Keemun black tea fermentation product as well as preparation method and application thereof

Abstract

The invention discloses a Keemun black tea fermentation product, a preparation method thereof and application of the Keemun black tea fermentation product in caenorhabditis elegans anti-aging research. Active components in the black tea can be more fully extracted by using the natural eutectic solvent, and then the active components are further fermented by sequentially using the lactobacillus and the thermophilic thermophilic bacteria, so that the Keemun black tea fermentation product is prepared. The Keemun black tea fermentation product prepared by the invention is applied to caenorhabditis elegans anti-aging research, and adverse effects caused by oxidative stress are relieved through the antioxidant effect of the Keemun black tea fermentation product, so that the antioxidant enzyme activity (SOD, CAT and GSH) of caenorhabditis elegans is remarkably improved, and the metabolite (MDA) is remarkably reduced; and the ROS level in the caenorhabditis elegans is also obviously reduced. Therefore, the Keemun black tea fermentation product prepared by the invention has better antioxidant activity, and can effectively improve the oxidative stress damage of the caenorhabditis elegans, thereby effectively prolonging the life of the caenorhabditis elegans.

Description

Technical Field

[0001] The invention relates to the technical field of tea extracts, and in particular to a Keemun black tea fermentation product and a preparation method and application thereof. Background Art

[0002] Tea is one of the three major beverages in the world. Due to its unique flavor and pharmacological functions, more and more people love to drink tea. According to different processing techniques, tea can be divided into six categories: green tea, white tea, yellow tea, oolong tea, black tea and dark tea. Among them, black tea is the largest tea produced and consumed in the world, and black tea consumption accounts for about 78% of the world's tea industry. Studies have shown that black tea can prevent or treat a variety of chronic diseases such as various cancers, cardiovascular diseases, metabolic syndrome and neurodegenerative diseases, and has potential anti-aging effects.

[0003] Since black tea has multiple biological activities, many tea skin care products are now available on the market. However, the only methods commonly used for the development of black tea products are direct extraction or multi-strain / single-strain direct fermentation. The comprehensive utilization of active ingredients in black tea is limited. At present, only preliminary research has been conducted on its antioxidant and anti-tyrosinase activities. Its anti-aging effect is only limited to the possibility of evaluating the efficacy of skin care products. There is a lack of scientific verification theory for in-depth exploration of its specific mechanism. Therefore, in the context of the era of functional skin care, how to further enhance the active efficacy of tea and conduct anti-aging verification is a key issue that still needs to be solved. Summary of the invention

[0004] The purpose of the present invention is to solve the problem of limited utilization of active ingredients in black tea in the prior art, and to provide a Keemun black tea fermentation product and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: a method for preparing a Keemun black tea fermentation product, comprising the following steps:

[0006] S1. Grind the Keemun black tea into powder, mix and extract with a natural low eutectic solvent, the extraction time is 20 to 40 minutes, the extraction temperature is 40 to 50° C., and a crude extract is obtained;

[0007] S2, adding deionized water to the crude extract at a solid-liquid ratio of 1:20 to 1:40, and then inoculating the lactobacillus seed solution, and culturing at 24°C to 28°C in a shaking incubator for 12 to 16 hours to complete the first fermentation step;

[0008] S3. The first step fermentation liquid is added to the thermophilic Thermus thermophilus seed liquid, and the temperature is raised to 60-80°C for secondary fermentation for 12-16 hours. The secondary fermentation liquid is then ultrasonically crushed, centrifuged to remove impurities, filtered through a membrane, and concentrated to obtain the Keemun black tea fermentation product.

[0009] Preferably, the Keemun black tea is crushed into powder of 20-120 mesh.

[0010] Preferably, the ratio of the Keemun black tea powder to the natural deep eutectic solvent is 0.1 to 0.5 g / ml (w / v).

[0011] Preferably, the molar ratio of fructose, glycerol and betaine in the natural low eutectic solvent is 1:1:2, and the water content is 10% to 15%.

[0012] Preferably, the inoculation amount of the lactobacillus seed solution is 0.2% to 0.5% (v / v).

[0013] More preferably, the inoculation amount of the Thermus thermophilus seed solution is 0.2% to 0.5% (v / v).

