Composite tea extract as well as preparation method and application thereof

Abstract

The invention discloses a composite tea extract, which is prepared by adopting an enzymolysis-physical field assisted combined method to extract and prepare the composite tea extract through the processes of material preparation, enzymolysis, enzyme deactivation, shearing, homogenization, centrifugation, suction filtration, concentration and freeze drying. Meanwhile, the invention further discloses application of the composite tea extract in food and health care products. According to the composite tea extract and the preparation method and application thereof, the overall 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 28%, the total sugar content is larger than or equal to 22%, and the yield is larger than or equal to 30%. The composite tea 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 technical field of tea utilization, and in particular relates to a composite tea extract and a preparation method and application thereof. Background Art

[0002] Disorders in glucose and lipid metabolism can easily induce a variety of metabolic diseases, including obesity, type 2 diabetes, non-alcoholic fatty liver disease, etc., which are often related to abnormal glucose and lipid metabolism. In patients with type 2 diabetes, insulin resistance leads to impaired glucose metabolism, and also causes lipid metabolism disorders, such as hypercholesterolemia and hypertriglyceridemia, which further aggravate the condition. Therefore, in the prevention, regulation and improvement of metabolic diseases such as type 2 diabetes, glucose and lipid metabolism is a very important regulatory pathway, but some commonly used drugs used in the treatment of these diseases, such as acarbose, orlistat and cholestyramine, can only play a therapeutic role through one of glucose metabolism or lipid metabolism, with a relatively single pathway of action and side effects to varying degrees. At present, finding and developing substances with good activity from natural products to replace these drugs has become a research hotspot, and a large number of studies have shown that drinking tea can reduce weight or reduce the risk of diabetes and its complications by regulating glucose and lipid metabolism, which also makes tea one of the research hotspots of functional foods.

[0003] As one of the three major beverages in the world, tea accounts for an increasingly larger share of the beverage market in China and even the world, which is mainly related to two reasons. On the one hand, it is because tea is rich in variety, not only can it quench thirst, but also has a rich taste and flavor. On the other hand, it is because tea has been proven by a large number of experimental results to contain 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. The traditional classification method divides tea 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. Different teas have different chemical compositions and different contents of active substances due to different processing techniques. Therefore, determining the main chemical components of tea and conducting targeted extraction and enrichment of its active components that can regulate glucose and lipid metabolism will help promote its application in the field of glucose control and lipid-lowering health care.

[0004] The traditional famous tea Phoenix Dancong belongs to the oolong tea system. It has a long history of planting and production and is native to the Fenghuang Mountain System in Chaozhou City, Guangdong Province. The quality of Dancong tea is greatly affected by the growing environment and has a long growing period. It can be harvested four to five times a year and is usually divided into spring tea, summer tea, autumn tea and winter tea according to the picking time. With the change of seasons, the ecological environment for the growth of tea trees also changes. The different environmental factors such as climate, temperature, humidity, and moisture lead to differences in the quality components of tea. Among them, spring tea has the highest sensory quality. Summer tea has a bitter taste due to the high temperature and the accumulation of tea polyphenols. Eventually, the situation of Dancong spring tea in short supply and Dancong summer tea in oversupply has formed. This directly leads to the underutilization of Dancong summer tea, which accounts for 20% of the total output of Dancong tea, and even Dancong summer tea is almost ignored at a low price. In fact, Dancong tea just has a poor flavor, but tea polyphenols, as one of the representative active substances of tea, have great development and utilization value in the field of functional activity. This also shows that Dancong summer tea may be able to find another way to enter the field of functional food, and while ensuring its activity, improve its flavor to a certain extent, so as to give full play to its advantages of low price and high tea polyphenols content and avoid its disadvantages of poor flavor.

