Method for increasing organic acid content and lipid-lowering activity of tea leaves

Abstract

The invention provides a method for increasing the organic acid content and lipid-lowering activity of tea leaves, and relates to the technical field of tea leaf processing and extracts. The method comprises the steps of enzyme deactivation, rolling, fermentation, extraction and the like, the content of part of organic acid in the extract can be remarkably increased through the method, and the content of triglyceride and the dgatmRNA level can be remarkably reduced. Meanwhile, the extract is derived from fresh tea leaves and has the characteristics of being green and healthy, and the risk of side effects can be reduced.

Description

Technical Field

[0001] The invention relates to the technical field of tea processing and extracts, and in particular to a method for increasing the organic acid content and lipid-lowering activity of tea. Background Art

[0002] Obesity is a complex metabolic disease caused by an imbalance between energy intake and expenditure, which leads to excessive fat accumulation in the body. It is affected by genetics, diet, exercise and complex biology. The body mass index (BMI) is between 25.0-29.9 kg / m 2 Overweight is diagnosed when BMI>30 kg / m 2 Reports show that more than half of adults in my country are overweight or obese.

[0003] Obesity not only affects a person's appearance, but also brings the risk of many other diseases and health problems, including heart disease, diabetes, high blood pressure, high cholesterol, liver disease, sleep apnea and certain cancers. At the same time, obesity can also have a significant impact on a person's quality of life, such as reduced sleep quality.

[0004] Bariatric surgery is the only effective way to reduce obesity. Drug treatments for obesity are generally divided into three categories: reducing food intake or suppressing appetite; changing metabolism or blocking fat absorption; and increasing calorie consumption. Currently, there are only two FDA-approved drugs for long-term treatment of obesity: the fat absorption blocker orlistat and the appetite suppressant sibutramine (MERIDIA). However, these drugs can cause serious side effects and have limited weight loss effects.

[0005] CN105873576A discloses a method for treating or preventing obesity in a subject in need thereof, wherein the subject is administered a therapeutically effective amount of a carbamate compound to achieve the effect of reducing weight and / or reducing food intake. However, this method still relies on drug action, and therefore still has the risk of side effects.

[0006] Therefore, there is an urgent need to develop new obesity treatment methods in order to achieve good obesity prevention, improvement or treatment effects while ensuring health. Summary of the invention

[0007] To solve the above problems, the present invention provides a method for increasing the organic acid content and lipid-lowering activity of tea leaves. The method significantly increases the content of organic acids and phenolic acids in tea extracts through processes such as withering, rolling, fermentation, and extraction of fresh tea leaves, thereby achieving a good effect of reducing triglyceride content and dgatmRNA levels.

[0008] The method for increasing the organic acid content and lipid-lowering activity of tea leaves of the present invention specifically comprises the following steps:

[0009] S1, picking fresh tea leaves, roasting and sterilizing them to obtain sterilized leaves, and then gently pressing and rolling the sterilized leaves to obtain rolled leaves;

[0010] S2, subjecting the rolled leaves to closed fermentation;

[0011] S3, drying the fermented tea leaves and then crushing them, passing them through a 40-mesh sieve, removing the ethanol after ethanol reflux extraction, and freeze-drying the extract to obtain a high-organic acid tea leaf extract.

[0012] Furthermore, the water content of the withered leaves is 64%-72%.

[0013] Furthermore, the kneading time is 5-10 minutes.

[0014] Furthermore, the sealed fermentation temperature is 20°C-27°C, and the sealed fermentation time is 6 months.

[0015] Furthermore, the drying temperature is 100°C-120°C, and the average moisture content of the tea leaves after drying is 5%-8%. Preferably, the drying temperature is 110°C, and the average moisture content of the tea leaves after drying is 6.5%.

[0016] Furthermore, the ethanol concentration is 70% and the extraction time is 2 hours.

[0017] The present invention also provides a high-organic acid tea extract prepared according to the method.

[0018] Another object of the present invention is to provide the use of the high-organic acid tea leaf extract in the preparation of lipid-lowering drugs or lipid-lowering functional foods.

[0019] Compared with the prior art, the beneficial technical effects of the present invention are:

[0020] The present invention can optimize the composition and distribution of organic acids in the extract and increase the content of certain organic acids. The method uses fresh tea leaves as raw materials and processes them through processes such as withering, rolling, fermentation and drying. No harmful substances are produced during the entire production process, reducing the risk of side effects. The tea extract prepared by the method of the present invention can significantly reduce the triglyceride content and dgat mRNA level of Caenorhabditis elegans fed a high-sugar diet, and has application value in preparing lipid-lowering drugs and lipid-lowering functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below in conjunction with the accompanying drawings.

