Oolong tea extract with multi-target sugar-controlling and lipid-lowering activity as well as preparation method and application of oolong tea extract

Abstract

The invention discloses an oolong tea extract with multi-target sugar-controlling and lipid-lowering activity as well as a preparation method and application of the oolong tea extract. The oolong tea extract is extracted and prepared by adopting an enzymolysis-physical field assisted combined method; the preparation method comprises the following steps: crushing, sieving, carrying out enzymolysis on cellulase and pectinase, extracting at medium temperature, deactivating enzyme, cooling, shearing at high speed, homogenizing at high pressure, centrifuging, taking supernatant, carrying out suction filtration, concentrating and freeze-drying. The preparation process is simple, compared with a traditional water extraction method, the sample yield and the extraction efficiency of tea polyphenol and tea polysaccharide can be improved at the same time, the total phenol content of the prepared extract is larger than or equal to 33%, the total sugar content is larger than or equal to 20%, and the yield is larger than or equal to 35%. The tea leaf extract has the capacity of inhibiting alpha-glucosidase, alpha-amylase, pancrelipase and cholesterol esterase and the capacity of cholate adsorption and cholesterol micelle dissolution inhibition at the same time, and can be applied to food and health care products.

Description

Technical Field

[0001] The invention belongs to the field of tea processing, and in particular relates to an oolong tea extract with multi-target glucose-controlling and lipid-lowering activities, and a preparation method and application thereof. Background Art

[0002] Obese people have a greatly increased risk of developing fatty liver, atherosclerosis, cardiovascular disease, and diabetes, and these diseases often cross-influence each other through pathways such as glucose and lipid metabolism. Some commonly used drugs used in the clinic to treat these diseases, such as acarbose, orlistat, and cholestyramine, have relatively single target types and have varying degrees of side effects. Therefore, more and more researchers are beginning to look for alternatives to these drugs from natural products, and epidemiological studies have shown that drinking tea is beneficial for weight loss or reducing the incidence of obesity, and reducing the risk of diabetes and its complications, which makes tea one of the research hotspots of functional foods.

[0003] Tea is processed from the buds or leaves of the tea tree belonging to the Theaceae family and the Camellia genus. The traditional classification method divides it into six categories according to different processing techniques (fermentation degree from low to high): unfermented green tea, lightly fermented white tea, slightly fermented yellow tea, semi-fermented oolong tea (also known as green tea), fully fermented black tea and post-fermented black tea. Tea can not only quench thirst, but also help meditation, soothe nerves or relax the body and mind, and has a sedative effect. A large number of experimental results have shown that tea contains many active substances that are beneficial to mental health, among which tea polyphenols and tea polysaccharides with multiple functions such as anti-cancer activity, antioxidant activity, anti-cardiovascular activity, antibacterial activity, anti-hyperglycemia activity, anti-obesity activity and improvement of intestinal health are the most representative. However, different teas have great differences in chemical composition due to different processing techniques, and the content of active substances is also different. Therefore, determining the main chemical components of tea and directional extraction and enrichment of its main active components is conducive to improving the sugar control and lipid-lowering activity of tea and promoting its application in the field of health care.

[0004] As the only semi-fermented tea among the six major teas, oolong tea has a stronger aroma than green tea and is fresher than black tea. In recent years, not only has the consumption of oolong tea increased, but more and more studies have reported that oolong tea has good blood sugar and lipid-lowering activities due to its tea pigments and tea polyphenols (Zhang, H. and R. Qi, et al. (2019). "The impact of oolong and black tea polyphenols on human health." Food Bioscience 29: 55-61). The traditional famous tea Phoenix Dancong belongs to the oolong tea system. It has a long history of planting and production. It is native to the Phoenix Mountain System in Chaozhou City, Guangdong Province. Its production process mainly includes picking, sun drying, air drying, touching, killing, rolling, baking, selecting and aromatizing. This process is regarded as the most sophisticated tea making process in the oolong tea production process. The quality of Dancong tea is greatly affected by the growing environment, and the growing period is long. It can be harvested four to five times a year. It is usually divided into spring tea, summer tea, autumn tea and winter tea according to the picking time. As the seasons change, the ecological environment in which tea trees grow also changes. The differences in environmental factors such as climate, temperature, humidity, and water content lead to differences in the quality components of tea. Among them, single-bush summer tea is affected by high temperatures, which leads to the synthesis and accumulation of tea polyphenols. This also causes its bitter taste to be frustrated in the promotion of traditional tea beverages. Tea farmers are even reluctant to pick it, resulting in waste. For this reason, single-bush summer tea, a type of oolong tea, has the unique advantage of exerting its blood sugar and lipid-lowering activity due to its accumulation of tea polyphenols. On the other hand, it has not been effectively utilized and is cheap, making it a highly potential food ingredient for blood sugar and lipid-lowering functions.

[0005] At present, only a small number of studies in China focus on the active effects of Phoenix Dancong tea. The research on Phoenix Dancong mainly focuses on its flavor substances. There are few reports on the research on active substances in Dancong tea, especially Dancong summer tea. In fact, the presence of polyphenols makes Dancong tea have excellent antioxidant activity, hypoglycemic activity and anti-obesity activity, indicating that Phoenix Dancong tea may be a potential source of anti-diabetic compounds, once again suggesting that Phoenix Dancong has great potential in the health care field of human health and disease prevention. In addition, the traditional way of drinking tea by directly brewing tea leaves has the defects of low dissolution rate of tea active substances and incomplete absorption and utilization. Therefore, it is necessary to consider the preparation of tea extracts with more concentrated active substances through processing and extraction of tea raw materials.

[0006] At present, the conventional extraction methods of tea extracts reported in domestic literature are mainly traditional water extraction methods. Traditional water extraction methods, such as the commonly used high-temperature water extraction method, have the advantages of being convenient and fast, but the yield of this method is low and the sample loss is large during the preparation process. The extraction and preparation of active substances in tea reported in the literature are mostly focused on the separation and preparation of active ingredients. For example, the separation and preparation methods of tea polyphenols include three types: solvent extraction, precipitation and resin methods. However, these methods have limitations: (1) The content and extraction rate of active ingredients in the extraction method are low; (2) A variety of organic solvents are used in large quantities, and some are even harmful solvents (such as chloroform) that are difficult to be accepted by the food and pharmaceutical industries; (3) Multiple heating and distillation steps are required, the operation steps are complicated and the cost is high. (4) The precipitation method and resin method are simpler and greener than the solvent extraction method, but their industrial application is not mature enough, and the active action targets of the separated and prepared tea extracts are relatively single. Therefore, bypassing separation and directly focusing on how to reduce the loss in the preparation process of tea extracts to obtain a tea extract with high yield and good activity is a very promising method for preparing tea extracts with broad market prospects and good economic benefits. Summary of the invention

[0007] The purpose of the present invention is to provide an oolong tea extract with multi-target glucose-controlling and lipid-lowering activities and a preparation method thereof. The tea extract can effectively exert the ability to inhibit α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, as well as the ability to inhibit bile salt adsorption and cholesterol micelle dissolution.

