claim 10, characterized in that, The crude polysaccharide purification steps include: The crude polysaccharide extract was dissolved in 0.1 mol / ml NaCl solution, centrifuged, and insoluble matter was removed. The supernatant was added to a equilibrated DEAE-52 column. After injection, linear elution was first performed with 0.3 mol / ml NaCl solution, followed by gradient elution with 0.1, 0.2, and 0.4 mol / ml NaCl solutions according to the position of the elution peak, until no sugar was detected. After concentration of the eluent, residual salts were removed by dialyzing with flowing distilled water, and the product was then freeze-dried under vacuum to obtain purified Lactobacillus reuteri extracellular polysaccharide.

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