Tea culture medium for improving antioxidant activity of plant lactobacillus and application of tea culture medium

Abstract

The invention discloses a tea culture medium capable of improving antioxidant activity of lactobacillus plantarum, which is prepared by the following steps: soaking sour tea in boiling water, filtering to obtain tea water, dissolving 1 / 5-1 / 10 MRS culture medium in the tea water, and sterilizing at high temperature and high pressure, or 38-42 g / L of peptone, 18-22 g / L of glucose, 0.08-0.12 g / L of magnesium sulfate, 0.4-0.6 g / L of manganese sulfate, 1-3 g / L of dipotassium phosphate and 1 L of tea water are mixed and sterilized to prepare the culture medium. Through a DPPH clearance rate experiment, the tea culture medium disclosed by the invention is found to be capable of improving the antioxidant activity of the plant lactobacillus SC75-2-2; the method comprises the following steps: culturing the plant lactobacillus SC75-2-2 by adopting a tea culture medium, crushing a culture bacterium solution to obtain an acellular supernatant, smearing the acellular supernatant on a rat full-thickness skin wound, collecting a skin tissue sample, and carrying out HE analysis, Masson analysis and immunohistochemical analysis, and the result shows that the acellular supernatant has the capability of promoting wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to the application of a tea culture medium for improving the antioxidant activity of Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) SC 75-2-2 and its culture products. Background Art

[0002] Tea has a long history in China and is one of the most popular beverages in the world. Tea is generally divided into three categories: fermented tea (such as black tea), non-fermented tea (such as green tea), and semi-fermented tea (such as oolong tea). The chemical components of tea mainly include tea polyphenols, proteins, tea polysaccharides, chlorophyll, and alkaloids. A large number of in vivo and in vitro medical and clinical studies have shown that tea polyphenols play an important role in human health, and fermentation significantly affects the biological activity of tea polyphenols. Currently, there are studies indicating that cultivating edible fungi with tea residues as the culture substrate can significantly increase the amino acid content. Wu et al. used tea powder as the substrate and added potato extract, glucose, and peptone to culture low-abundance "unculturable microorganisms" that grow by separating the tea inclusions.

[0003] Sour tea is a fermented tea from the De'ang ethnic group. The most significant feature of sour tea is that it undergoes complete anaerobic fermentation during the processing. The fermented tea not only has the tea fragrance of the tea itself but also a special sour taste. Through the determination of the content of tea polyphenols and catechins and the metabolome analysis of sour tea and unfermented tea, it was found that some phenolic substances (protocatechuic acid, coumaric acid) are the main differential metabolites with significant increase, and the antioxidant activity after fermentation is also significantly improved. In recent years, it has been found that its antioxidant ability is closely related to the metabolism of the bacteria, and although traditional culture media (such as MRS medium) can support its growth, it is difficult to efficiently induce antioxidant activity. Therefore, it is of great significance to develop a new culture medium that can not only promote the proliferation of the bacteria but also enhance its antioxidant activity.

[0004] Currently, there are no relevant reports on using sour tea as a culture medium component to culture Lactiplantibacillus plantarum to improve its performance. Summary of the Invention

[0005] The present invention provides the application of a tea culture medium for improving the antioxidant activity of Lactiplantibacillus plantarum ( Lactobacillus plantarum ) SC 75-2-2 and its culture products. The in vivo experimental results show that the cell-free supernatant of Lactiplantibacillus plantarum SC75-2-2 cultured with the tea culture medium of the present invention can promote wound healing.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: 1. Soak the sour tea in boiling water, filter to obtain tea water, dissolve 1 / 5 - 1 / 10 MRS medium with the tea water, and sterilize it by high temperature and high pressure to obtain the tea leaf medium; wherein the MRS medium is 10 g / L peptone, 8 g / L beef powder, 4 g yeast powder, 20 g / L glucose, 1 mL Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate; Or mix 38 - 42 g / L peptone, 18 - 22 g / L glucose, 0.08 - 0.12 g / L magnesium sulfate, 0.4 - 0.6 g / L manganese sulfate, 1 - 3 g / L dipotassium hydrogen phosphate and 1 L tea water, and sterilize it by high temperature and high pressure to obtain; 2. Through the DPPH scavenging rate experiment, the results show that the tea leaf medium of the present invention can improve the antioxidant activity of Lactobacillus plantarum SC75 - 2 - 2; 3. For the cell - free supernatant prepared by breaking the culture solution of Lactobacillus plantarum SC75 - 2 - 2 cultured with the above - mentioned tea leaf medium, smear the cell - free supernatant on the full - thickness skin wound of rats, collect skin tissue samples for HE analysis, Masson analysis and immunohistochemical analysis, and the results show that the cell - free supernatant has the ability to promote wound healing.