[0008] The technical innovation and characteristics of the present invention lie in that the yield, active substance content and extraction efficiency of oolong tea extracts under six methods, namely, high-temperature water extraction (H), enzymatic assisted extraction (E), physical field assisted extraction (P), high-temperature water extraction-enzymatic combined extraction (HE), high-temperature water extraction-physical field assisted combined extraction (HP) and enzymatic hydrolysis-physical field assisted combined extraction (EP) are compared at the same time, and the in vitro glucose-controlling and lipid-lowering activities of the samples prepared by the six methods in six pathways related to sugar digestion, fat digestion and fat absorption are evaluated, thereby broadening the application scope of oolong tea in functional foods, and providing a tea extract with glucose-controlling and lipid-lowering activities having good economic benefits, as well as a preparation method and application thereof.

[0009] The specific technical solutions of the present invention are as follows.

[0010] The present invention provides a method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities, comprising the following steps:

[0011] Preparation of single-bush tea powder, enzymatic hydrolysis with cellulase and pectinase, enzyme inactivation and cooling, high-speed shearing, high-pressure homogenization, centrifugal filtration, concentration and freeze-drying.

[0012] Furthermore, the specific conditions for extracting the oolong tea extract are as follows:

[0013] (1) taking oolong tea raw material, placing it in a grinder for grinding, and obtaining oolong tea powder for later use;

[0014] (2) adding water to tea powder in a certain material-liquid ratio, mixing in a round-bottom flask, and adding enzymes in a certain proportion for enzymolysis, and shaking on a shaker to obtain a suspension 1; the enzymes include cellulase and pectinase for enzymolysis;

[0015] (3) The flask containing suspension 1 was transferred to a constant temperature water bath, and after high temperature enzyme inactivation, suspension 2 was obtained after cooling;

[0016] (4) Before homogenizing suspension 2, the suspension 2 is treated with a shearing machine to obtain suspension 3;

[0017] (5) homogenizing the suspension 3 under high pressure;

[0018] (6) The homogenized suspension 3 is transferred into a centrifuge cup and then centrifuged in a centrifuge. After the centrifugation time is over, the supernatant after centrifugation is taken and placed in a Buchner funnel for suction filtration to obtain a filtrate;

[0019] (7) The filtrate is placed in a rotary evaporator for vacuum concentration, and then the solution is transferred to a freeze dryer for freeze drying. After drying, the powder is recovered to obtain oolong tea extract powder.

[0020] Furthermore, in step (1), the powder is sieved through a 60-mesh sieve after being crushed; and the oolong tea is a single-bush summer tea.

[0021] Furthermore, in step (2), the material-liquid ratio is 1:10-1:20 g / mL; the cellulase added to the mixture is 0.25%-0.75% of the total mass of the tea powder; the mass of the pectinase added to the mixture is 0.25%-0.75% of the total mass of the tea powder; the shaking table is oscillated at a rotation speed of 100-160 r / min; the enzymolysis temperature is 50-60°C; and the enzymolysis time is 2-6 h.

[0022] Furthermore, in step (3), the enzyme inactivation is carried out at a temperature of 90 to 100° C.; the enzyme inactivation time is 10 to 20 minutes; and the cooling time is 5 to 15 minutes.

[0023] Furthermore, in step (4), the shearing machine shears at 1000 to 5000 r / min for 5 to 10 min.

[0024] Furthermore, in step (5), the homogenization pressure is 10 MPa to 30 MPa; and the homogenization times are 1 to 3 times.

[0025] Furthermore, in step (6), the centrifugation is performed at a centrifugal force of 8000 to 10000 g for 20 to 30 min.

[0026] Furthermore, in step (7), the reduced pressure concentration is carried out at 50 to 60°C to concentrate to 10% to 20% of the original volume; and the freeze drying is carried out at -10 to -20°C for 20 to 30 hours.

[0027] Furthermore, the enzyme is added in an amount of 0.8-1.2% of the total mass of the tea powder, preferably 1%.

[0028] The present invention also discloses an oolong tea extract with glucose-controlling and lipid-lowering activity prepared by the above-mentioned preparation method, wherein the total phenol content of the oolong tea extract is ≥33%, the total sugar content is ≥20% and the yield is ≥35%, and the oolong tea extract has the ability to inhibit α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, as well as the ability to inhibit bile salt adsorption and cholesterol micelle dissolution.

[0029] The oolong tea extract provided by the invention has a high sample yield under specified process conditions, and the process conditions are simple, mild, green and environmentally friendly.

[0030] The invention also discloses the use of the oolong tea extract with glucose-controlling and lipid-lowering activity prepared by the preparation method in the preparation of food or health products.

[0031] Compared with the prior art, the present invention has the following advantages and effects:

[0032] (1) The oolong tea raw material used in the present invention can be summer tea from the single-bush tea system produced in Chaozhou City, Guangdong Province, which has not been effectively utilized due to the bitter taste caused by the accumulation of tea polyphenols. On the one hand, the present invention utilizes its low price to develop and utilize its extracts, and on the other hand, it focuses on its active functions in a different way, using cellulase and pectinase enzymolysis to destroy the cell wall structure, promote the dissolution of active ingredients such as tea polysaccharides and tea polyphenols, and perform enzymolysis and extraction under medium temperature conditions, which has little effect on its active ingredients, has low requirements on production equipment and facilities, does not involve the use of toxic and harmful organic solvents, is environmentally friendly, and complies with food processing standards. In addition, the high-pressure homogenization method is used to further destroy its cell structure, further promote the dissolution of its active ingredients, significantly improve the utilization rate of raw materials, and reduce production costs.

