CN113303386A - Fermented tea helpful for delaying organism aging and preparation method and application thereof - Google Patents
Fermented tea helpful for delaying organism aging and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/16—Tea extraction; Tea extracts; Treating tea extract; Making instant tea
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
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Abstract
The invention discloses fermented tea helpful for delaying nematode senescence and a preparation method thereof. According to the invention, Aspergillus niger RAF106 with high safety separated from Pu' er tea is used for fermenting 0.5% -10% of green tea soup for 2-8 days to prepare fermented tea. Compared with unfermented tea, the fermented tea prepared by the method can prolong the average life of nematodes by more than 17 percent and prolong the maximum life by 2 days; the accumulation of fat in the body of the nematode is obviously reduced, the content of Malondialdehyde (MDA) is reduced by 24.37 percent, the content of maximum Glutathione (GSH) and the enzymatic activity of superoxide dismutase (SOD) are respectively increased by 104.13 percent and 47.21 percent, which shows that the fermented tea can obviously delay the aging of the nematode. Therefore, the functional fermented tea for delaying the nematode aging can be prepared by the method, a novel tea beverage or food ingredients related to the tea can be developed, and the method has good application prospects in the fields of tea processing and food processing.
Description
Technical Field
The invention relates to the technical field of fermented functional foods, in particular to fermented tea helpful for delaying senescence and a preparation method and application thereof.
Background
With the rising proportion of the population of the global aging population, health and social problems brought by aging and the accompanying huge costs have gradually become the hot spots of concern. According to statistics, in 2019, the population number of 65 years old and above is 7.03 hundred million in the world, the proportion of the population is increased from 6% to 9% in 199 years, and the number of the old people is doubled to 15 hundred million and further increased to 16% in 2050. Under the combined action of internal and external environments, aging can cause harmful and irreparable changes in biomolecules, cells, tissues and organs in human bodies, and can cause the attenuation of multiple organ functions, thereby causing the occurrence and development of various aging-related metabolic, nervous and cardiovascular diseases. Therefore, the search for food or medicine which can safely and effectively delay the aging process, reduce the incidence rate of age-related diseases and prolong the life time is a hot problem in the current medical research of the elderly.
The tea water contains a plurality of bioactive components beneficial to human health, including tea polyphenol, tea polysaccharide, tea pigment and the like, and has physiological effects of refreshing, promoting urination, removing toxicity, resisting mutation, resisting tumor, reducing blood fat, resisting anaphylactic reaction and the like. With the global demand for functional health drinks growing and consumers tending to believe more that the products traditionally associated with health are, there is an increasing demand for functional or health tea beverages in the market, and one such type is the annual composite growth rate of probiotic tea beverages, such as yeast, bacteria co-fermented Kangpu's market from 2016 to 2020 of up to 25%, with a global market share of 2025 being expected to grow to $ 54.5 billion in the year. Research shows that after the tea is subjected to liquid fermentation by eurotium coronarium, the activities of cellulase, pectinase, polyphenol oxidase, amylase and protease of tea water are obviously enhanced, and the contents of main biochemical components including amino acid, tea polyphenol and the like are improved (research on technological parameters of Liuqiaolin. liquid fermented tea and related enzyme activities [ D ] of Sichuan university of agriculture, 2007 ]); the scavenging activity of the tea beverage fermented by lactic acid bacteria on ABTS free radicals is improved (Liujiaqi, the research and functional research of the lactic acid bacteria fermented tea beverage [ D ]. Nanchang university, 2017 ]); the total phenol content and DPPH clearance of fermented tea-oolong tea are higher than those of unfermented tea-green tea (Mahui et al. 4 kinds of tea aqueous extracts and in-vitro antioxidant performance research of tea polyphenol [ J ]. food research and development, 2019.); in addition, the bioavailability of active ingredients such as tea polyphenol of tea leaves or tea water is increased after the tea leaves or tea water is fermented by microorganisms or enzyme systems thereof, and the physiological activities such as tumor resistance, oxidation resistance, immunoregulation and the like are enhanced (Du Ming et al. Therefore, the health efficacy of the tea leaves or the tea water after microbial fermentation is more obvious, and the tea water meets the requirements of current consumers. Through information retrieval, the current patents on the fermented tea mainly focus on the development and preparation of fermented tea products with the effects of retaining active substances, stabilizing quality, improving antioxidation, antivirus, reducing blood sugar, improving immunity and the like, and the preparation process of the fermented tea for delaying the aging of the organism aims at achieving the aim by adding the substances such as aizoon stonecrop, okra and loquat leaves, and related reports that the fermented tea for delaying the aging of the organism is prepared by fermenting green tea water only through microorganisms are not found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a preparation method of fermented green tea.