[0014] Further preferably, in S3, the secondary fermentation liquid is placed in an ultrasonic crusher and ultrasonicated at an ultrasonic power of 300-400 W for 30-40 min, and then centrifuged at a speed of 8000-10 000 rpm for 10-20 min, and the upper clear layer is filtered through a 0.22 μm filter membrane and concentrated under reduced pressure to obtain a solid fermentation product.

[0015] The present invention also provides a Keemun black tea fermentation product prepared by the preparation method and its application in anti-aging research of Caenorhabditis elegans.

[0016] The beneficial effects of the present invention are:

[0017] (1) The present invention utilizes a natural low eutectic solvent to more fully extract the active ingredients in black tea, and then further ferments the extract using Lactobacillus and Thermus thermophilus to significantly increase the active factors, while reducing the molecular weight for easier absorption.

[0018] (2) The Keemun black tea fermentation product prepared by the present invention is applied to the anti-aging research of Caenorhabditis elegans. Through the antioxidant effect of the Keemun black tea fermentation product, the adverse effects of oxidative stress are effectively alleviated, so that the antioxidant enzyme activity (SOD, CAT, GSH) of Caenorhabditis elegans is significantly improved, while the metabolite (MDA) is significantly decreased, and the ROS level in Caenorhabditis elegans is also significantly decreased. Therefore, the Keemun black tea fermentation product prepared by the present invention has good antioxidant activity, can effectively improve the damage of oxidative stress in Caenorhabditis elegans, and thus effectively prolong the life span of Caenorhabditis elegans. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of ingestion of Keemun black tea fermentation products on the survival of Caenorhabditis elegans under different stress conditions; 1-A is 30% hydrogen peroxide and 1-B is 5mM paraquat.

[0020] Figure 2 The effects of Keemun black tea fermentation products on the antioxidant enzyme activities and metabolite (MDA) content of Caenorhabditis elegans; including the relative levels of SOD (2-A), CAT (2-B), GSH (2-C) and MDA (2-D).

[0021] Figure 3 Effects of Keemun black tea fermentation products on ROS in Caenorhabditis elegans.

[0022] In the present invention, Lactobacillus plantarum is stored in China National Center for Microbiological Culture Collection with the number CGMCC No. 24962 and purchased from Shanghai Guyan Industrial Co., Ltd.; Thermus thermophilus is purchased from Shanghai Guyan Industrial Co., Ltd. with the item number GOYJ9883.

[0023] Escherichia coli OP50: purchased from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota 。

[0024] ROS-specific fluorescent probe DCFH-DA ： Purchased from Sigma-Aldrich (USA).

[0025] The Caenorhabditis elegans comes from Professor Wu's team at Huazhong University of Science and Technology, and is published in the literature "Wang Gang, Song Bingbing, Jia Xuejing et al. Caenorhabditis elegans Ceramides from Sea Red Rice Bran Improve Health Indicators and Increase Stress Resistance of through Insulin / IGF-1 Signaling (IIS) Pathway and JNK-1. [J]. J Agric Food Chem, 2022, 70: 15080-15094." DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0027] Example 1 Preparation of Keemun black tea fermentation product

[0028] (1) Preparation of strain seed solution

[0029] YPD medium: Take 10g of glucose, 10g of peptone, 5g of yeast extract, add 1L of deionized water, and then sterilize at 105℃ for 30min. Then inoculate Lactobacillus and Thermus thermophilus respectively and place them in a shaking incubator at 26 / 75℃ for 24h before use.

[0030] (2) Preparation of black tea extract using natural deep eutectic solvent

[0031] First, a certain amount of fructose, glycerol, and betaine were weighed in a molar ratio of 1:1:2 to prepare a natural low eutectic solvent (water content of 10%). Then, the Keemun black tea was crushed into a powder of 20 to 120 meshes, and the black tea powder and the natural low eutectic solvent were accurately weighed in a ratio of 0.2 g / ml (w / v) of Keemun black tea powder to the natural low eutectic solvent, and the mixture was placed in an extraction device and mixed, and then extracted. The extraction time was 30 minutes; the extraction temperature was 45°C, and a crude extract was obtained after extraction.

[0032] (3) Further fermentation of black tea extract

[0033] After adding deionized water to the crude extract at a solid-liquid ratio of 1:20, Lactobacillus seed liquid was inoculated at an inoculation amount of 0.2% (v / v), and cultured in a shaking incubator at 26°C for 14 hours for the first fermentation; then 0.2% (v / v) Thermus thermophilus seed liquid was inoculated, and the temperature was raised to 75°C for secondary fermentation for 14 hours to obtain a fermentation liquid.