[0033] (2) The present invention adopts the synergistic extraction of oolong tea extract by using cellulase and pectinase enzymolysis combined with physical field assisted method. The extraction efficiency of active substances represented by tea polyphenols and tea polysaccharides in oolong tea is greatly improved through the synergy of parameters such as enzymolysis ratio, enzymolysis time, homogenization pressure, and homogenization times. The total phenol content of the obtained oolong tea extract is ≥33%, the total sugar content is ≥20%, and the yield is ≥35%, and the content of active substances is high.

[0034] (3) The oolong tea extracts obtained by the present invention have good glucose-controlling and lipid-lowering activities, and can improve glucose and lipid metabolism by inhibiting α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, adsorbing bile salts and inhibiting cholesterol micelle dissolution.

[0035] (4) The preparation method provided by the present invention is simple to operate, and the extraction efficiency of active substances is significantly improved. It can promote the dissolution of tea polyphenols and tea polysaccharides in oolong tea. The prepared oolong tea extract shows the ability to exert multi-target glucose-controlling and lipid-lowering activities, and lays a theoretical foundation for promoting the processing and utilization of oolong tea in a new way. The entire process is simple, time-consuming, and does not involve the use of toxic and harmful organic solvents. It is environmentally friendly and meets food processing standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The total phenol and total sugar contents and yields of the oolong tea extract prepared in Example 1 of the present invention and six oolong tea extracts prepared in Comparative Examples 1-5 are shown.

[0037] Figure 2 This is a graph showing the inhibition rate of α-glucosidase of the oolong tea extract prepared in Example 1 of the present invention and six oolong tea extracts prepared in Comparative Examples 1-5 at the same concentration.

[0038] Figure 3 This is a graph showing the inhibition rate of α-amylase of the oolong tea extract prepared in Example 1 of the present invention and six kinds of oolong tea extracts prepared in Comparative Examples 1-5 at the same concentration.

[0039] Figure 4 This is a graph showing the inhibition rate of pancreatic lipase of the oolong tea extract prepared in Example 1 of the present invention and six kinds of oolong tea extracts prepared in Comparative Examples 1-5 at the same concentration.

[0040] Figure 5 This is a graph showing the inhibition rate of cholesterol esterase of the oolong tea extract prepared in Example 1 of the present invention and six oolong tea extracts prepared in Comparative Examples 1-5 at the same concentration.

[0041] Figure 6This is a graph showing the bile salt adsorption rate and cholesterol micelle dissolution inhibition rate of the oolong tea extract prepared in Example 1 of the present invention and six oolong tea extracts prepared in Comparative Examples 1-5 at the same concentration.

[0042] Figure 7 The total phenol and total sugar contents and yields of three oolong tea extracts prepared in Examples 1, 2 and 3 of the present invention are shown. DETAILED DESCRIPTION

[0043] The specific implementation and technical effects of the present invention are described in detail below in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

[0044] Example 1

[0045] The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to Example 1 of the present invention comprises extracting and preparing the oolong tea extract by using a cellulase and pectinase enzymatic hydrolysis method combined with a physical field-assisted method, and the specific steps are as follows:

[0046] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0047] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was added with 0.25% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker (120 r / min) at a temperature of 55° C. for 4 h to obtain a suspension 1.

[0048] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0049] (4) High-speed shearing: Before homogenizing the suspension 2, shearing was performed at 5000 r / min for 10 min using a shearing machine to obtain an extract 2;

[0050] (5) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 20 MPa, repeat once, and obtain the extract 3;

[0051] (6) Centrifugal filtration: The homogenized suspension 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0052] (7) Concentration and freeze drying: The filtrate was concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate was transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder was recovered to obtain oolong tea extract powder OTEP1.

[0053] Example 2

[0054] The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to Example 2 of the present invention comprises extracting and preparing the oolong tea extract by using a cellulase and pectinase enzymatic hydrolysis method combined with a physical field-assisted method, and the specific steps are as follows:

[0055] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0056] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was added with 0.25% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker (120 r / min) at a temperature of 55° C. for 2 h to obtain a suspension 1.

[0057] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0058] (4) High-speed shearing: Before homogenizing the suspension 2, shearing was performed at 5000 r / min for 10 min using a shearing machine to obtain an extract 2;

[0059] (5) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 10 MPa, repeat once, and obtain the extract 3;

[0060] (6) Centrifugal filtration: The homogenized suspension 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0061] (7) Concentration and freeze drying: The filtrate was concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate was transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder was recovered to obtain oolong tea extract powder OTEP2.

[0062] Example 3

[0063] The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to Example 1 of the present invention comprises extracting and preparing the oolong tea extract by using a cellulase and pectinase enzymatic hydrolysis method combined with a physical field-assisted method, and the specific steps are as follows:

[0064] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0065] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was enzymatically hydrolyzed by cellulase and 0.25% of the total mass of the mixture was added for enzymatic hydrolysis. The mixture was hydrolyzed for 6 h on a shaker (120 r / min) at a temperature of 55° C. to obtain a suspension 1.

[0066] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0067] (4) High-speed shearing: Before homogenizing the suspension 2, shearing was performed at 5000 r / min for 10 min using a shearing machine to obtain an extract 2;

[0068] (5) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 30 MPa, repeat once, and obtain the extract 3;

[0069] (6) Centrifugal filtration: The homogenized suspension 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0070] (7) Concentration and freeze drying: The filtrate was concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate was transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder was recovered to obtain oolong tea extract powder OTEP3.

[0071] Comparative Example 1

[0072] A method for preparing an oolong tea extract (high temperature water extraction method, H), specifically comprising the following steps:

[0073] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0074] (2) High temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, extracted for 2 h on a shaker (120 r / min) at 100 °C, and cooled to room temperature to obtain extract 1;

[0075] (3) Centrifugal filtration: The extract 1 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0076] (4) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder (OTH).

[0077] Comparative Example 2

[0078] A method for preparing an oolong tea extract (enzymatic hydrolysis, E), specifically comprising the following steps:

[0079] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0080] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was added with 0.25% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker (120 r / min) at a temperature of 55° C. for 4 h to obtain a suspension 1.

[0081] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0082] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0083] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder (OTE).

[0084] Comparative Example 3

[0085] A method for preparing an oolong tea extract (physical field assisted method, P), specifically comprising the following steps:

[0086] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0087] (2) Medium temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, and extracted for 2 h on a shaker at 120 r / min and 55°C, then cooled to room temperature to obtain extract 1;

[0088] (3) High-speed shearing: Before homogenizing the extract 1, shearing was performed at 5000 r / min for 10 min to obtain the extract 2;

[0089] (4) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 20 MPa, repeat once, and obtain the extract 3;

[0090] (5) Centrifugal filtration: The extract 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0091] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55°C to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20°C for 24 hours. After drying, the powder is recovered to obtain oolong tea extract powder (OTP).