[0022] Figure 1 It is the test result of organic acid components of the product;

[0023] Figure 2The results of Oil Red O staining of Caenorhabditis elegans in the normal group and diet group are as follows:

[0024] Figure 3 This is the result of Oil Red O staining of Caenorhabditis elegans in the high-sugar diet group;

[0025] Figure 4 The results of Oil Red O staining of Caenorhabditis elegans in the high sugar + extract of Example 1 diet group;

[0026] Figure 5 Results of relative lipid composition testing of Caenorhabditis elegans fed with different tea extracts;

[0027] Figure 6 is the quantitative analysis result of glyceride;

[0028] Figure 7 This is the result of quantitative analysis of phospholipids;

[0029] Figure 8 This is the detection result of relative expression of dgat mRNA. DETAILED DESCRIPTION

[0030] The technical solution provided by the present invention is further described below in conjunction with embodiments.

[0031] Example 1 A method for increasing the organic acid content and lipid-lowering activity of tea extract, comprising the following steps:

[0032] S1, picking fresh leaves of Yunkang No. 10 with one bud and two or three leaves, and roasting and sterilizing them at 250° C. to obtain sterilized leaves with a moisture content of 68±4%, and then gently pressing and rolling the sterilized leaves for 10 minutes to obtain rolled leaves;

[0033] S2, fermenting the rolled leaves in a sealed manner at room temperature (25° C.) for 6 months;

[0034] S3. Dry the fermented tea leaves at 100° C. until the average moisture content of the tea leaves is 6.5%, then grind them, pass them through a 40-mesh sieve, extract them with 70% ethanol under reflux for 2 hours, remove the ethanol, and freeze-dry the extract to obtain a high organic acid tea leaf extract.

[0035] Comparative Example 1 A method for processing a raw tea extract, comprising the following steps:

[0036] S1, picking fresh leaves of Yunkang No. 10 with one bud and two or three leaves, and then killing them to obtain killed leaves with a water content of 64%-72%, and then gently pressing and rolling the killed leaves for 10 minutes to obtain rolled leaves;

[0037] S2, drying the rolled leaves until the average moisture content of the tea leaves is 6.5%, thereby obtaining sun-dried green tea (raw tea);

[0038] S3, crushing the raw tea, passing through a 40-mesh sieve, extracting with 70% ethanol under reflux for 2 hours, removing the ethanol, and freeze-drying the extract to obtain a raw tea extract.

[0039] Comparative Example 2 A method for processing a cooked tea extract, the steps are as follows:

[0040] S1, picking fresh leaves of Yunkang No. 10 with one bud and two or three leaves, and then killing them to obtain killed leaves with a water content of 64%-72%, and then gently pressing and rolling the killed leaves for 10 minutes to obtain rolled leaves;

[0041] S2, drying the rolled leaves until the average moisture content of the tea leaves is 6.5% to obtain sun-dried green tea (raw tea), and fermenting them in piles to obtain cooked tea;

[0042] The method of pile fermentation is as follows: the raw tea is dampened to an average moisture content of 30%, placed in a wooden frame, covered with wet linen cloth for fermentation, and the pile is turned over once every 7 days for a total of 42 days;

[0043] S3, drying the cooked tea at 50° C. until the average water content of the tea leaves is 6.5%, then crushing the tea leaves, passing through a 40-mesh sieve, refluxing and extracting the tea leaves with 70% ethanol for 2 h, removing the ethanol, and freeze-drying the extract to obtain a cooked tea extract.

[0044] Test Example 1 Component Detection

[0045] The extracts of Example 1 and Comparative Examples 1-2 were tested for components as follows: 100 mg of sample was accurately weighed into a centrifuge tube, 0.5 mL of 80% methanol aqueous solution (containing 0.2% vitamin C) was added, vortexed and mixed, ultrasonically extracted for 30 min at room temperature, centrifuged at 12000 rpm for 10 min, the supernatant was taken, the extraction was repeated twice, the supernatants of the two times were combined and mixed, diluted several times according to the actual situation, and then tested on the machine, and the data was normalized. The results are shown in FIG. Figure 1 shown.

[0046] Depend on Figure 1 It can be seen that the content of organic acids in the tea extract obtained by the method of the present invention is significantly increased. Among them, the increase in the content of gallic acid, salicylic acid, shikimic acid, quinic acid, lactic acid and succinic acid, catechin and epicatechin is particularly significant. It can be seen that a specific fermentation process is helpful to increase the content of organic acids in tea extracts.

[0047] Test Example 2 Nematode Experiment

[0048] In normal medium (normal diet) and high-sugar medium (100 mM glucose, high-sugar diet), 100 mg / L of the extracts of Example 1, Comparative Example 1 and Comparative Example 2 were used to intervene and culture Caenorhabditis elegans for 72 h (i.e., from the larval stage to the adult stage), and then Oil Red O staining was performed ( Figure 2-4), image pro plus (IPP) was used to measure the integrated optical density of lipids and the total area (Area) of the worm after staining, and the average optical density was used to represent the fat content of the worm: MOD = IOD / Area, and the data were normalized. The results are shown in Figure 5 shown.