[0034] (4) The fermentation liquid was placed in an ultrasonic crusher and ultrasonicated at an ultrasonic power of 400 W for 30 min, and then centrifuged at a speed of 10,000 rpm for 10 min. The upper clear layer was filtered through a 0.22 μm filter membrane and concentrated under reduced pressure to obtain a solid black tea fermentation product.

[0035] Example 2 Application of Keemun black tea fermentation products in anti-aging research of Caenorhabditis elegans

[0036] (1) Oxidative stress experiments under different conditions

[0037] First, Caenorhabditis elegans (from Professor Wu's team at Huazhong University of Science and Technology) was inoculated on NGM plates containing Escherichia coli OP50 and grown in a medium at 20°C. Then, Caenorhabditis elegans was rinsed with M9 buffer after the L4 stage and lysed with bleach to obtain synchronized nematodes. In order to evaluate the protective effect of Keemun black tea fermentation products on nematodes, 30% hydrogen peroxide and 5mM paraquat were applied to NGM plates in this experiment. Then, the number of days the nematodes survived after exposure to different concentrations of Keemun black tea fermentation products (0.10, 0.20 and 0.50 mg / mL) was observed. The reversal effect was then evaluated by exposure to stressors. The nematodes were transferred to fresh extracts or control plates containing hydrogen peroxide or paraquat every other day and counted until they all died. The nematodes were gently probed with platinum wire every day to observe the extent of nematode death and exclude abnormal worms. The number of nematodes in each group is detailed in Table 1.

[0038] (2) Antioxidant enzyme activity assay

[0039] The vitality of the nematodes was examined by measuring the levels of antioxidant enzymes and metabolites (MDA). Enzymes associated with oxidative stress in vivo include superoxide dismutase (SOD) and catalase (CAT); as well as glutathione (GSH) and malondialdehyde (MDA) metabolites. In this experiment, C. elegans was stressed with 30% hydrogen peroxide and then exposed to different concentrations of Keemun black tea fermentation products (0.10, 0.20, and 0.50 mg / mL), and then samples were collected and the activity of antioxidant enzymes was determined using a kit purchased from Shanghai Bioengineering Yuanye.

[0040] (3) ROS level measurement

[0041] ROS is crucial in the oxidative system and is measured by relative fluorescence quantification. In this experiment, Caenorhabditis elegans was stressed with 30% hydrogen peroxide and then exposed to different concentrations of Keemun black tea fermentation products (0.10, 0.20 and 0.50 mg / mL). The ROS-specific fluorescent probe DCFH-DA (100 μM) was added to a liquid culture medium containing 0.5% NaCl. The content of ROS was measured by stirring at a final concentration of 500 μM for 1 hour in the dark. The nematodes were crushed using an ultrasonic pulverizer, and the supernatant (50 μL) was mixed with DCFH-DA (50 μL) in a 96-well plate. The fluorescence intensity of each sample was measured using a plate reader (Filter Max F5) (excitation: 485 nm; emission: 535 nm).

[0042] Test results:

[0043] 1. The intake of Keemun black tea fermentation products increases the resistance of Caenorhabditis elegans to different stressors

[0044] like Figure 1 As shown in Supplementary Table 1, the results showed that the intake of Keemun black tea fermented products prolonged the average lifespan of C. elegans in a dose-dependent manner compared with the control, and the concentration of 0.50 mg / mL resulted in the highest average lifespan extension of 87.9% under hydrogen peroxide. Secondly, paraquat was used as a neurotoxin in chronic oxidative stress experiments to induce chronic toxic damage in nematodes. After consuming different concentrations of Keemun black tea fermented products for 5 days, we exposed the nematodes to NGM containing 10 mM paraquat and evaluated the mortality rate over time. Compared with the control group, an extension of the lifespan of nematodes was observed after taking paraquat, where the intake of Keemun black tea fermented products at concentrations of 0.10–0.50 mg / mL increased paraquat resistance by 9.7–33.3%. These results indicate that the intake of Keemun black tea fermented products alleviated the cell damage caused by paraquat and hydrogen peroxide to varying degrees.