[0092] Comparative Example 4

[0093] A method for preparing an oolong tea extract (high temperature water extraction-enzymatic hydrolysis method, EH), specifically comprising the following steps:

[0094] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0095] (2) Enzymatic hydrolysis with cellulase and pectinase: water was added to tea powder at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was enzymatically hydrolyzed by cellulase and 0.25% of the total mass of the mixture was added for enzymatic hydrolysis. The mixture was hydrolyzed for 4 h on a shaker at 120 r / min and 55° C. to obtain a suspension 1.

[0096] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0097] (4) High temperature extraction: transfer suspension 2 to a shaker, extract for 2 h at 100 °C under shaking conditions (120 r / min), and cool to room temperature to obtain extract 1;

[0098] (5) Centrifugal filtration: The extract 1 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0099] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder (OTEH).

[0100] Comparative Example 5

[0101] A method for preparing an oolong tea extract (high temperature water extraction-physical field assisted combination method, HP), specifically comprising the following steps:

[0102] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0103] (2) High temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, and extracted for 2 h on a shaker at 120 r / min and 100 °C, then cooled to room temperature to obtain extract 1;

[0104] (3) High-speed shearing: Before homogenizing the extract 1, shearing was performed at 5000 r / min for 10 min to obtain the extract 2;

[0105] (4) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 20 MPa, repeat once, and obtain the extract 3;

[0106] (5) Centrifugal filtration: The extract 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0107] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OTHP.

[0108] Comparative Example 6

[0109] A method for preparing an oolong tea extract comprises the following steps:

[0110] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0111] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.6% of the total mass of the mixture was added with 0.4% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker at 120 r / min and at 55° C. for 4 h to obtain suspension 1.

[0112] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0113] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0114] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT1.

[0115] Comparative Example 7

[0116] A method for preparing an oolong tea extract comprises the following steps:

[0117] (1) Preparation of single-bush tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0118] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.5% of the total mass of the mixture was added with cellulase and 0.5% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker at 120 r / min and 55° C. for 4 h to obtain a suspension 1.

[0119] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0120] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0121] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT2.

[0122] Comparative Example 8

[0123] A method for preparing an oolong tea extract comprises the following steps:

[0124] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0125] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.4% of the total mass of the mixture was added with 0.6% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker at 120 r / min and at 55° C. for 4 h to obtain suspension 1.

[0126] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0127] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0128] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT3.

[0129] Comparative Example 9

[0130] A method for preparing an oolong tea extract comprises the following steps:

[0131] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0132] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.25% of the total mass of the mixture was added with 0.75% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker at 120 r / min and at 55° C. for 4 h to obtain suspension 1.

[0133] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0134] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0135] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT4.

[0136] Comparative Example 10

[0137] A method for preparing an oolong tea extract comprises the following steps:

[0138] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0139] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was added with 0.25% of the total mass of the mixture for enzymatic hydrolysis. The mixture was hydrolyzed on a shaker at 120 r / min and at 55° C. for 2 h to obtain suspension 1.

[0140] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0141] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0142] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT5.

[0143] Comparative Example 11

[0144] A method for preparing an oolong tea extract comprises the following steps:

[0145] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0146] (2) Enzymatic hydrolysis with cellulase and pectinase: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. At the same time, 0.75% of the total mass of the mixture was enzymatically hydrolyzed with cellulase and 0.25% of the total mass of the mixture. The mixture was hydrolyzed on a shaker at 120 r / min and 55° C. for 6 h to obtain a suspension 1.

[0147] (3) Inactivation of enzyme and cooling: The flask containing suspension 1 was transferred to a constant temperature water bath, and the enzyme was inactivated for 15 min at 95°C, and then cooled for 5 min to obtain suspension 2;

[0148] (4) Centrifugal filtration: Transfer the suspension 2 into a centrifuge cup, place it in a centrifuge, and centrifuge it for 25 min at a centrifugal force of 12,000 g. Then, take the supernatant after centrifugation and place it in a Buchner funnel for filtration to obtain a filtrate.

[0149] (5) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT6.

[0150] Comparative Example 12

[0151] A method for preparing an oolong tea extract comprises the following steps:

[0152] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0153] (2) Medium temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, extracted for 2 h on a shaker (120 r / min) at 55 °C, and cooled to room temperature to obtain extract 1;

[0154] (3) High-speed shearing: Before homogenizing the extract 1, shearing was performed at 5000 r / min for 10 min to obtain the extract 2;

[0155] (4) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 10 MPa, repeat once, and obtain the extract 3;

[0156] (5) Centrifugal filtration: The extract 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0157] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT8.

[0158] Comparative Example 13

[0159] A method for preparing an oolong tea extract comprises the following steps:

[0160] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0161] (2) Medium temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, extracted for 2 h on a shaker (120 r / min) at 55 °C, and cooled to room temperature to obtain extract 1;

[0162] (3) High-speed shearing: Before homogenizing the extract 1, shearing was performed at 5000 r / min for 10 min to obtain the extract 2;

[0163] (4) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 30 MPa, repeat once, and obtain the extract 3;

[0164] (5) Centrifugal filtration: The extract 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0165] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT10.

[0166] Comparative Example 14

[0167] A method for preparing an oolong tea extract comprises the following steps:

[0168] (1) Preparation of oolong tea powder: Take single-bush summer tea raw materials, put them into a grinder for grinding, and pass them through a 60-mesh sieve to obtain oolong tea powder for later use;

[0169] (2) Medium temperature extraction: tea powder was added with water at a solid-liquid ratio of 1:10 g / mL, mixed in a round-bottom flask, extracted for 2 h on a shaker (120 r / min) at 55 °C, and cooled to room temperature to obtain extract 1;

[0170] (3) High-speed shearing: Before homogenizing the extract 1, shearing was performed at 5000 r / min for 10 min to obtain the extract 2;

[0171] (4) High-pressure homogenization: homogenize the extract 2 at a homogenization pressure of 20 MPa, repeat twice, and obtain the extract 3;

[0172] (5) Centrifugal filtration: The extract 3 was transferred into a centrifuge cup, placed in a centrifuge, and centrifuged for 25 min at 12,000 g. The supernatant after centrifugation was then placed in a Buchner funnel for filtration to obtain a filtrate.

