Application of lightly fermented tea in preparation of medicine for treating intestinal inflammation and / or mental diseases related to inflammation

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of lightly fermented tea in preparation of medicines for treating intestinal inflammation and / or mental diseases related to inflammation. In the invention, the extract of the lightly fermented tea can directly reduce the accumulation amount of LPS in the blood of rats, so that the inflammatory response and depression-like behavior caused by LPS are reduced, which indicates that the intake of the lightly fermented tea reduces inflammation by reducing the concentration of LPS, so that the organism does not generate strong immune regulation and intestinal flora regulation response.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of lightly fermented tea in the preparation of drugs for treating intestinal inflammation and / or inflammation-related mental diseases. Background Art

[0002] Catechins mainly prevent or delay the development of inflammation by regulating the intestinal flora and interfering with the inflammatory pathway, and have a relieving effect on diseases such as inflammation, obesity, cardiovascular diseases, neurodegenerative diseases, and diabetes. However, the low bioavailability of catechins has become an important factor restricting the exertion of their efficacy. Only a small part of tea catechins are absorbed by the human small intestine, and most tea catechins are carried into the large intestine, where they are glycosylated by bacterial enzymes in the colon and then dehydroxylated and demethylated by intestinal microorganisms into intermediate metabolites, which are further transformed into small molecular compounds and enter the enterohepatic circulation or systemic circulation to exert various physiological functions. The lightly fermented tea in the present invention is obtained by the transformation of green tea by microorganisms, so that the ester-type catechins that are difficult to pass through the biological membrane are transformed into oligomeric non-ester-type catechins that can be absorbed by intestinal cells and enter the systemic circulation.

[0003] The immune system, metabolic system, and endocrine system participate in the bidirectional communication between the intestine and the brain, and these communications are regulated by microorganisms through microbiota-derived neurochemicals and metabolites. Although the intestinal flora is far from the brain, its metabolites such as short-chain fatty acids (SCFAs), endotoxins, etc. play an important role in the development and maturation of the central nervous system. The possible ways of interaction between the brain and the intestine with the microbiota include neural (vagus nerve and intestine), endocrine (hypothalamic-pituitary-adrenal axis and enteroendocrine), metabolic (bacterial metabolites and host metabolism), and immune (innate immunity and adaptive immunity). Therefore, chronic inflammation may cause depressive-like emotions and behaviors in organisms through the "gut-brain axis".

[0004] Tea polyphenols, as potential intervention means for improving the intestinal microecology and preventing inflammation, have attracted extensive attention. The catechins in green tea are mainly ester-type catechins. Although such catechins are known to have good anti-inflammatory effects, their bioavailability is low. In addition, most of the current studies on the anti-inflammatory effects of tea polyphenols or catechins focus on their reduction of inflammatory responses by regulating the intestinal flora. Lipopolysaccharide (LPS) is a key factor directly causing inflammatory responses, and there are few reports on the scavenging effect of non-ester-type catechins on LPS in the body, and there is also little research on the alleviating effect of non-ester-type catechins on depressive-like behaviors caused by LPS. The lightly fermented tea in the present invention is obtained by fermenting green tea with Aspergillus niger, decomposing part of the ester-type catechins into non-ester-type catechins and gallic acid with higher bioavailability, and exploring its intervention effect on LPS-induced inflammation and the resulting depressive-like behaviors. Summary of the Invention

[0005] To overcome the disadvantages and deficiencies in the prior art, the primary object of the present invention is to provide an application of a lightly fermented tea in the preparation of a drug for treating intestinal inflammation and / or inflammation-related mental diseases; the extract of the lightly fermented tea can directly reduce the accumulation amount of LPS in the blood of rats, thereby down-regulating the inflammatory response and depressive-like behavior caused by LPS, indicating that the intake of the lightly fermented tea down-regulates inflammation by reducing the LPS concentration, so that the body does not produce strong immune regulation and intestinal flora regulation responses.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] An application of a lightly fermented tea in the preparation of a drug for treating intestinal inflammation and / or inflammation-related mental diseases, wherein the lightly fermented tea is obtained by solid-state aerobic light fermentation using an Aspergillus niger strain.