Another object of the present invention is to provide fermented green tea produced by the above production method.
It is a further object of the present invention to provide the use of said fermented green tea.
The above object of the present invention is achieved by the following technical solutions:
a preparation method of fermented green tea comprises the following steps:
s1, leaching: soaking tea water containing 0.5-10 wt% of green tea at 80-85 ℃ for 10-14 min, taking filtrate, repeating the steps, and taking tea water mixed liquor as fermented tea water stock solution;
s2, fermentation: taking the tea water stock solution obtained in the step S1, pasteurizing, adding 1% v/v Aspergillus niger RAF106 bacterial suspension, and culturing at 28-32 ℃ for 2 d-8 d at 180-220 r/min; the concentration of the Aspergillus niger RAF106 bacterial suspension is 1 x 107spore/mL; the Aspergillus niger RAF106 is preserved in the China general microbiological culture Collection center in 2014, 8 and 27 months, and the preservation number is CGMCC NO. 9608.
Aspergillus niger RAF106 is an important fermentation industrial strain which is automatically separated in the early stage of the experiment of the inventor. According to the invention, the Aspergillus niger RAF106 with high safety separated from the Pu' er tea is used for fermenting the green tea water to prepare the fermented tea, and the result shows that the average service life of nematodes of the prepared fermented tea can be prolonged by more than 17%, and the maximum service life of the nematodes can be prolonged by 2 d; the fat accumulation in the nematode body is obviously reduced, the MDA content is reduced by 24.37 percent, the maximum GSH content and the SOD enzyme activity are respectively increased by 104.13 percent and 47.21 percent, the service life of the nematode can be obviously prolonged, the fat accumulation is reduced, the antioxidant enzyme activity is improved, and the oxidative damage is reduced, thereby achieving the effect of delaying the organism aging.
Preferably, the fermentation time of step S2 is 2-6 d.
Preferably, the content of green tea in the tea water of the green tea in the step S1 is 0.5 wt%, and the cultivation time of the step S2 is 6 d. After the fermented green tea prepared under the condition is fed with nematodes for 72 hours at 25 ℃, the service lives of the nematodes are obviously prolonged, the average service life is prolonged by 17.85%, and the maximum service life is prolonged by 2 d; the fat content in the nematode is reduced; the content of MDA in the nematode body is respectively and obviously reduced by 24.37 percent, the SOD activity is obviously increased by 47.21 percent, and the content of GSH is obviously increased by 104.13 percent.
Preferably, the content of green tea in the tea water of the green tea in the step S1 is 1.0 wt%, and the cultivation time of the step S2 is 4 d. After the fermented green tea prepared under the condition is fed with nematodes for 72 hours at 25 ℃, the service lives of the nematodes are obviously prolonged, the average service life is prolonged by 12.86%, and the maximum service life is prolonged by 1 d; the fat content in the nematode is reduced; the content of MDA in the nematode is obviously reduced by 14.55%, the SOD activity is respectively obviously increased by 31.25%, and the content of GSH is obviously increased by 42.20%.
Preferably, the content of the green tea in the tea water of the green tea in the step S1 is 10 wt%, the culture time in the step S2 is 2d, the average life span is respectively prolonged by 11.55%, and the maximum life span is prolonged by 1 d; the fat content in the nematode is reduced; the content of MDA in the nematode is obviously reduced by 12.49%, the SOD activity is respectively and obviously increased by 28.66%, and the GSH content is obviously increased by 39.58%.