[0007] The Lactobacillus plantarum ( Lactobacillus plantarum ) SC75 - 2 - 2 has been disclosed in 202211499533X.

[0008] The present invention has the following beneficial effects: (1) The tea leaf medium of the present invention can improve the antioxidant activity of Lactobacillus plantarum. Oxidative stress is closely related to many chronic diseases, and continuous oxidative stress will lead to the deterioration of the disease. Antioxidants can inhibit the development of the disease. Lactobacillus plantarum not only has antioxidant function but also has the characteristics of being natural and harmless, so it is obviously more suitable for human use; (2) The cell - free supernatant prepared from Lactobacillus plantarum SC75 - 2 - 2 cultured with the tea leaf medium of the present invention has the effect of promoting wound healing, effectively weakening oxidative stress injury, reducing inflammatory response, and promoting the formation of skin appendages and dense collagen deposition; At the same time, as a natural edible product, Lactobacillus plantarum has a wide range of beneficial activities for human health. The method of the present invention provides a better application prospect for the application of Lactobacillus plantarum. Description of the Drawings

[0009] Figure 1 are the DPPH free - radical scavenging rate results of the cell - free supernatants prepared by culturing Lactobacillus plantarum with three kinds of tea leaf media, * p value < 0.05, ** p value < 0.01, ***p The value < 0.001, **** p The value < 0.0001; Figure 2 It is the treatment effect diagram of the wound tissue by the cell-free supernatant prepared by culturing Lactobacillus plantarum with tea medium; Figure 3 It is the wound tissue section diagram of hematoxylin and eosin (H&E) staining and Masson staining of different experimental groups on the 11th day (blue represents collagen fibers, and red represents muscle fibers and red blood cells); Figure 4 It is the fluorescence staining diagram of IL-6 and TGF-β in the skin tissue of different experimental groups on the 11th day; Figure 5 It is the statistical result of the expression level of IL-6 in the skin tissue of different experimental groups on the 11th day; Figure 6 It is the statistical result of the expression level of TGF-β in the skin tissue of different experimental groups on the 11th day. Detailed implementation manners

[0010] The content of the present invention will be further explained below through examples, but these examples do not limit the protection scope of the present invention. The methods in the examples are all conventional methods without special instructions. The reagents used are all conventional commercially available reagents or reagents prepared according to conventional methods without special instructions; Example 1: Preparation of tea medium 1. Weigh 3 g of De'ang sour tea (purchased from Santai Mountain De'ang Ethnic Township, Yunnan Province, China), place it in 100 mL of boiling water and let it stand for 10 min, filter out the tea leaves to obtain tea water; dissolve the components of MRS medium (10 g / L peptone, 8 g / L beef powder, 4 g / L yeast powder, 20 g / L glucose, 1 mL Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate) in 100 mL of tea water according to 1 / 5 of the addition amount, sterilize it at 115 °C for 20 min in a high-pressure sterilizer to obtain MRSTea 5 medium; 2. Dissolve the components of MRS medium in 100 mL of tea water according to 1 / 10 of the addition amount to obtain MRS Tea 10 medium, and the rest is the same as step 1; 3. Prepare MRS B medium by mixing 40 g / L peptone, 20 g / L glucose, 0.1 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 2 g / L dipotassium hydrogen phosphate and 1 L of tea water and sterilize it at 115 °C for 20 min; 4. Prepare according to 10 g / L peptone, 8 g / L beef powder, 4 g yeast powder, 20 g / L glucose, 1 mL Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L ammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1 L water, and sterilize to obtain the ordinary MRS medium.

[0011] Example 2: Cultivation of Lactobacillus plantarum SC75-2-2 and Preparation of Cell-Free Supernatant 1. Inoculate the activated Lactobacillus plantarum SC75-2-2 into the ordinary MRS medium at an inoculation amount of 4‰, and anaerobically culture it statically at 37 °C for 12 h as the control group; 2. Inoculate the activated Lactobacillus plantarum SC75-2-2 into the MRS Tea 5 medium, MRSTea 10 medium, and MRS B medium respectively at an inoculation amount of 2%, and anaerobically culture it statically at 37 °C for 48 h as the experimental group; 3. Take the bacterial suspension (10 7 CFU / mL) obtained after the cultivation in steps 1 and 2, place it in a cell disruptor, ultrasonicate it at 150 Hz for 5 s, and turn it off for 5 s; after 40 min of treatment, centrifuge it at 8000 rpm / min for 10 min to obtain the cell-free supernatant (CFS).