[0173] (6) Concentration and freeze drying: The filtrate is concentrated under reduced pressure at 55° C. to 15% of the original volume, and then the concentrate is transferred to a freeze dryer and freeze-dried at -20° C. for 24 h. After drying, the powder is recovered to obtain oolong tea extract powder OT11.

[0174] The oolong tea extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 14 were measured using the following protocol.

[0175] 1. Calculation of yield

[0176] The yield of oolong tea extract was calculated according to formula (1).

[0177]

[0178] 2. Determination of the content of main active chemical components

[0179] (1) Determination of total phenol content

[0180] The total phenol content of the sample was determined by the Folin phenol method (refer to GB / T 8313-2018). Weigh 5 mg of gallic acid standard, dilute to 50 mL with deionized water, and prepare a 0.1 mg / mL gallic acid standard solution. Accurately measure 0.00, 0.05, 0.10, 0.20, 0.30, and 0.40 mL of gallic acid solution in a 5 mL centrifuge tube, add distilled water to 3 mL and shake well, then add 0.25 mL of Folin phenol reagent, mix well, add 0.75 mL of 20% sodium carbonate solution, then add 1 mL of deionized water, shake well, and react in a 40 ° C water bath for 2 hours. After the reaction is completed, cool quickly, measure its absorbance at 760 nm, and obtain a standard curve. Accurately weigh 10 mg of sample, add 1 mL of deionized water to prepare 10 mg / mL extract mother solution, dilute the single-bush summer tea extract mother solution 600 times, determine the sample absorbance value according to the above steps, and then convert the sample gallic acid equivalent according to the standard curve.

[0181] (2) Determination of total sugar content

[0182] The total sugar content in the sample was determined by the phenol-sulfuric acid method (reference GB / T 15672-2009). Weigh 10 mg of glucose standard, dilute to 100 mL with deionized water, and prepare a 0.1 mg / mL glucose standard solution. Accurately measure 0.1, 0.2, 0.3, 0.4, and 0.5 mL of glucose standard solution in a test tube, and add deionized water to 0.5 mL. Add 0.5 mL of phenol solution (5%, w / v), mix well, and immediately add 2.5 mL of concentrated sulfuric acid. Mix well again and let stand at room temperature for 30 minutes. Measure the absorbance at 490 nm to obtain a standard curve. Accurately weigh 10 mg of the sample, add 1 mL of deionized water to prepare a 10 mg / mL extract mother solution. Dilute the extract solution 100 times and take 0.5 mL. Measure the absorbance value according to the above steps, and convert the glucose equivalent in the sample according to the standard curve.

[0183] 3. Determination of in vitro glucose and lipid digestion enzyme inhibitory activity

[0184] Preparation of sample stock solution: Take the sample and prepare it into 20 mg / mL stock solution with deionized water for later use.

[0185] (1) α-glucosidase inhibitory activity assay: 10 U / mL α-glucosidase solution and 10 mmol / L p-NPG solution were prepared with 0.1 mol / L pH 6.8 phosphate buffer; and the sample solution was diluted to 10-50 μg / mL.

[0186] Take 30 μL of sample solution and α-glucosidase solution respectively and mix them evenly in a 96-well plate, incubate at 37°C for 10 min, add 30 μL of reaction substrate (p-NPG solution), react at 37°C for 15 min, and finally add 0.1 mol / L Na 2 CO 3 100 μL of the solution was added to terminate the reaction, and its absorbance at 405 nm was measured using an ELISA reader. Blank: phosphate buffer was used instead of the sample solution; sample control: phosphate buffer was used instead of the α-glucosidase solution; blank control: phosphate buffer was used instead of the sample and α-glucosidase solution. The absorbance values ​​of sample / blank / sample control / blank control were respectively recorded as A 1 / A 10 / A 11 / A 00 , the α-glucosidase inhibition rate was calculated according to formula (1).

[0187] α-Glucosidase inhibition rate (%) = [1-(A1-A11) / (A10-A00)] × 100% (1)

[0188] (2) Determination of α-amylase inhibitory activity: 3700 U / mL α-amylase solution and 10 mg / mL soluble starch solution were prepared with 0.1 mol / L pH 6.8 phosphate buffer; and the sample solution was diluted to 1-20 mg / mL.

[0189] Take 200 μL of sample solution and 200 μL of α-amylase solution, mix them thoroughly, incubate at 37°C for 15 minutes, add 200 μL of substrate soluble starch solution, mix thoroughly and incubate at 37°C for 5 minutes. Then, add 200 μL of DNS reagent solution, heat the resulting mixture with boiling water for 5 minutes, and cool it in ice water for 5 minutes to terminate the reaction. Finally, add 1.2 mL of PBS to the resulting mixture, mix well, and measure the absorbance at 540 nm using an enzyme reader. Blank: replace the sample solution with phosphate buffer; sample control: replace the α-amylase solution with phosphate buffer; blank control: replace the sample and α-amylase solution with phosphate buffer. The absorbance values ​​of sample / blank / sample control / blank control are respectively calculated as A 1 / A 10 / A 11 / A 00 , the α-glucosidase inhibition rate was calculated according to formula (2).

[0190] α-Amylase inhibition rate (%) = [1-(A1-A11) / (A10-A00)] × 100% (2)

[0191] (3) Determination of pancreatic lipase inhibition activity: Intestinal electrolytes consisted of 6.8 mmol / L KCl, 0.8 mmol / L KH 2 PO 4 、85mmol / LNaHCO 3 , 38.4mmol / LNaCl, 0.33mmol / LMgCl 2 , 8.4mmol / LHCl, 10mmol / L bile salt and 0.6mmol / L CaCl 2 Prepared by dissolving, adjusting pH to 7.0. Prepare 2mg / mL pancreatic lipase solution and 2mg / mL p-NPB solution with intestinal electrolytes; dilute the sample solution to 1-20mg / mL. The pancreatic lipase solution needs to be centrifuged at 4000g for 10 minutes before use and filtered through a 0.22μm filter membrane.