[0008] The lightly fermented tea is specifically prepared by the following method:

[0009] (1) Add an Aspergillus niger spore suspension to the distilled water used for moistening the tea leaves, with the moisture content of the tea leaves being 30% - 45%, and then evenly spray the distilled water added with the Aspergillus niger spore suspension on the sun-dried green tea leaves of Yunnan big-leaf variety. On average, 1 mL of the Aspergillus niger spore suspension is used for every 100 g of the sun-dried green tea leaves of Yunnan big-leaf variety. After spraying, mix evenly and put it into a fermentation bag;

[0010] (2) Place the fermentation bag containing the sun-dried green tea leaves of Yunnan big-leaf variety in step (1) in a fermentation box with the temperature set at 40°C - 45°C, and ferment for 2 - 6 days under moisturizing conditions; take out the fermented sun-dried green tea leaves and dry them at 80°C until the water content is less than 5% to obtain the lightly fermented tea. The contents of gallic acid and non-esterified catechins in the tea leaves increase, the content of esterified catechins decreases, and at the same time, the content of free amino acids increases.

[0011] The Aspergillus niger strain is Aspergillus niger RAF106;

[0012] The concentration of the Aspergillus niger spore suspension is 1 - 2×10 8 CFU / mL.

[0013] The intestinal inflammation is lipopolysaccharide (LPS)-induced intestinal inflammation.

[0014] The inflammation-related mental disease is depression, specifically depressive-like behavior caused by LPS.

[0015] Use of a lightly fermented tea water extract in the preparation of a drug for treating intestinal inflammation and / or inflammation-related mental disorders, wherein the lightly fermented tea water extract is prepared by the following method: Weigh 40 g of tea leaves, add boiling distilled water according to a material-liquid ratio of 1 g:20 mL, extract in a boiling water bath for 10 min, and filter with 8 layers of gauze; then add an equal volume of boiling distilled water to the filter residue, repeat the above extraction and filtration steps, and combine the filtrates; Concentrate the collected filtrates by vacuum rotary evaporation at 50-55 °C to 1 / 3 of the original volume, pre-cool the concentrated solution at -60 °C for 24 h, and then freeze-dry at -50 °C and 0.04 mbar for 72-84 h to obtain a freeze-dried powder of the lightly fermented tea water extract.

[0016] The present invention has the following advantages and beneficial effects compared with the prior art:

[0017] (1) In the invention, the intervention of lightly fermented tea significantly down-regulated the increase in the LPS accumulation amount in the blood caused by LPS, down-regulated the levels of pro-inflammatory factors in the blood, and up-regulated the levels of anti-inflammatory factors, indicating that the intake of lightly fermented tea down-regulates the inflammatory response by reducing the LPS concentration, so that the body does not produce a strong immune stress.

[0018] (2) Lightly fermented tea contains a high content of non-esterified catechins, and the long-term intervention of lightly fermented tea prevents rats from developing depressive-like behaviors and reduced exploratory desires caused by LPS. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the grouping of animal experiments.

[0020] Figure 2 It is a graph of the blood inflammation indexes of rats; Note: n = 6, the results are expressed as Mean ± SEM, #p < 0.0001 indicates a very extremely significant difference compared with the Control group; **p < 0.01 and ****p < 0.0001 indicate a very significant and very extremely significant difference compared with the Model group.

[0021] Figure 3 It is the result of the open field test, where (a) is the total movement distance of the rats in the open field; (b) is the proportion of the movement distance of the rats in the central area to the total distance; (c) is the movement trajectory of the rats in the open field; Note: n = 3, the results are expressed as Mean ± SEM, *p < 0.05 and **p < 0.01 indicate a significant difference compared with the Model group.

[0022] Figure 4 It is the light-dark box shuttle test; where (a) is the number of times the rats shuttle between the light box and the dark box; (b) is the time the rats stay in the light area; (c) is the movement trajectory of the rats in the light-dark box; Note: n = 3, the results are expressed as Mean ± SEM. Detailed implementation manners

[0023] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0024] Embodiment 1

[0025] Prepare lightly fermented tea according to the preparation method of the novel fermented sour tea disclosed in the Chinese patent with the patent number ZL202211709015.6. Specifically, the steps are as follows:

[0026] (1) Preparation of improved PDA medium: Weigh 10 g of sun-dried green tea and boil it in boiling water for 10 min. After cooling, make up the volume to 200 mL, add 4.5 g of potato extract powder, 0.3 g of sucrose, and 4 g of agar powder. After autoclaving at 121 °C for 15 min, 200 mL of improved PDA plate medium is prepared.