[0012] Example 3: Determination of DPPH Scavenging Rate of Cell-Free Supernatant Mix 1 mL of the cell-free supernatant prepared in Example 2 and 1 mL of tea with 2 mL of DPPH (0.5 mM) methanol solution respectively. After dark incubation at room temperature for 30 min, centrifuge and take the supernatant, and detect the absorbance value at 517 nm. Calculate the DPPH radical scavenging rate through the following formula: DPPH scavenging rate = 1 - [ (A 样 - A 空 ) / A 对 ] × 100%, where A 样 : cell-free supernatant or tea + DPPH; A 空 : cell-free supernatant or tea + methanol; A 对 : water + DPPH As Figure 1 The results showed that the DPPH radical scavenging rates of the cell-free supernatants (CFS) prepared by culturing Lactobacillus plantarum in the MRS Tea 5 medium, MRS Tea 10 medium, and MRS B medium were 84.63%, 74.27%, and 76.02% respectively, which were all significantly higher than those of tea (56.29%) and the CFS (45.55%, 46.10%, 49.82%) prepared by culturing Lactobacillus plantarum in the ordinary MRS medium (control group).

[0013] Example 4: Application of cell-free supernatant prepared by culturing Lactiplantibacillus plantarum SC75-2-2 in MRS Tea 5 medium in promoting wound healing 1. Aliquot the cell-free supernatant prepared by culturing Lactiplantibacillus plantarum SC75-2-2 in the above MRS Tea 5 medium into 15 mL per tube, freeze-dry it in a vacuum freeze dryer for 48 h, then dissolve it in ultrapure water to 3 mL, and store it at -20 °C.

[0014] 2. Establishment of full-thickness skin injury model Male SD rats (body weight 300 ± 20 g) were purchased from Kunming Medical University and allowed 1 week for adaptation. Subsequently, all rats were randomly divided into a control group, an iodine tincture group, and a supernatant group according to different treatment methods, with 3 rats in each group. The rats were anesthetized with isoflurane, the hair on the back of the rats was removed, and a circular defect with a diameter of 10 mm was formed on the left and right sides of the back using a skin punch. The wound surface of the rats in the supernatant group was smeared with 500 μL of the cell-free supernatant dissolution solution prepared in step 1, covered once a day, the control group was not treated, and the iodine tincture group was smeared with 500 μL of iodine tincture as a positive control group; on the 12th day, the rats were euthanized, and skin tissue samples were collected and stored in paraformaldehyde fixative. During this period, the wounds were photographed with a digital camera every other day.

[0015] The results are as Figure 2 shown. Compared with the control group and the iodine tincture group, the wound area treated with the supernatant group was significantly reduced, especially on the 11th day, indicating that the cell-free supernatant of the present invention has the effect of promoting wound healing.

[0016] 3. HE staining of skin tissue The skin tissue was embedded in paraffin and cut into thin slices. The slices were successively placed in environment-friendly dewaxing solution I for 20 min, environment-friendly dewaxing solution II for 20 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 75% alcohol for 5 min, and washed with tap water. The frozen sections were taken out from the -20 °C refrigerator and restored to room temperature, fixed with tissue fixative for 15 min, and then rinsed with running water. The sections were treated with a high-definition constant staining pretreatment solution for 1 min. The sections were stained with hematoxylin stain for 3 - 5 min, washed with tap water, differentiated with a differentiation solution, washed with tap water, blued with a bluing solution, and rinsed with running water. The sections were dehydrated in 95% alcohol for 1 min and stained with eosin stain for 15 s. The sections were successively placed in absolute ethanol I for 2 min, absolute ethanol II for 2 min, absolute ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, and xylene II for 2 min for transparency, and sealed with neutral balsam. Microscopic examination was performed and image acquisition and analysis were carried out.

[0017] The histological evaluation of the wound healing effects of different groups was performed by H&E staining, and the results are shown in Figure 3In the above figure, new epidermis and dermis layers were formed in all groups. Compared with the control group and the iodine tincture group, more skin appendages, including hair follicles, blood vessels, and sweat glands, appeared in the wounds treated with the supernatant on the 11th day.