[0192] Take 50 μL of sample and pancreatic lipase solution respectively and mix them evenly in a 96-well plate, incubate at 37°C for 10 min, add 50 μL of reaction substrate (p-NPB solution), react at 37°C for 25 min, and measure the absorbance at 405 nm with an enzyme marker. Blank: replace the sample solution with intestinal electrolytes; sample control: replace the pancreatic lipase solution with intestinal electrolytes; blank control: replace the sample and pancreatic lipase solution with intestinal electrolytes. The absorbance values ​​of sample / blank / sample control / blank control are respectively calculated as A 1 / A 10 / A 11 / A 00 , the pancreatic lipase inhibition rate was calculated according to formula (3).

[0193] Pancreatic lipase production rate (%) = [1-(A1-A11) / (A10-A00)] × 100% (3)

[0194] (3) Cholesterol esterase inhibitory activity assay: The method is the same as pancreatic lipase assay, except that the cholesterol esterase concentration is 10 μg / mL. Blank: replace the sample solution with intestinal electrolytes; sample control: replace the cholesterol esterase solution with intestinal electrolytes; blank control: replace the sample and cholesterol esterase solution with intestinal electrolytes. The absorbance values ​​of sample / blank / sample control / blank control are calculated as A1 / A10 / A11 / A00, respectively, and the pancreatic lipase inhibition rate is calculated according to formula (4).

[0195] Cholesterol esterase efficiency (%) = [1-(A1-A11) / (A10-A00)] × 100% (4)

[0196] 4. In vitro simulated digestion-lipid absorption determination

[0197] The bile salt adsorption rate and cholesterol micelle dissolution inhibition rate were determined based on the in vitro static simulated gastrointestinal food digestion model INFOGEST. In short, the simulation of digestion includes oral, gastric and intestinal digestion stages. The preparation of simulated saliva (SSF), gastric juice (SGF) and intestinal juice (SIF) was referred to Brodkorb (2019), and the configuration of cholesterol micelles was prepared according to the previous study of the research group Chen (2023). The sample (final concentration of 10 mg / mL) was dissolved in 2 mL of SSF, 6.25 μL of CaCl2(H2O)2 (0.3 mol / L) was added, the pH was adjusted to 7, and then the total volume was made up to 2.5 mL with deionized water. In the gastric stage, 2 mL of SGF, 1.25 μL of CaCl2(H2O)2 (0.3 mol / L) and 0.4 mL of pepsin solution (16.7 mg / mL dissolved in SGF) were added, and the total volume was made up to 5 mL with deionized water, and then incubated in a 37°C water bath with shaking (120 r / min) for 2 h. In the intestinal stage, 4 mL of trypsin solution (2.5 mg / mL dissolved in cholesterol micelles) and 10 μL of CaCl2(H2O)2 were added. 2 (H 2 O) 2 (0.3 mol / L), and make up the total volume to 10 mL with deionized water, then adjust the pH to 7 and incubate at 37°C for 2 h. Finally, cool the mixture in ice water and centrifuge (10000g, 10 min, 4°C) to collect the supernatant. Take a part of the supernatant and dilute it 100 times to determine the total bile acid content using a bile acid determination kit. The other part of the supernatant was extracted three times with methanol (1:1, v / v) and the cholesterol content was determined by the OPA method. The blank group is a reaction system without adding a sample. The bile acid or cholesterol content of the blank group and the sample group is calculated as B10 and B1, respectively. The bile salt adsorption rate and the cholesterol micelle dissolution inhibition rate are calculated according to formula (1)

[0198] Bile salt adsorption rate / cholesterol micelle dissolution inhibition rate (%) = [1-B1 / B10] × 100% (1)

[0199] After measurement, the following analysis was made.

[0200] A. Analysis of components of oolong tea extract prepared by cellulase and pectinase hydrolysis

[0201] The total phenol content and sample yield of oolong tea extracts prepared by enzymatic hydrolysis with different ratios of cellulase and pectinase and different enzymatic hydrolysis time conditions are shown in Table 1 (including Comparative Examples 6-11 and Comparative Example 2).

[0202] Dancong summer tea belongs to the oolong tea family, and oolong tea has a higher polyphenol content among the six major teas. In addition, Dancong summer tea is affected by high temperature, which leads to the accumulation of more tea polyphenols. Therefore, the total phenol content can be used as an indicator to optimize the process of preparing Dancong summer tea extract. On the other hand, process production needs to consider costs, and reducing raw material losses is an important part, so the sample yield is also used as the main indicator for optimizing the process of preparing Dancong summer tea extract.

[0203] Cellulase and pectinase can promote the release of intracellular substances by destroying pectin and cellulose in the cell wall of plant cells, respectively. Therefore, the type of enzyme can significantly affect the final result of enzymolysis. Comparison of OT1, OT2, OT3, OT4 and OTE (Comparative Examples 6-9 and Comparative Example 2) shows that the total phenol content and yield of oolong tea extracts prepared by enzymolysis-assisted extraction with cellulase and pectinase in different proportions are significantly different, and the total phenol content of the obtained samples is greater than 30%. Among them, OTE has the highest total phenol content, and although the sample yield of OT4 is the highest, it is not significantly different from OTE. Therefore, considering the total phenol content and sample yield, the enzymolysis ratio for preparing OTE is optimal. For the enzymolysis method, in addition to the type of enzyme, the enzymolysis time can also significantly affect the final result of enzymolysis, so the enzymolysis time is further optimized to prepare OT6 and OT7. It is found that OT6 has the highest total phenol content but no significant difference with OTE, while OTE has a higher yield and is significantly different from OT6. In summary, the enzymatic hydrolysis parameters of cellulase and pectinase for preparing OTE are optimal, which significantly improves the sample yield while ensuring a high total phenolic content in the sample.

[0204] Table 1 Total phenol content and yield of oolong tea extracts prepared by cellulase and pectinase hydrolysis under different conditions

[0205]

[0206] Note: Different letters indicate significant differences between samples in the same index (p<0.05)

[0207] B. Component analysis of oolong tea extract prepared by physical field assisted method

[0208] The total phenol content and sample yield of the oolong tea extract prepared by the physical field assisted method with different homogenization pressures and homogenization times are shown in Table 2 (including Comparative Examples 12-14 and Comparative Example 3).

[0209] Under the action of high pressure, the material in the suspended liquid state flows at high speed through a cavity with a special internal structure (high-pressure homogenization cavity), which can fully destroy the material, promote the dissolution of substances in the material and achieve a homogenization effect. The homogenization pressure and the number of homogenization times significantly affect the homogenization effect. Furthermore, the homogenization pressure and the number of homogenization times in the process of preparing oolong tea extract by the physical field-assisted method are optimized and improved.