Preferably, the soaking in step S1 is 85 ℃ soaking for 12 min.
Preferably, the cultivation in step S2 is performed in a constant temperature shaking table with 30 ℃ and 200r/min of rotation speed.
The invention also claims the fermented green tea prepared by any one of the methods.
The fermented green tea prepared by the invention can obviously prolong the service life of nematodes, reduce the accumulation of fat, improve the activity of antioxidant enzyme and reduce oxidative damage, thereby achieving the effect of delaying the aging of organisms; accordingly, the present invention also provides the following uses of the fermented green tea:
the fermented green tea is applied to preparing an antioxidant product.
The use of said fermented green tea in the manufacture of a product for reducing fat accumulation in the body.
The fermented green tea is applied to preparing products for resisting body aging.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides fermented tea beneficial to delaying organism aging, which is prepared by fermenting green tea soup by using Aspergillus niger RAF106 which is separated from Pu' er tea and has high safety. Compared with unfermented tea, the fermented tea prepared by the method can prolong the average life of nematodes by more than 17 percent and prolong the maximum life by 2 days; the accumulation of fat in the body of the nematode is obviously reduced, the malondialdehyde content is reduced by 24.37%, the maximum glutathione content and the enzymatic activity of superoxide dismutase are respectively increased by 104.13% and 47.21%, which shows that the fermented tea can obviously prolong the service life of the nematode, reduce the accumulation of fat, improve the activity of antioxidant enzyme and reduce oxidative damage, thereby achieving the effect of delaying the aging of organisms. Therefore, the method can be used for preparing the functional fermented tea for delaying the aging of the organism, developing novel tea drinks or food ingredients related to the tea, and has good application prospect in the fields of tea processing and food processing.
Drawings
FIG. 1 shows the survival rate of nematodes after tea water treatment after fermentation of 0.5% green tea for various periods of time.
FIG. 2 is a graph showing the survival rate of nematodes after treatment of tea water after fermentation of 1% green tea for various periods of time.
FIG. 3 is a graph showing the survival rate of nematodes after treatment of tea water after fermentation of 10% green tea for various periods of time.
Fig. 4 shows the accumulation of fat after treatment of nematodes for 3d in tea water obtained after fermentation of control group, 0.5% green tea for 6d, 1% green tea for 4d, and 10% green tea for 2 d.
FIG. 5 shows the MDA content of the tea water after treatment of nematodes for 3 days in the control group, 0.5% green tea fermented for 6 days, 1% green tea, and 1% green tea fermented for 4 days.
FIG. 6 shows the SOD activity of nematodes treated with tea water of control group, 0.5% green tea fermented for 6 days, 1% green tea, and 1% green tea fermented for 4 days.
Fig. 7 shows GSH content of tea water treated with nematodes 3d after tea water treatment of control, 0.5% green tea fermented for 6d, 1% green tea, and 1% green tea fermented for 4 d.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Caenorhabditis elegans is wild type N2.
The caenorhabditis elegans has simple structure, short service life, moderate test period, easy propagation and large passage number, is convenient to observe, and is the most classical model organism for researching aging. The genetic background is clear, and nematode genes and human genes have large conserved sequences. Meanwhile, with the prolonging of the life span of the nematode, the phenomena of aging similar to human beings, such as slow action, various tissue damages, increase of oxidized protein and the like, occur. Therefore, caenorhabditis elegans was chosen as an animal model for studying the anti-aging effect of fermented tea in the following examples.
Aspergillus niger (Aspergillus niger) RAF106 strain, the preservation number is CGMCC NO.9608, and the preservation unit is China general microbiological culture Collection center; the address of the preservation unit is No. 3 Xilu No. 1 of Beijing province of China, Chaoyang district.
Green tea (Maojian) was purchased from Meitan, Guizhou, Lanxin tea.