[0018] 4. Masson staining of skin tissue Put the sections into environment-friendly dewaxing solution Ⅰ for 20 min, environment-friendly dewaxing solution Ⅱ for 20 min, absolute ethanol Ⅰ for 5 min, absolute ethanol Ⅱ for 5 min, 75% alcohol for 5 min in sequence, and wash with tap water. For frozen sections, rewarm and fix: Take out the frozen sections from the -20°C refrigerator and let them return to room temperature. Fix with tissue fixative for 15 min and then rinse with running water. Immerse the sections in Masson A solution overnight and wash with running tap water. Put the sections into the staining solution which is a mixture of Masson B solution and Masson C solution in equal proportion, stain for 1 min, wash with tap water, differentiate with the differentiating solution for several seconds, and wash with tap water. Immerse the sections in Masson D solution for 6 min and rinse with tap water. Immerse in Masson E solution for min. Without washing, drain slightly and directly stain with Masson F solution for 2 - 30 s. Rinse and differentiate the sections with 1% acetic acid, dehydrate with two cylinders of absolute ethanol. Put the sections into the third cylinder of absolute ethanol for 5 min, make them transparent with xylene for 5 min, and mount with neutral gum. Examine under a microscope and collect and analyze the images. Collagen is the main component of the skin and plays a key role in promoting wound healing by providing appropriate physical support and promoting cell migration, proliferation, and differentiation.

[0019] The results of Masson staining analysis are shown in Figure 3 the following figure. Dense collagen depositions were formed in the wounds of all three groups.

[0020] 5. Fluorescent staining of IL-6 and TGF-β in skin tissue The slices were successively placed in environment-friendly dewaxing solution Ⅰ for 10 min, environment-friendly dewaxing solution Ⅱ for 10 min, environment-friendly dewaxing solution Ⅲ for 10 min, absolute ethanol Ⅰ for 5 min, absolute ethanol Ⅱ for 5 min, absolute ethanol Ⅲ for 5 min, and then washed with distilled water. The tissue sections were placed in a repair box filled with EDTA antigen repair buffer (pH 8.0) and antigen repair was carried out in a microwave oven. Medium heat for 8 min, stop for 8 min, then turn to medium-low heat for 7 min. During this process, buffer over-evaporation should be prevented and the slices must not be dried. After repair, let it cool naturally. The slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for washing 3 times, 5 min each time. After the slices were slightly dried by shaking, a histochemical pen was used to draw a circle around the tissue, and BSA was added dropwise (10% donkey serum was used for blocking when the primary antibody was from goat, and 3% BSA was used for blocking when the primary antibody was from other sources), and blocked for 30 min. The prepared primary antibody was added dropwise, and the slices were placed flat in a wet box and incubated overnight at 4°C. The slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for washing 3 times, 5 min each time. The corresponding secondary antibody was added and incubated at room temperature in the dark for 50 min. The slides were placed in PBS (pH 7.4) and shaken on a decolorizing shaker for washing 3 times, 5 min each time. DAPI staining solution was added and incubated at room temperature in the dark for 10 min. The slides were placed in PBS (pH 7.4) and washed on a decolorizing shaker 3 times, 5 min each time. Spontaneous fluorescence quenching agent B solution was added for 5 min and rinsed with running water for 10 min. Mount with anti-fluorescence quenching mounting medium. The slices were placed under a fluorescence microscope, and the positive area ratios of IL-6 and TGF-β were calculated based on an automated image analysis software (positive area ratio = total positive area / tissue pixel area); On the 11th day, the expressions of IL-6 and TGF-β in each group were detected to evaluate the recovery of infected wounds, and the results are shown in Figures 4 - 6 , it can be seen from the figure that compared with other groups, the expression of IL-6 in the supernatant group was the lowest (4.29% lower than 20.44% in the control group and 24.65% in the iodine tincture group). In contrast, the average expression of TGF-β in the supernatant group was 12.93%, higher than that in the control group and comparable to that in the iodine tincture group. The experimental results indicate that the cell-free supernatant of the present invention can effectively reduce oxidative stress damage and decrease the inflammatory response.

Claims

1. A tea culture medium for improving the antioxidant activity of Lactiplantibacillus plantarum ( Lactobacillus plantarum ), characterized in that: The tea solution is obtained by soaking and filtering sour tea in boiling water. Dissolve 1 / 5 - 1 / 10 MRS medium with the tea solution and sterilize it to prepare; or mix 38 - 42 g / L peptone, 18 - 22 g / L glucose, 0.08 - 0.12 g / L magnesium sulfate, 0.4 - 0.6 g / L manganese sulfate, 1 - 3 g / L dipotassium hydrogen phosphate and 1 L tea solution, and sterilize it to prepare.

2. Application of the cell-free supernatant prepared by culturing Lactobacillus plantarum SC75-2-2 with the tea leaf medium described in claim 1 in the preparation of a wound healing promoting preparation.

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