[0005] Anhua dark tea is the ancestor of Chinese dark tea. It is a general term for dry tea made from fresh leaves of tea trees grown in the local population of Anhua, Hunan, through processes such as withering, rolling, fermentation, and baking and drying with pinewood fire, or compressed tea made from dry tea as raw materials by steaming, stepping, and pressing. It has now become a national geographical indication product in China. Fuzhuan tea is the most representative type of tea in the Anhua dark tea series. It is a compressed dark tea with a long history and unique craftsmanship. Tea leaves are easily produced during the extrusion process. These tea leaves are only slightly inferior in appearance, and the chemical composition is not significantly different from the tea that has been successfully extruded into bricks, but they are often ignored and wasted. In addition, the characteristic golden yellow particles of Fuzhuan tea are mainly produced by the fermentation metabolism of Aspergillus niger, which not only gives Fuzhuan tea a unique "fungus flower fragrance" flavor, but also brings it a mellow, delicate and sweet pleasant taste. More importantly, the study of active substances in Anhua dark tea shows that it contains tea polysaccharides and special secondary metabolites, which can enhance lipid metabolism, promote insulin secretion and insulin sensitivity in vivo, and regulate intestinal microbial metabolism, which may play a certain role in improving glucose and lipid metabolism disorders. Obviously, Fuzhuan tea is not only sweet, but also has been reported to have many applications in many active functional fields such as anti-oxidation and hypoglycemic.

[0006] Compound tea is a compound tea with new flavor and function created by mixing different types of tea or tea with other plant ingredients (such as herbs, petals, fruits, etc.). This mixture can be tea from different origins, different types or different processing techniques. The current market environment is relatively mature and there is still a lot of room for development due to the rich variety of tea. 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.

[0007] At present, the improvement of the bitter taste of single-bush summer tea reported in domestic literature is based on reducing the content of tea polyphenols to reduce its bitterness, but this undoubtedly reduces its functional activity. In addition, the conventional extraction method of tea extract is the traditional water extraction method, such as the commonly used high-temperature water extraction method. Although it has the advantages of convenience and speed, the yield of this method is low and the sample loss is large. In addition, after the single-bush summer tea is extracted by water extraction, tea polyphenols are still the main component. Unless the extraction rate is reduced, the bitterness is still obvious. In addition, the extraction and preparation of active substances in tea in the existing literature are mostly focused on the separation and preparation of active ingredients. For example, the separation and preparation of tea polyphenols adopts three methods: solvent extraction, precipitation and resin method. However, these methods have limitations: (1) The extraction method has low content of active ingredients and extraction rate; (2) It uses a variety of organic solvents and the amount is large. Some are even harmful solvents (such as chloroform) and are difficult to be accepted by the food and pharmaceutical industries; (3) It requires multiple heating and distillation processes, and the operation steps are complicated and the cost is high. (4) Compared with solvent extraction, precipitation and resin methods are simpler and greener, but their industrial applications are not yet mature, and the active targets of the separated and prepared tea extracts are relatively single.

[0008] Therefore, bypassing separation and directly focusing on how to reduce the loss in the preparation process of tea extracts, how to prepare tea extracts with more concentrated active substances, and after clarifying that both Dancong summer tea and Anhua dark tea have the ability to regulate glucose and lipid metabolism, and confirming their respective characteristics in chemical composition and flavor, from the perspective of compound tea, using these two types of tea with sufficient sources and low prices to compound, to obtain compound tea extracts with both flavor and high yield and good activity is a very promising research direction with broad market prospects and good economic benefits. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a composite tea extract with the function of controlling sugar and reducing lipids, high yield and good activity, and a preparation method and application thereof.