[0049] Nematode culture conditions: temperature 20℃±0.5℃, humidity 40%-60%, food is inactivated E.coli OP50.

[0050] Triglyceride Quantification Analysis:

[0051] Ice bath ultrasound + Folch extraction of Caenorhabditis elegans lipids: first use ice bath ultrasound to break the nematodes (0.5 mg), then add the extract (chloroform: methanol: water = 8:4:3), vortex for 2 minutes and ice bath ultrasound for 15 minutes (frequency: 40 kHz), centrifuge at 4°C 10000 rpm for 5 minutes, and leave the lower layer. Transfer the lower layer to a new centrifuge tube, blow dry with nitrogen, add 1 mL (isopropanol: acetonitrile: water = 2:1:1) solvent, vortex for 2 minutes, centrifuge at 4°C, 10000 rpm for 5 minutes, and pass the supernatant through a membrane into a sample bottle for use.

[0052] Liquid chromatography conditions: ACE 3C18 column (150mmx3.0 mm id, 3.0μm), mobile phase A: isopropanol: acetonitrile (90:10, v / v), B: acetonitrile: water (60:40, v / v), 5mmol / of ammonium formate was added to both mobile phases. Column temperature 50℃, flow rate 0.2mL / min, injection volume 1μL. Elution gradient 0-3.00min: 30%A, 3.01-5.00min: 30-50%A, 5.01-12.00min: 50%-82%A, 12.01-28.00min: 82%-99%A, 28.01-30.00min: 30%A. All samples were randomly injected, and one QC was inserted every nine injections.

[0053] Mass spectrometry conditions: The mass spectrometry data were acquired in full scan mode, with spray voltage: 4.0 kV (+), 3.5 kV (-); sheath gas flow rate: 45 (+), 40 (-); auxiliary gas flow rate: 15 (+), 10 (-); capillary transfer temperature: 350 ° C (+), 320 ° C (-); the mass-to-charge ratio (Mass-to-charge ratio, m / z) scanning range was set to 100-1200 m / z; high energy collision voltage: 15, 35 and 65 cV. When performing MS and MS / MS analysis, the AGC was lx106 and lx105, respectively, and the maximum injection time was 100 ms and 50 ms. The resolution was 35000 and 70000. The isolation window was set to 1 m / z. In addition, in order to obtain more MS / MS information. Improve the accuracy of qualitative analysis. The scanning range of QC samples was divided into five different scanning ranges, namely 100-300, 299-600, 599-760, 759--860 and 859-1200 m / z.

[0054] The data is normalized and the results are as follows Figure 6 , Figure 7 shown.

[0055] Determination of dgat mRNA:

[0056] 1 mL of Trizol solution was added to the centrifuge tube where the C. elegans sample was collected. Total DNA was extracted according to the instructions of the kit, and the integrity, purity and concentration of DNA were tested. Reverse transcription was performed according to the instructions of the reverse transcription kit. The total system of fluorescent quantitative PCR (RT-qPCR) reaction was 20 μL, including 10 μL of Takara SYBR Green supermix, 0.4 μL of ROX II, 0.8 μL of gene primers (0.4 μL each of upstream and downstream primers), 1 μL of cDNA (dilute the cDNA concentration to about 300 ng / uL before use) and 7.8 μL of sterile water; the reaction procedure was Hold (95°C for 10 min), Cycling 40 times (95°C, 30 s, 59°C, 30 s, 72°C, 20 s). After the reaction, the melting curve and amplification curve were analyzed, and the gene expression level was analyzed by the relative quantitative method 2-△△CT. The data were normalized and the results are shown in the figure. Figure 8 shown.

[0057] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for increasing the organic acid content and lipid-lowering activity of tea, characterized in that: The following steps are involved: S1, picking fresh tea leaves, roasting and sterilizing them to obtain sterilized leaves, and then gently pressing and rolling the sterilized leaves to obtain rolled leaves; S2, subjecting the rolled leaves to closed fermentation; S3, drying the fermented tea leaves and then crushing them, passing them through a 40-mesh sieve, removing the ethanol after ethanol reflux extraction, and freeze-drying the extract to obtain a high-organic acid tea leaf extract.

2. The method according to claim 1, characterized in that The water content of the blanched leaves is 64%-72%.

3. The method according to claim 1, characterized in that The kneading time is 5-10 minutes.

4. The method according to claim 1, characterized in that: The sealed fermentation temperature is 20° C.-27° C., and the sealed fermentation time is 6 months.

5. The method according to claim 1, characterized in that The drying temperature is 100° C.-120° C., and the average moisture content of the tea leaves after drying is 5%-8%.

6. The method according to claim 1, characterized in that The ethanol concentration is 70% and the extraction time is 2 hours.

7. A high-organic acid tea extract, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. The high-organic acid tea leaf extract according to claim 7 is used for preparing lipid-lowering drugs or lipid-lowering functional foods.

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