[0210] The traditional water extraction method for preparing tea extract has high losses and low sample yield. The extract after high-temperature water extraction is assisted by high-pressure homogenization after cooling, which is beneficial to improve the utilization rate of raw materials. Comparing OT8, OT9 and OTP (Comparative Examples 12-13 and Comparative Example 3), it is found that there are significant differences in the total phenol content and yield of oolong tea extracts prepared by high-pressure homogenization assisted extraction under different homogenization pressures, and the total phenol content of the obtained samples can reach more than 39%. Among them, OT9 has the highest total phenol content, but there is no significant difference with OTP, while the sample yield of OTP is significantly higher than that of OT9. This shows that too low homogenization pressure may lead to poor homogenization effect, while too high homogenization pressure may cause the equipment temperature to be too high, affecting the dissolution of substances in tea cells, and placing too much burden on the equipment. Therefore, considering the total phenol content and sample yield, the homogenization pressure for preparing OTP is the optimal parameter. Further optimization of the homogenization times to prepare OT10 revealed that the more homogenization times, the higher the sample yield and total phenol content. Comparison revealed that although there was no significant difference in the yields of OT10 and OTP, the total phenol content of OTP was higher, and the fewer times, the lower the equipment maintenance requirements. In summary, the physical field-assisted method for preparing OTP has the best parameters, which significantly improves the sample yield while ensuring a high total phenol content in the sample.

[0211] Table 2 Total phenol content and yield of oolong tea extracts prepared by physical field assisted method under different conditions

[0212]

[0213] Note: Different letters indicate significant differences between samples in the same index (p<0.05)

[0214] C. Analysis of components of oolong tea extract prepared by cellulase and pectinase enzymatic hydrolysis combined with physical field assisted method and other process methods

[0215] The active substance content and sample yield of oolong tea extracts prepared by different processes (including Example 1 and Comparative Examples 1-5) are as follows: Figure 1 In the figure, capital letters are used to indicate significant differences between samples at the same high concentration or significant differences between the bile salt adsorption rates of each sample, lowercase letters indicate significant differences between samples at the same low concentration or significant differences between the cholesterol micelle dissolution inhibition rates of each sample, and different letters indicate significant differences between samples (p<0.05).

[0216] By comparing with the traditional high temperature water extraction method (H), enzymatic assisted extraction method (E), physical field assisted extraction method (P), high temperature water extraction-enzymatic combined extraction method (EH), and high temperature water extraction-physical field assisted combined extraction method (HP) after parameter optimization, the significant advantages of enzymatic combined extraction method (EP) in improving sample yield and active substance extraction rate are clarified. The H method in comparative example 1 has been optimized and can reflect the actual dissolution of active substances under the traditional tea drinking method to the greatest extent. Although this method has the advantages of simplicity and convenience, the utilization rate of raw materials in the preparation process is not high, which indirectly reflects the defects of the traditional tea drinking method of directly brewing tea leaves. The oolong tea extract prepared by the EP method (Example 1) has a sample yield increased by 87% compared with the H method (Comparative Example 1). Although the total phenol content under the H method is the highest, it is only 16% higher than the total phenol content under the EP method, and the EP method not only has a higher yield, but also has the highest total sugar content. Therefore, considering the active substance content and sample yield comprehensively, the EP method has more advantages than the H method.

[0217] Both the E method (Comparative Example 2) and the P method (Comparative Example 3) are beneficial to destroying tea cells, thereby achieving the purpose of promoting the dissolution of water-soluble active substances such as tea polyphenols and tea polysaccharides in tea leaves, and removing water-insoluble impurities by centrifugation. Compared with the H method, both methods can improve the sample yield and active substance extraction rate to varying degrees. Further, it is considered to combine the E method and the P method and respectively combine them with the H method to obtain the EP method, the EH method (Comparative Example 4) and the HP method (Comparative Example 5). Figure 1 , compared with the H method, the sample yields of the EH method and the HP method are not significantly different, and some even decrease. In comparison, the EP method retains the mild medium-temperature extraction process, which is green and environmentally friendly, and significantly reduces the loss of active substances such as tea polyphenols and tea polysaccharides in oolong tea. Compared with the other five methods, the prepared oolong tea extract sample has the highest yield, which can reach 43%, and has a higher content of active substances, with the contents of total phenols and total sugars reaching 34.59% and 22.27%, respectively. In summary, the present invention innovatively applies the cellulose enzymatic hydrolysis-assisted extraction method and the physical field-assisted extraction method to the preparation of oolong tea extracts. Compared with the samples prepared by the traditional water extraction method, the sample yield is significantly increased, and the extraction efficiency of active substances (the product of the yield and the content) is also higher. In addition, from Figure 7 It can be seen that the extracts prepared by the EP method within the scope defined by the present invention can satisfy the requirements of total phenol content ≥ 33%, total sugar content ≥ 20% and yield greater than 35%.

[0218] D. Analysis of the glucose-controlling and lipid-lowering activity of oolong tea extract prepared by cellulase and pectinase enzymatic hydrolysis combined with physical field-assisted method and other process methods

[0219] The inhibition rates of oolong tea extract prepared by cellulase and pectinase enzymatic hydrolysis combined with physical field assisted method on α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase at different concentrations, as well as the inhibition rates of bile salt adsorption and cholesterol micelle dissolution were as follows Figure 2-6 (Different letters indicate significant differences among samples (p < 0.05)).

[0220] Glucose and lipid metabolism diseases are often complicated. Glucose and lipids, as important nutrients, interact with each other in the human body through different metabolic pathways. α-glucosidase and α-amylase are the most important enzymes in the digestion of carbohydrates. Inhibition of these two enzymes has become a widely used clinical strategy for the treatment of type II diabetes. Cholesterol esterase and pancreatic lipase jointly hydrolyze a variety of lipid substrates, including phospholipids, triglycerides, fat-soluble vitamins and dietary cholesterol esters, and are key enzymes in the lipid digestion and absorption pathway. Therefore, oolong tea extract has an inhibitory effect on the above four enzymes ( Figure 3-6 ) can reflect its glucose-controlling and lipid-lowering activity. The inhibitory activity of oolong tea extracts prepared by the six methods on α-glucosidase can reach μg level ( Figure 3 ), when the sample concentration was 80μg / mL and 100μg / mL, there was no significant difference in the inhibition rate of α-glucosidase between the samples prepared by the EP method and the H method, but they were the highest among the six methods, all reaching more than 80%. Although the inhibition rate of α-amylase, pancreatic lipase and cholesterol esterase of the H method was the highest among the six methods at the set sample concentration, it was only 3%-16% higher than the inhibition rate of the EP method on these three enzymes, which was not significant in terms of the improvement rate. In addition, considering the influence of the yield, comparing the total active efficacy of the extract samples prepared by the six methods with the same quality of oolong tea raw materials, it is not difficult to find that the total active efficacy of the extract prepared by the EP method is the highest (the product of the yield and the inhibition rate), which is more in line with the needs of actual industrial production. Therefore, compared with the other five methods, the oolong tea extract prepared by the EP method performed best in inhibiting the activity and total activity efficacy of glucosidase, and had the best total activity efficacy in inhibiting α-amylase, pancreatic lipase and cholesterol esterase. By inhibiting the above glycolipid digestive enzymes, the absorption and metabolism of glycolipids can be delayed, thereby achieving the purpose of controlling blood sugar and lowering blood lipids.