[0027] (2) Pure culture of Aspergillus niger: Take out the glycerol tube of Aspergillus niger RAF106 strain stored at -20 °C, and use a pipette to aspirate 100 μL of Aspergillus niger spore suspension and coat it on the improved PDA plate medium. Incubate at a constant temperature of 30 °C for 4 - 5 d for activation. Use it after the plate is covered with spores.

[0028] (3) Preparation of Aspergillus niger spore suspension: Take out the plate covered with spores and operate in a super clean bench. Use a pipette to aspirate 0.1% Tween 80 onto the plate, use a spreader to scrape the spores on the plate, and use a pipette to transfer the spore suspension to a 10 mL centrifuge tube and shake well to obtain the spore suspension. Use a hemocytometer to count, and adjust the spore concentration to obtain an Aspergillus niger spore suspension with a concentration of 1.18×10 8 spores / mL.

[0029] (4) Mix the spore suspension and sun-dried green tea in a certain proportion: Weigh 500 g of Yunnan big-leaf sun-dried green tea (from Menghai County, Yunnan Province). It takes 225 mL of distilled water to rehydrate the tea leaves to 45%. Therefore, mix 5 mL of the Aspergillus niger spore suspension obtained in the above step (3) with 220 mL of distilled water, add it to a spray bottle, shake well and spray it onto the Yunnan big-leaf sun-dried green tea; after spraying, cover the bag mouth with six layers of gauze and tie it with a rubber band.

[0030] (5) High-temperature fermentation: Put the fermentation bag containing Yunnan big-leaf sun-dried green tea into a fermentation incubator at 45 °C, and place a fresh-keeping box filled with water under the fermentation bag to maintain the humidity inside the fermentation incubator. Turn the leaves every day and ferment for 6 days.

[0031] (6) Drying and packaging: Take out the fermented tea leaves, spread them out on a tray, untangle the tea leaves entangled due to mycelial growth to make the tea leaves in a loose state, put them in an 80°C oven and dry for 1 - 2 hours, turning them over every half hour during this period. The water content of the obtained tea leaves is less than 5%, and lightly fermented tea is obtained.

[0032] Example 2:

[0033] Preparation of freeze-dried powder of water extract of lightly fermented tea: Weigh 40 g of the lightly fermented tea obtained in Example 1, add 800 mL of boiling distilled water, extract in a boiling water bath for 10 minutes, and filter with 8 layers of gauze; then add the same volume of boiling distilled water to the filter residue, extract again in a boiling water bath for 10 minutes, and filter with 8 layers of gauze. Combine the filtrates from the two filtrations; rotate and evaporate the collected filtrate under vacuum at 50°C to 1 / 3 of the original volume. The concentrated solution is precooled at -60°C for 24 hours, and then freeze-dried at -50°C and 0.04 mbar for 72 hours to obtain the freeze-dried powder of water extract of lightly fermented tea (LFT), and store it sealed at low temperature.

[0034] Put the sun-dried green tea of Yunnan big-leaf variety into an 80°C oven and dry for 1 - 2 hours, turning it over every half hour during this period. The water content of the obtained tea leaves is less than 5%, and unfermented tea is obtained. The obtained unfermented tea is processed according to the same steps as the preparation of the freeze-dried powder of water extract of lightly fermented tea above to obtain the freeze-dried powder of water extract of unfermented tea (GT).

[0035] Example 3

[0036] Respectively prepare the freeze-dried powder of water extract of lightly fermented tea and the freeze-dried powder of water extract of unfermented tea prepared in Example 2 into 5 mg / mL aqueous solutions, filter them with a 0.45 μm water-based filter membrane, and determine their catechin content by high performance liquid chromatography.