[0010] The technical solution of the present invention to solve the above technical problems is:

[0011] A method for preparing a composite tea extract, using an enzymatic hydrolysis-physical field assisted method, comprises the following steps:

[0012] S1: prepare materials, take oolong summer tea and black tea pieces, put them into a grinder for grinding, and obtain oolong tea powder and black tea powder for later use;

[0013] S2 enzymolysis, taking oolong tea powder and dark tea powder in proportion, mixing them, adding deionized water according to the material-liquid ratio, and adding cellulase and pectinase to perform enzymolysis in a shaking process, and obtaining an enzymolysis solution after the enzymolysis is completed;

[0014] S3: inactivating the enzyme, inactivating the enzyme by high temperature in a constant temperature water bath, and obtaining a suspension after cooling;

[0015] S4 shearing, shearing the suspension using a shearing machine to obtain a sheared liquid;

[0016] S5 homogenization, placing the shearing liquid into a homogenizer for homogenization to obtain a homogenized liquid;

[0017] S6 centrifugation, placing the homogenate in a centrifuge for centrifugation, and taking the supernatant after centrifugation;

[0018] S7: filtering by suction, placing the supernatant in a Buchner funnel for filtering by suction, and obtaining a filtrate;

[0019] S8: Concentration: placing the filtrate in a rotary evaporator for concentration under reduced pressure to obtain a concentrated solution;

[0020] S9 freeze drying, transferring the concentrated solution to a freeze dryer for freeze drying, and recovering the concentrated solution after drying, and the obtained powder is the composite tea extract;

[0021] The oolong summer tea is Dancong summer tea, and the black tea broken tea is Fuzhuan broken tea. Dancong summer tea has a bitter taste due to the accumulation of tea polyphenols and has not been effectively utilized. Fuzhuan broken tea is broken tea produced in the process of processing Fuzhuan tea produced in Anhua County, Hunan Province into bricks. These broken teas are only slightly inferior in appearance, and the chemical composition is not significantly different from the tea successfully squeezed into bricks, but they are often ignored and wasted. On the one hand, the present invention utilizes the characteristics of low price of single-bush summer tea and the characteristics of low price of Fuzhuan broken tea as "processing waste", combines the characteristics that Fuzhuan broken tea and Fuzhuan tea have no significant difference in chemical composition but have widely reported blood sugar and lipid-lowering activities and sweet taste, and mixes the two to develop a specific base material for a composite tea extract. On the other hand, the present invention takes a different approach to focus on its active functions, utilizes cellulase and pectinase enzymatic hydrolysis to destroy the cell wall structure, promotes the dissolution of the active ingredient tea polyphenols, and performs enzymatic hydrolysis and extraction under medium temperature conditions, which has little impact 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 specifications. 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 raw material utilization rate, and reduce the production cost.

[0022] Specifically, in the step of preparing materials S1, the oolong tea powder and the dark tea powder are crushed by a grinder and then passed through a 60-mesh sieve to obtain the powder.

[0023] Specifically, in the S2 enzymatic hydrolysis step, the weight ratio of oolong tea powder to black tea powder is 1:1 to 1:4; the material-liquid ratio is 1:10 to 1:20 g / mL; the amount of cellulase added is 0.25% to 0.75% of the total mass of tea powder; the amount of pectinase added is 0.25% to 0.75% of the total mass of tea powder; the enzymatic hydrolysis temperature is 50 to 60°C; the enzymatic hydrolysis time is 2 to 6 hours; the shaking process is shaking on a shaking table at a speed of 100 to 160 r / min. The cell wall structure is destroyed by the co-enzymatic hydrolysis of cellulase and pectinase, promoting the dissolution of the active ingredient tea polyphenols. Pectinase can specifically decompose the pectin component in the plant cell wall, making the cell wall structure loose, and cellulase can decompose cellulose into smaller units and destroy the plant cell wall. Both can promote the release of effective ingredients in the cell. In addition, enzymatic extraction is carried out at a moderate temperature of 50 to 60°C, which has little impact on its active ingredients and reduces the requirements for production equipment and facilities.

[0024] Preferably, the total amount of the cellulase and the pectinase added is 0.8-1.2% of the total mass of the tea powder.

[0025] Specifically, in the S3 enzyme inactivation step, the constant temperature water bath method is carried out in an environment of 90 to 100° C., the reaction time is 10 to 20 minutes, and the cooling time after the reaction is 5 to 15 minutes.