[0221] In addition to sugar and lipid digestion, the inhibition of these two lipid absorption pathways by bile salt adsorption and cholesterol micelle dissolution can also exert glucose control and lipid-lowering activity. The final system concentration of the oolong tea extract prepared by the six methods in the simulated digestion in vitro was 10 mg / mL. Figure 7As shown in the figure, all samples have the ability to adsorb bile salts and inhibit the dissolution of cholesterol micelles. The sample under the EP method has the highest bile salt adsorption rate, and is the only sample with a bile salt adsorption rate >15% among the six methods, but it is only significantly different from the extract samples under the P method and the H method. Except for the samples prepared by the E method, the inhibition rate of cholesterol micelle dissolution of the obtained oolong tea extracts is >40%, but there is no significant difference. This may be related to the fact that there are more types of active substances that exert lipid absorption and more diverse methods, such as binding to micelles to directly destroy their stability, or absorbing bile acids during digestion to inhibit the formation of micelles. Therefore, the samples prepared by the six methods can all exert glucose-controlling and lipid-lowering activities through the lipid absorption pathway, and the oolong tea extract prepared by the EP method has the strongest bile salt adsorption capacity, and the inhibition rate of cholesterol micelle dissolution is not much different.

[0222] In summary, by comparing the embodiments of the present invention with the comparative examples, it can be seen that: considering the sample yield and the content of active substances, the oolong tea extract prepared by the cellulase and pectinase enzymatic hydrolysis method combined with the physical field assisted method is the best, and has multi-target glucose-controlling and lipid-lowering activities involving sugar digestion, fat digestion and fat absorption, and can improve glucose and lipid metabolism by inhibiting the activities of α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, as well as bile salt adsorption and cholesterol micelle dissolution inhibition, and exert glucose-controlling and lipid-lowering activities. Breaking the boundary from the traditional beverage field to the functional food field, improving the situation that the single-bush summer tea has not been effectively utilized due to its high polyphenol content and bitter taste, and expanding the market demand for the single-bush summer tea in a new way to improve its economic benefits. This shows that the technical solution of the present invention can be used to obtain a type of single-bush summer tea extract with multi-target glucose-controlling and lipid-lowering activities through a better preparation process.

[0223] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities, characterized in that: The specific steps include: (1) taking oolong tea raw materials, placing them in a grinder for grinding, and obtaining oolong tea powder; (2) adding water to oolong according to the material-liquid ratio, and adding enzymes for enzymolysis, and shaking to obtain a suspension 1, wherein the enzymes include cellulase and pectinase for enzymolysis; (3) Transfer suspension 1 to a constant temperature water bath, inactivate the enzyme at high temperature, and then cool to obtain suspension 2; (4) Before homogenizing suspension 2, the suspension 2 is treated with a shearing machine to obtain suspension 3; (5) homogenizing the suspension 3 under high pressure; (6) Centrifuge the homogenized suspension 3 in a centrifuge. After the centrifugation time is over, take the supernatant after centrifugation and place it in a Buchner funnel for suction filtration to obtain a filtrate; (7) The filtrate is placed in a rotary evaporator for vacuum concentration, and then the solution is transferred to a freeze dryer for freeze drying. After drying, the powder is recovered to obtain oolong tea extract powder.

2. The tea extract prepared by the method for preparing an oolong tea extract with multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: In step (1), the oolong tea powder is obtained by crushing and passing through a 60-mesh sieve; the oolong tea is a single-bush summer tea.

3. The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: In step (2), the material-liquid ratio is 1:10-1:20 g / mL; the cellulase added to the mixture is 0.25%-0.75% of the total mass of the tea powder; the mass of the pectinase added to the mixture is 0.25%-0.75% of the total mass of the tea powder; the shaking is performed on a shaking table at a rotation speed of 100-160 r / min; the enzymolysis temperature is 50-60°C; and the enzymolysis time is 2-6 h.

4. The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: In step (3), the enzyme inactivation is carried out at a temperature of 90 to 100°C; the enzyme inactivation time is 10 to 20 minutes; and the cooling time is 5 to 15 minutes.

5. The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: In step (4), the shearing machine shears at 1000-5000 r / min for 5-10 min; in step (5), the homogenization pressure is 10 MPa-30 MPa; and the homogenization times are 1-3 times.

6. The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: In step (6), the centrifugation is carried out at a centrifugal force of 8000 to 10000 g for 20 to 30 min; in step (7), the reduced pressure concentration is carried out at 50 to 60° C. to concentrate to 10% to 20% of the original volume; and the freeze drying is carried out at -10 to -20° C. for 20 to 30 h.

7. The method for preparing an oolong tea extract having multi-target glucose-controlling and lipid-lowering activities according to claim 1, characterized in that: The enzyme addition amount accounts for 0.8-1.2% of the total mass of the tea powder.

8. An oolong tea extract with multi-target glucose-controlling and lipid-lowering activities, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8.

9. The oolong tea extract with multi-target glucose-controlling and lipid-lowering activities according to claim 8, characterized in that: The oolong tea extract has a total phenol content of ≥33%, a total sugar content of ≥20% and a yield of ≥35%. The multi-target glucose-controlling and lipid-lowering activities include the ability to inhibit α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, as well as the ability to inhibit bile salt adsorption and cholesterol micelle dissolution.

10. Use of the oolong tea extract with multi-target glucose-controlling and lipid-lowering activities according to claim 8 in the preparation of food or health products.

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