[0037] Liquid phase analysis conditions:

[0038] Table 1.1 Mobile phase ratio

[0039]

[0040] Table 1.2 HPLC elution program

[0041] Time (min) Mobile Phase A Mobile Phase B 0 100％ 0％ 5 100％ 0％ 25 65％ 35％ 25.1 0％ 100％ 30 0％ 100％ 30.1 100％ 0％ 35 100％ 0％

[0042] The chromatographic column is Waters XBridge C18 (250×4.6 mm, 5 μm), the column temperature is set at 35°C, the detection wavelength is 280 nm, and the injection volume is 10 μL.

[0043] Catechin content before and after fermentation: After fermentation by Aspergillus niger, most of the esterified catechins (EGCG, ECG) in tea leaves were converted into non-esterified catechins (EGC, EC) and gallic acid. The total amount of non-esterified catechins in the fermented tea (LFT) increased from 6.27% to 17.51%. Among them, EGC was about 3.8 times that before fermentation (GT), and EC was about 2.1 times that before fermentation. The total amount of esterified catechins decreased from 16.31% to 2.5%. Among them, EGCG decreased by 83.0%, and ECG decreased by 86.3%.

[0044] Table 2.1 Catechin component contents in LFT and GT

[0045] Component Name LFT Content (%) GT Content (%) GA 11.73 0.57 EGC 9.57 2.47 C 1.35 1.75 EC 7.94 3.80 EGCG 1.37 8.05 ECG 1.13 8.26

[0046] Example 4:

[0047] (1) Establish a rat inflammation model with LPS

[0048] After 7 days of adaptive feeding of 30 SD rats, they were divided into 5 groups: control group, model group, low-dose lightly fermented tea group (LLFT), high-dose lightly fermented tea group (HLFT), and unfermented tea group (LGT). LPS with a daily intraperitoneal injection dose of 300 μg / kg (bw) was used, and different tea extracts were used for gavage. The grouping experimental plan is as Figure 1 shown. The control group and the model group were treated with normal saline by gavage. The low-dose lightly fermented tea group (LLFT) was gavaged with the freeze-dried powder of the lightly fermented tea water extract obtained in Example 2 at a dose of 33.3 mg / kg (bw). The high-dose lightly fermented tea group (HLFT) was gavaged with the freeze-dried powder of the lightly fermented tea water extract obtained in Example 2 at a dose of 100 mg / kg (bw). The unfermented tea group (LGT) was gavaged with the freeze-dried powder of the unfermented tea water extract obtained in Example 2 at a dose of 33.3 mg / kg (bw).

[0049] After 49 days of the experiment, the rats were fasted but allowed to drink water for 12 h, anesthetized with pentobarbital, sacrificed by injecting an overdose of anesthetic after cardiac blood collection, and then dissected. The blood samples were left standing at 4 °C for 4 - 6 h, centrifuged at 3000 rpm for 10 min at 4 °C, the upper serum or plasma was carefully collected, and aliquoted into small tubes and stored in a -80 °C refrigerator for later use.

[0050] (2) Determination of rat blood inflammation indicators

[0051] The plasma LPS content was determined by the dynamic turbidity method using horseshoe crab reagent. The contents of inflammatory factors (TNF-α, IL-6, IL-1β, IL-10) and lipopolysaccharide-binding protein (LBP) in the serum were determined using an ELISA kit, and all operations were carried out according to the reagent instructions.

[0052] (3) Changes in rat inflammation levels

[0053] Continuous intragastric administration of LFT can reduce the accumulation of LPS in the plasma of rats. The LPS levels in the plasma of rats in each group are as shown in Figure 2 (a) of the figure. Injecting 300 μg / kg (bw) / d LPS into rats for 49 days increased the LPS content in the plasma of rats by 24 times. Intake of both LFT and GT can significantly reduce the accumulation of LPS. The LPS concentration in the plasma of rats in the low-dose LFT group (LLFT group) (0.298 ± 0.096 EU / mL) was significantly reduced by 75.1% compared with the Model group (1.197 ± 0.431 EU / mL), the HLFT group (0.192 ± 0.090 EU / mL) was significantly reduced by 84.0%, and the LGT group (0.341 ± 0.120 EU / mL) was significantly reduced by 71.5%. When the concentration of LPS entering the blood is reduced, LBP will decrease accordingly. Compared with the model group (40.407 ± 1.141 ng / mL), the LBP in the serum of rats in the LLFT group (28.159 ± 0.513 ng / mL) was reduced by 30.31% ( Figure 2 (b) of the figure).