[0026] Specifically, in the S4 shearing step, the speed of the shearing machine is 1000-5000r / min, and the shearing time is 5-10min; in the S5 homogenizing step, the homogenizing pressure is 10Mpa-30Mpa, and the number of homogenizations is 1-3 times. The suspension is sheared by the shearing machine, so that the suspended matter in the suspension is further crushed and then dispersed into the suspension, so that the particle size of the suspended matter in the obtained sheared liquid is smaller and the distribution is more uniform. When the sheared liquid flows through the cavity with a special internal structure (high-pressure homogenization cavity) at high speed under high pressure, the cell structure of the material in the sheared liquid can be fully destroyed, which promotes the further dissolution of the material in the material and achieves the effect of homogenization.

[0027] Specifically, in the S6 centrifugation step, the centrifugal force of the centrifuge is set to 8000-10000g, and the centrifugation time is 20-30min; in the S8 step, the reduced pressure concentration is carried out at a temperature of 50-60°C, and concentrated to 10%-20% of the original volume; in the S9 step, the freeze-drying temperature of the freeze dryer is -10--20°C, and the drying time is 20-30h.

[0028] The invention also discloses a composite tea extract, which is prepared by the preparation method of the composite tea extract.

[0029] Specifically, the yield of active ingredients in the composite tea extract is ≥30%, the content of tea polyphenols is ≥28%, and the content of tea polysaccharides is ≥22%. The composite 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.

[0030] The invention also discloses an application of the composite tea extract, which is used as an additive in food or health products.

[0031] The present invention has the following beneficial effects: (1) The raw materials used in the present invention are used to develop and utilize composite tea extracts, because of their own conditions, and their own characteristics of being either cheap or neglected and wasted. Under the specified process conditions, the dissolution of tea polyphenols and tea polysaccharides in the composite tea can be promoted. The prepared samples have the characteristics of high yield and good activity, and thus show good blood sugar and lipid-lowering effects. A theoretical foundation is laid for promoting the processing and utilization of oolong tea and Anhua black tea in a new way. In addition, the process is relatively simple, has low requirements on production equipment and facilities, does not involve the use of harmful and toxic organic solvents, is good for the environment, and complies with food processing filing.

[0032] (2) The present invention utilizes cellulase and pectinase enzymatic hydrolysis to destroy the cell wall structure, promote the dissolution of the active ingredient tea polysaccharide, and performs enzymatic hydrolysis and extraction under medium temperature conditions, which has little effect on the active ingredients. In addition, the high-pressure homogenization method is combined to further destroy the cell structure, further promote the dissolution of the active ingredients, significantly improve the raw material utilization rate, and reduce the production cost.

[0033] (3) The present invention adopts the cellulase and pectinase enzymatic hydrolysis method combined with the physical field assisted method for synergistic extraction to prepare the composite tea extract. Through the synergy of parameters such as enzymatic hydrolysis ratio, enzymatic hydrolysis time, homogenization pressure, and homogenization times, the extraction efficiency of active substances represented by tea polyphenols and tea polysaccharides in oolong tea and Anhua dark tea is greatly improved.

[0034] (4) The composite 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, and can be used in food and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The total phenol and total sugar contents and yields of eight composite tea extracts prepared in Examples 1 and 4-6 of the present invention and Comparative Examples 1-4 are shown in the table.

[0036] Figure 2 The table shows the inhibition rates of α-glucosidase and α-amylase of eight kinds of composite tea extracts prepared in Examples 1, 4-6 of the present invention and Comparative Examples 1-4 at the same concentration.

[0037] Figure 3 The table shows the inhibition rates of pancreatic lipase and cholesterol esterase of eight kinds of composite tea extracts prepared in Examples 1, 4-6 of the present invention and Comparative Examples 1-4 at the same concentration.

[0038] Figure 4 The table shows the bile salt adsorption rate and cholesterol micelle dissolution inhibition rate of eight kinds of composite tea extracts prepared in Examples 1, 4-6 of the present invention and Comparative Examples 1-4 at the same concentration.