[0045] Table 1 Effects of Keemun black tea fermentation products on the lifespan of Caenorhabditis elegans under different stressors

[0046]

[0047] 2. Keemun black tea fermentation products improve the effects of oxidative stress

[0048] like Figure 2 As shown, MDA and GSH content and the activities of antioxidant enzymes SOD and CAT are important indicators of oxidative stress, which can better understand the relevant dynamic changes in oxidative stress resistance, which leads to increased resistance in C. elegans after ingesting Keemun black tea fermentation products. Compared with the control group, SOD and CAT activities were significantly increased by 1.52-2.12 times and 1.26-2.83 times, respectively, GSH increased by 1.15-1.34 times, and the metabolite MDA decreased by 64.7%. Therefore, the above results show that nematodes treated with Keemun black tea fermentation products show significant improvements in resistance and antioxidant enzyme activities. It induces stimulation and produces endogenous responses through long-term intake, accompanied by a corresponding increase in the relative expression of enzyme activity in the body when encountering external stimuli. Taken together, these results indicate that C. elegans is able to use its defense system to detoxify, develop resistance to external stimuli, and maintain healthy longevity and survival.

[0049] 3. Keemun black tea fermentation products reduce ROS levels

[0050] like Figure 3As shown in Figure 2, ROS plays an important role in apoptosis through DNA damage, amino acid oxidation, and lipid peroxidation, leading to the aging of organisms. Therefore, the level of ROS is often used to assess the degree of aging and the level of oxidative damage in organisms. In this experiment, nematodes were treated with different concentrations of Keemun black tea fermentation products for 5 days. Compared with the blank control, the level of ROS in nematode cells was reduced by 70.21–31.2% (p<0.05) after ingestion of 0.10–0.50 mg / mL of Keemun black tea fermentation products. It is worth noting that within the effective range of Keemun black tea fermentation products, the level of ROS gradually decreased with increasing concentration, indicating that the intake of Keemun black tea fermentation products may slow down the expression of NADPH oxidase on the plasma membrane of the mitochondrial procytosis of nematodes.

[0051] The specification and drawings of the present invention are considered to be illustrative rather than restrictive. On the basis of the present invention, those skilled in the art can make some substitutions and modifications to some of the technical features according to the disclosed technical content without creative labor, all of which are within the protection scope of the present invention.

Claims

1. A method for preparing a Keemun black tea fermentation product, characterized in that: The following steps are involved: S1. Grind the Keemun black tea into powder, mix and extract with a natural low eutectic solvent, the extraction time is 20 to 40 minutes, the extraction temperature is 40 to 50° C., and a crude extract is obtained; S2, adding deionized water to the crude extract at a solid-liquid ratio of 1:20 to 1:40, and then inoculating the lactobacillus seed solution, and culturing at 24°C to 28°C in a shaking incubator for 12 to 16 hours to complete the first fermentation step; S3. The first step fermentation liquid is added to the thermophilic Thermus thermophilus seed liquid, and the temperature is raised to 60-80°C for secondary fermentation for 12-16 hours. The secondary fermentation liquid is then ultrasonically crushed, centrifuged to remove impurities, filtered through a membrane, and concentrated to obtain the Keemun black tea fermentation product.

2. The preparation method according to claim 1, characterized in that: The Keemun black tea is crushed into powder of 20-120 meshes.

3. The preparation method according to claim 1, characterized in that: The ratio of the Keemun black tea powder to the natural deep eutectic solvent is 0.1-0.5 g / ml (w / v).

4. The preparation method according to claim 1, characterized in that: The molar ratio of fructose, glycerol and betaine in the natural low eutectic solvent is 1:1:2, and the water content is 10% to 15%.

5. The preparation method according to claim 1, characterized in that: The inoculation amount of the lactobacillus seed liquid is 0.2% to 0.5% (v / v).

6. The preparation method according to claim 1, characterized in that: The inoculation amount of the Thermus thermophilus seed solution is 0.2% to 0.5% (v / v).

7. The preparation method according to claim 1, characterized in that: In S3, the secondary fermentation liquid is placed in an ultrasonic crusher and ultrasonicated at an ultrasonic power of 300-400 W for 30-40 minutes, and then centrifuged at a speed of 8000-10 000 rpm for 10-20 minutes. The supernatant is filtered through a 0.22 μm filter membrane and concentrated under reduced pressure to obtain a solid fermentation product.

8. A Keemun black tea fermentation product prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the Keemun black tea fermentation product as claimed in claim 8 in anti-aging research of Caenorhabditis elegans.

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