[0054] Inflammatory factors are an important class of cytokines involved in immune regulation. Detection of inflammatory factors in serum can reflect the body's inflammatory level. The contents of TNF-α, IL-1β, IL-6, and IL-10 are as shown in Figure 2 (c-f) of the figure. In the serum of Model rats with an activated inflammatory response induced by LPS, the contents of TNF-α (84.827 ± 3.948 pg / mL), IL-1β (164.790 ± 11.259 pg / mL), and IL-6 (259.829 ± 4.377 pg / mL) were 138.06%, 99.39%, and 159.07% higher than those in the Control group, indicating a very significant increase in the inflammatory level in the rats. Compared with the Model group, the TNF-α (51.157 ± 2.926 pg / mL), IL-1β (115.599 ± 4.888 pg / mL), and IL-6 (185.905 ± 3.621 pg / mL) in the LLFT group of rats decreased by 39.69%, 29.85%, and 28.45% respectively. IL-10 is an important anti-inflammatory factor that plays a negative regulatory role in the expression of pro-inflammatory cytokines. The content of IL-10 in the Model group (10.538 ± 0.767 pg / mL) was significantly reduced by 80.07%, while the LLFT group (27.327 ± 0.788 pg / mL) was upregulated by 159.31%. The above results show that lightly fermented tea can reduce the accumulation of LPS in the blood of rats, thereby alleviating the inflammatory stress response caused by LPS and ultimately downregulating the inflammatory level in the rats.

[0055] Example 5

[0056] After 7 days of adaptive feeding, 30 SD rats were divided into 5 groups: control group, model group, low-dose lightly fermented tea group (LLFT), high-dose lightly fermented tea group (HLFT) and unfermented tea group (LGT). They were intraperitoneally injected with 300 μg / kg (bw) LPS every day and gavaged with different tea extracts. The experimental scheme of grouping was as follows: Figure 1 As shown, the control group and the model group were treated with normal saline by gavage, the low-dose lightly fermented tea group (LLFT) was treated with 33.3 mg / kg (bw) of the freeze-dried powder of the lightly fermented tea water extract obtained in Example 2 by gavage, the high-dose lightly fermented tea group (HLFT) was treated with 100 mg / kg (bw) of the freeze-dried powder of the lightly fermented tea water extract obtained in Example 2 by gavage, and the unfermented tea group (LGT) was treated with 33.3 mg / kg (bw) of the freeze-dried powder of the unfermented tea water extract obtained in Example 2 by gavage. After 49 days of the experiment, the rats were subjected to behavioral analysis.

[0057] 1. Experimental methods for behavioral analysis of rats

[0058] (1) Open-Field Test

[0059] The open field apparatus (50cm*50cm*45cm) was covered with black cardboard at the bottom, and the test was conducted in a quiet environment. The activity area was divided into 16 small squares of equal area. The four squares in the middle were called the central area, and the rest were called the edge area. At the beginning of the test, the rats were placed in the center of the open field. After 5 minutes of free activity, the rats were returned to their respective cages. The video tracking software Super maze was used to record the total activity distance and time of the rats, as well as the activity in each area. When a rat completed the test, the floor of the open square was wiped with 75% alcohol and evaporated naturally before the next rat was tested to avoid the bias effect of the odor clues left by the previous rat.

[0060] (2) Light-dark transition test

[0061] The light box and the dark box each occupy one-half of the light-dark box (50cm*25cm*25cm), and there is a 7.5cm*7.5cm doorway in the partition wall between the two boxes for the animals to pass through. At the beginning of the experiment, the rat was placed in the center of the light box, and the video tracking software Super maze was used to record the number of box crossings and the residence time in the light box and the dark box within 10 minutes. When a rat completed the test, the floor of the open square was wiped with 75% alcohol and evaporated naturally before the next rat was tested.