[0039] Figure 5 This is a table showing the QDA sensory results of eight kinds of composite tea extracts prepared in Examples 1, 4-6 of the present invention and Comparative Examples 1-4 at the same concentration.

[0040] Figure 6 This is a table showing the total phenolic and total sugar contents and yields of the three composite tea extracts prepared in Examples 1-3 of the present invention. DETAILED DESCRIPTION

[0041] The present invention is described in detail below with reference to the embodiments.

[0042] Embodiment 1:

[0043] A method for preparing a composite tea extract according to Example 1 of the present invention comprises the following steps:

[0044] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0045] S2 enzymatic hydrolysis, take oolong tea powder and black tea powder in a ratio of 1:3, mix them and pour them into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:10g / mL to fully mix the tea powder and water, and at the same time add 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder. Then, place the round-bottom flask on a shaker with a rotation speed of , and perform enzymatic hydrolysis at a temperature of 55°C for 4 hours. After the enzymatic hydrolysis is completed, an enzymatic hydrolyzate is obtained.

[0046] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0047] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0048] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 20 MPa, homogenizing twice to obtain a homogenized liquid.

[0049] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0050] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0051] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0052] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP1.

[0053] Embodiment 2:

[0054] A method for preparing a composite tea extract according to Example 2 of the present invention comprises the following steps:

[0055] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0056] S2 enzymatic hydrolysis, take oolong tea powder and black tea powder in a ratio of 1:3, mix them and pour them into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:10g / mL to fully mix the tea powder and water, and at the same time add 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder. Then, place the round-bottom flask on a shaker with a rotation speed of , and perform enzymatic hydrolysis at a temperature of 55°C for 2h. After the enzymatic hydrolysis is completed, an enzymatic solution is obtained.

[0057] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0058] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0059] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 10 MPa, homogenizing twice to obtain a homogenized liquid.

[0060] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0061] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0062] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0063] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP2.

[0064] Embodiment 3:

[0065] A method for preparing a composite tea extract according to Example 3 of the present invention comprises the following steps:

[0066] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0067] S2 enzymatic hydrolysis, take oolong tea powder and black tea powder in a ratio of 1:3, mix them and pour them into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:10g / mL to fully mix the tea powder and water, and at the same time add 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder. Then, place the round-bottom flask on a shaker with a rotation speed of , and perform enzymatic hydrolysis at a temperature of 55°C for 6h. After the enzymatic hydrolysis is completed, an enzymatic hydrolyzate is obtained.

[0068] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0069] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0070] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 30 MPa, homogenizing twice to obtain a homogenized liquid.

[0071] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0072] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0073] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0074] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP3.

[0075] Embodiment 4:

[0076] A method for preparing a composite tea extract according to Example 4 of the present invention comprises the following steps:

[0077] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0078] S2 enzymatic hydrolysis: oolong tea powder and black tea powder are mixed in a ratio of 1:1 and poured into a round-bottom flask. Deionized water is added in a solid-liquid ratio of 1:10 g / mL to fully mix the tea powder and water. At the same time, 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder are added. The round-bottom flask is then placed on a shaker with a rotation speed of , and enzymatic hydrolysis is performed at a temperature of 55°C for 4 hours. After the enzymatic hydrolysis is completed, an enzymatic solution is obtained.

[0079] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0080] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0081] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 20 MPa, homogenizing twice to obtain a homogenized liquid.

[0082] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0083] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0084] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0085] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP4.

[0086] Embodiment 5:

[0087] A method for preparing a composite tea extract according to Example 5 of the present invention comprises the following steps:

[0088] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0089] S2 enzymatic hydrolysis, take oolong tea powder and black tea powder in a ratio of 1:2, mix them and pour them into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:10g / mL to fully mix the tea powder and water, and at the same time add 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder. Then, place the round-bottom flask on a shaker with a rotation speed of , and perform enzymatic hydrolysis at a temperature of 55°C for 4 hours. After the enzymatic hydrolysis is completed, an enzymatic solution is obtained.