Example 1 Effect of fermented tea on nematode longevity
Preparation of fermented tea
Accurately weighing 5g of tippy tea, adding 500mL of 85 ℃ drinking water, soaking for 12min under the condition of 85 ℃ water bath, stirring once every 3min, filtering by using a filter screen, recovering the filtrate into a sterilized blue-mouth bottle, immediately screwing a cover, adding 500mL of 85 ℃ drinking water into the rest tea residue, repeating the steps, uniformly mixing the two filtrates, taking the high temperature into consideration to destroy structures such as tea polyphenol and the like, performing membrane filtration sterilization by using a 0.22 mu m filter head to obtain 0.5% green tea water, and storing at-20 ℃ for later use after subpackaging. According to the experimental method, the same mass of tea leaves is weighed to obtain the green tea water with the concentration of 1% and 10% respectively.
Placing 150mL of the above tea water with each concentration in 250mL conical flask, pasteurizing in 60 deg.C constant temperature water bath for 30min, cooling, and adding 1.5mL Aspergillus niger RAF106 bacterial suspension (1 × 10) into 150mL of tea water7spores/mL). Placing the prepared tea water containing the bacterial suspension in a constant temperature shaking table with the temperature of 30 ℃ and the rotating speed of 200r/min for culturing for 8d, sampling at fixed points every 2d to obtain tea with different degrees of fermentation, and taking out and storing at-20 ℃ for later use.
Second, measurement of lifetime
1. Experimental methods
Equal amounts of e.coli OP50 with an OD600 value of 0.5 and different concentrations (0.5%, 1%, 10%) of fermented tea were added to NGM agar plates that were coated with e.coil OP50 as controls. Caenorhabditis elegans growing to the L4 stage after the synchronization treatment are selected to be on the treated NGM solid culture dishes, each culture dish is respectively provided with 20-25 nematodes, each treatment group is provided with 3 plates, and the caenorhabditis elegans are cultured in a constant temperature incubator at the temperature of 20 ℃. Recording the death number of the nematodes at the same time every day, counting the survival rate, when the number of eggs laid by the nematodes in the previous period is large, replacing the plate every day and transferring the plate to a new NGM flat plate added with the same sample, and replacing the new plate every 1-2 days until the nematodes die completely, and drawing a life curve.
2. Results of the experiment
The results are shown in FIGS. 1 to 3: too short or too long fermentation time is not beneficial to prolonging the service life of the nematodes, and the service life of the nematodes can be remarkably prolonged when 0.5% of green tea water is fermented for 6 days, 1% of green tea water is fermented for 4 days and 10% of green tea water is fermented for 2 days; compared with a control, the average service life is respectively prolonged by 17.85%, 12.86% and 11.55%, and the maximum service life is prolonged by 2 d; the semi-fermented tea is more beneficial to prolonging the service life of the nematodes, wherein the tea water concentration is 0.5-10%, the effect of prolonging the service life of the nematodes is remarkable when the fermentation time is 2 d-6 d, and the average service life is prolonged by 11.55-17.85%.
Example 2 Effect of fermented tea on nematode fat content
1. Experimental methods
Transferring nematodes growing to L4 stage in the same period to NGM culture medium containing the treatment group of the same example 1, culturing the nematodes at 20 ℃ for 3 days, washing and collecting the nematodes in a centrifuge tube by using M9 buffer solution and 0.01% Triton X-100 solution, centrifuging at 2000r/min for 1min, taking the supernatant, repeatedly washing for 3 times, adding 1mL of stationary liquid for fixing for 30min, removing the supernatant, transferring the nematode to-80 ℃, standing for 30min, taking the nematode out, immediately dissolving in a 43 ℃ water bath, putting the nematode into a-80 ℃ refrigerator, repeating the operations, repeatedly freezing and thawing, finally washing by using M9 buffer solution, adding 500 mu L of Nile red dye mixed solution into each tube, incubating for 2h, centrifuging at 2000r/min for 1min, and removing the supernatant. mu.L of nematodes were placed on the slide and covered with a cover slip, observed under a microscope and photographed.