[0062] 2. Mitigation effect of LFT on LPS-induced depressive-like behavior in rats

[0063] Communication between the immune system and the brain plays an important role in enabling host organisms to engage in temporary and specific defensive behaviors, such as reducing locomotor and exploratory activities. A growing body of evidence supports the association between various mental disorders, such as depression, anxiety, schizophrenia, etc., and central or peripheral inflammation. Moreover, depression promotes inflammation, and the increased pro-inflammatory factors caused by inflammation stimulate microglial cells in the brain, exacerbating depressive symptoms. Studies have shown that LPS can induce depressive-like behavior in mice. In this experiment, the open field test and the light-dark box shuttle test were used to explore the effects of LPS on the locomotor ability and exploratory desire of rats, as well as the antidepressant effect of catechin dietary intervention.

[0064] (1) Open field test

[0065] In this test, the open field was divided into a central area and a peripheral area. According to the habits of rats, rats with an exploratory desire can shuttle and stay in the central area, while rats with depressive-like behavior tend to move along the periphery. Therefore, the longer the rats stay in the central area, the less severe their depressive-like state. The test results are as Figure 3 shown. There was no significant difference in the total locomotor distance of rats in each test group, indicating that LPS did not limit the locomotor ability of rats. However, the proportion of the locomotor distance in the central area of the LPS intervention group decreased, and the intake of LFT significantly restored this inhibition (P<0.05), and the high-dose group had a better antidepressant effect (p<0.01).

[0066] (2) Light-dark box shuttle test

[0067] The light-dark box shuttle test was used to evaluate the anxiety status of rats. In the light-dark box, rats prefer to move in the dark box, but their exploratory nature prompts them to try to explore the light box. However, the bright light in the light box inhibits the exploratory activities of animals in the light box. The test results are as Figure 4 shown. The number of times and the duration of stay of rats in the LPS-induced model group in the light box decreased, indicating a decline in their exploratory ability in the light box. Rats intervened with LFT or GT recovered their exploratory ability to varying degrees, especially the HLFT group had the best recovery effect.

[0068] In summary, LPS inhibited the exploratory desire of rats, resulting in depressive and anxiety-like emotions. The intervention with LFT, a high non-esterified catechin, can significantly alleviate this depressive-like behavior.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a lightly fermented tea in the preparation of a medicament for treating intestinal inflammation and / or inflammation-related mental disorders, characterized in that: The light-fermented tea is obtained by solid-state aerobic light fermentation using Aspergillus niger strains.

2. The application according to claim 1, characterized in that: The light-fermented tea is specifically prepared according to the following method: (1) Add the Aspergillus niger spore suspension to the distilled water used for moistening the tea leaves, with the moisture content of the tea leaves being 30% - 45%. Then evenly spray the distilled water containing the Aspergillus niger spore suspension on the sun-dried green tea of Yunnan big-leaf variety. On average, 1 mL of the Aspergillus niger spore suspension is used for every 100 g of the sun-dried green tea of Yunnan big-leaf variety. After spraying, mix evenly and put it into a fermentation bag. (2) Place the fermentation bag containing the sun-dried green tea of Yunnan big-leaf variety in step (1) into a fermentation chamber with the temperature set at 40°C - 45°C and ferment for 2 - 6 days under moisturizing conditions. Take out the fermented sun-dried green tea and dry it at 80°C until the water content is lower than 5% to obtain the light-fermented tea. The contents of gallic acid and non-ester catechins in the tea leaves increase, the content of ester catechins decreases, and at the same time, the content of free amino acids increases.

3. The application according to claim 1, characterized in that: The Aspergillus niger strain is Aspergillus niger RAF106; the concentration of the Aspergillus niger spore suspension is 1-2×10 8 CFU / mL.

4. Use of a lightly fermented tea water extract in the preparation of a medicament for treating intestinal inflammation and / or inflammation-related mental diseases, characterized in that: The water extract of the light-fermented tea is prepared according to the following method: Weigh 40 g of tea leaves and add boiling distilled water according to the material-liquid ratio of 1 g:20 mL, extract in a boiling water bath for 10 min, and filter with 8 layers of gauze. Then add the same volume of boiling distilled water to the filter residue, repeat the above extraction and filtration steps, and combine the filtrates. Vacuum rotary evaporate and concentrate the collected filtrate at 50 - 55°C to 1 / 3 of the original volume. Pre-cool the concentrated solution at -60°C for 24 h, and then freeze-dry at -50°C and 0.04 mbar for 72 - 84 h to obtain the freeze-dried powder of the water extract of the light-fermented tea.

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