[0090] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0091] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0092] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 30 MPa, homogenizing twice to obtain a homogenized liquid.

[0093] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0094] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0095] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0096] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP5.

[0097] Embodiment 6:

[0098] A method for preparing a composite tea extract according to Example 6 of the present invention comprises the following steps:

[0099] S1 prepares materials, takes Dancong summer tea and Fuzhuan broken tea, puts them into grinders respectively for grinding, and passes through a 60-mesh sieve to obtain oolong tea powder and black tea powder for later use.

[0100] S2 enzymatic hydrolysis, take oolong tea powder and black tea powder in a ratio of 1:4, mix them and pour them into a round-bottom flask, add deionized water at a solid-liquid ratio of 1:10g / mL to fully mix the tea powder and water, and at the same time add 0.75% cellulase and 0.25% pectinase based on the total mass of the tea powder. Then, place the round-bottom flask on a shaker with a rotation speed of , and perform enzymatic hydrolysis at a temperature of 55°C for 4 hours. After the enzymatic hydrolysis is completed, an enzymatic hydrolyzate is obtained.

[0101] S3 enzyme inactivation: transfer the round-bottom flask containing the enzymatic solution to a constant temperature water bath, inactivate the enzyme at high temperature at 95°C for 15 min, cool for 5 min, and obtain a suspension.

[0102] S4 shearing: the suspension was sheared at 5000 r / min for 10 min using a shearing machine to obtain a sheared liquid.

[0103] S5 homogenization, placing the shearing liquid in a homogenizer, homogenizing under a homogenizing pressure of 30 MPa, homogenizing twice to obtain a homogenized liquid.

[0104] S6 centrifugation, transfer the homogenate into a centrifuge cup, place it in a centrifuge, centrifuge at 12000g for 25 minutes, and take the supernatant after centrifugation.

[0105] S7 is filtered, and the supernatant is placed in a Buchner funnel for filtering to obtain a filtrate.

[0106] S8 concentration: placing the filtrate in a rotary evaporator and concentrating under reduced pressure at 55° C. until the filtrate is concentrated to 15% of the original volume to obtain a concentrated solution.

[0107] S9 freeze drying, transferring the concentrated solution to a freeze dryer, freeze drying at -20°C for 24 hours, and recovering after drying. The obtained powder is the composite tea extract, which is recorded as DOEP6.

[0108] Comparative Example 1:

[0109] A method for preparing a composite tea extract comprises the following steps:

[0110] (1) Preparation of composite tea powder: Take single-bush summer tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain oolong tea powder for later use, wash the grinder and dry it for later use; take Fuzhuan broken tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain black tea powder for later use;

[0111] (2) High temperature extraction: Oolong tea powder and dark tea powder were mixed in a ratio of 1:1 to obtain a mixed tea powder. Water was added to the mixed tea powder in a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. The mixture was extracted for 2 h on a shaker (120 r / min) at 100°C, and then cooled to room temperature to obtain an extract 1.

[0112] (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.

[0113] (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 the composite tea extract powder DOH1.

[0114] Comparative Example 2:

[0115] A method for preparing a composite tea extract comprises the following steps:

[0116] (1) Preparation of composite tea powder: Take single-bush summer tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain oolong tea powder for later use, wash the grinder and dry it for later use; take Fuzhuan broken tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain black tea powder for later use;

[0117] (2) High temperature extraction: Oolong tea powder and dark tea powder were mixed in a ratio of 1:2 to obtain a mixed tea powder. Water was added to the mixed tea powder in a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. The mixture was extracted for 2 h on a shaker (120 r / min) at 100°C, and then cooled to room temperature to obtain an extract 1.

[0118] (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.

[0119] (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 hours. After drying, the powder is recovered to obtain the composite tea extract powder DOH2.