2. Results of the experiment
Compared with unfermented tea, the fat content of the 3 rd nematodes of three groups of 0.5% fermented tea 6d, 1% fermented tea 4d and 10% fermented tea 2d is reduced, wherein the fat content of the 0.5% fermented tea 6d is the least, which shows that the fat accumulation in the organism is obviously reduced when the tea concentration range is 0.5% -10% and the fermentation time range is 2 d-6 d, thereby achieving the effect of delaying the organism aging.
Example 3 Effect of fermented tea on nematode antioxidant Capacity
1. Experimental methods
And (4) operating according to the instructions of a malondialdehyde kit, a superoxide dismutase kit and a total glutathione kit, and determining the oxidation resistance of the nematodes after the fermented tea is treated.
2. Results of the experiment
MDA is an intermediate product of lipid peroxidation, a common indicator of oxidative damage in cells; SOD can convert O2 -Decomposition into O2And H2O2Can protect cells from oxidative damage; GSH, together with glutathione transferase, is considered a cell detoxification enzyme system in which GSH content is somewhat of an important indicator of intracellular oxidative damage. SOD activity, GSH content are directly proportional to nematode longevity, while MDA content is inversely proportional to nematode longevity. The results of the monitoring of the 0.5 percent fermented tea water 6d, the 1 percent fermented tea water 4d and the 10 percent fermented tea water 2d in the invention on the MDA content, the SOD activity and the GSH content in the nematode body show that: compared with unfermented tea, the contents of MDA in the nematodes of 0.5%, 1% and 10% of the fermented tea water group are respectively and remarkably reduced by 24.37%, 14.55% and 12.49%, the SOD activities are respectively and remarkably increased by 47.21%, 31.25% and 28.66%, and the GSH contents are respectively and remarkably increased by 104.13%, 42.20% and 39.58%. The results show that the organism treated by the fermented tea with the fermentation time of 2 d-6 d in the tea water range of 0.5% -10% concentration has the best oxidation resistance and oxidation damage reduction capability.
Claims (10)
1. A preparation method of fermented green tea is characterized by comprising the following steps:
s1, leaching: soaking tea water containing 0.5-10 wt% of green tea at 80-85 ℃ for 10-14 min, taking filtrate, repeating the steps, and taking tea water mixed liquor as fermented tea water stock solution;
s2, fermentation: taking the tea water stock solution obtained in the step S1, pasteurizing, adding 1% v/v Aspergillus niger RAF106 bacterial suspension, and culturing at 28-32 ℃ for 2-8 d at 180-220 r/min; the Aspergillus niger RAF106 strainThe concentration of the suspension was 1X 107spore/mL; the Aspergillus niger RAF106 is preserved in the China general microbiological culture Collection center in 2014, 8 and 27 months, and the preservation number is CGMCC NO. 9608.
2. The method of claim 1, wherein the green tea is contained in the tea water of the green tea at step S1 in an amount of 0.5 wt%, and the cultivation time at step S2 is 6 days.
3. The method of claim 1, wherein the green tea is contained in the tea water of the green tea at step S1 in an amount of 1.0 wt%, and the cultivation time at step S2 is 4 d.
4. The method of claim 1, wherein the green tea is contained in the tea water of the green tea at step S1 in an amount of 10.0 wt%, and the cultivation time at step S2 is 2 d.
5. The method for preparing a nano-particle according to claim 1, wherein the soaking in step S1 is 85 ℃ soaking for 12 min.
6. The method according to claim 1, wherein the culturing in step S2 is carried out in a constant temperature shaker at 30 ℃ and a rotation speed of 200 r/min.
7. Fermented green tea produced by the method according to any one of claims 1 to 6.
8. Use of the fermented green tea of claim 7 for the preparation of an antioxidant product.
9. Use of the fermented green tea of claim 7 for the preparation of a product for reducing fat accumulation in the body.
10. Use of the fermented green tea of claim 7 for the preparation of a product for delaying aging in the body.
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