[0120] Comparative Example 3:

[0121] A method for preparing a composite tea extract comprises the following steps:

[0122] (1) Preparation of composite tea powder: Take single-bush summer tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain oolong tea powder for later use, wash the grinder and dry it for later use; take Fuzhuan broken tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain black tea powder for later use;

[0123] (2) High temperature extraction: Oolong tea powder and dark tea powder were mixed in a ratio of 1:3 to obtain a mixed tea powder. Water was added to the mixed tea powder in a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. The mixture was extracted for 2 h on a shaker (120 r / min) at 100°C, and then cooled to room temperature to obtain an extract 1.

[0124] (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.

[0125] (4) Concentration and freeze drying: The filtrate was concentrated under reduced pressure at 55°C to 15% of the original volume, and the concentrate was transferred to a freeze dryer and freeze-dried at -20°C for 24 hours. After drying, the powder was recovered to obtain the composite tea extract powder DOH3.

[0126] Comparative Example 4:

[0127] A method for preparing a composite tea extract comprises the following steps:

[0128] (1) Preparation of composite tea powder: Take single-bush summer tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain oolong tea powder for later use, wash the grinder and dry it for later use; take Fuzhuan broken tea raw material, put it into a grinder for grinding, pass it through a 60-mesh sieve to obtain black tea powder for later use;

[0129] (2) High temperature extraction: Oolong tea powder and dark tea powder were mixed in a ratio of 1:4 to obtain a mixed tea powder. Water was added to the mixed tea powder in a solid-liquid ratio of 1:10 g / mL, and the mixture was mixed in a round-bottom flask. The mixture was extracted for 2 h on a shaker (120 r / min) at 100°C, and then cooled to room temperature to obtain an extract 1.

[0130] (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.

[0131] (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 hours. After drying, the powder is recovered to obtain the composite tea extract powder DOH4.

[0132] The composite tea extracts prepared in Examples 1 to 6 and Comparative Examples 1 to 4 were tested using the following protocol.

[0133] 1. Calculation of yield

[0134] The yield of oolong tea extract was calculated according to formula (1).

[0135]

[0136] 2. Determination of the content of main active chemical components

[0137] (1) Determination of total phenol content

[0138] 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.

[0139] (2) Determination of total sugar content

[0140] 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.

[0141] 3. Determination of in vitro glucose and lipid digestion enzyme inhibitory activity

[0142] Preparation of sample stock solution: Take the sample and prepare it into 20 mg / mL stock solution with deionized water for later use.

[0143] (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.

[0144] 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 100 μL of 0.1 mol / L Na2CO3 solution to terminate the reaction. Use an enzyme reader to measure the absorbance at 405 nm. Blank: replace the sample solution with phosphate buffer; sample control: replace the α-glucosidase solution with phosphate buffer; blank control: replace the sample and α-glucosidase solution with phosphate buffer. The absorbance values ​​of sample / blank / sample control / blank control are calculated as A1 / A1, respectively. 10 / A 11 / A 00 , the α-glucosidase inhibition rate was calculated according to formula (2).

[0145] α-glucoside inhibition rate (%) = [1-(A1-A11) / (A10-A00)]×100% (2)

[0146] (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.

[0147] 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 calculated as A1 / A ... 10 / A 11 / A 00, the α-glucosidase inhibition rate was calculated according to formula (3).

[0148] α-amylase inhibition rate (%) = [1-(A1-A11) / (A10-A00)]×100% (3)

[0149] (3) Determination of pancreatic lipase inhibitory activity: Intestinal electrolytes are prepared from 6.8mmol / L KCl, 0.8mmol / L KH2PO4, 85mmol / L NaHCO3, 38.4mmol / L NaCl, 0.33mmol / L MgCl2, 8.4mmol / L HCl, 10mmol / L bile salts and 0.6mmol / L CaCl2. The pH is adjusted to 7.0 after dissolution. 2mg / mL pancreatic lipase solution and 2mg / mL p-NPB solution are prepared with intestinal electrolytes; the sample solution is diluted to 1-20mg / mL. The pancreatic lipase solution needs to be centrifuged at 4000g for 10min and filtered through a 0.22μm filter membrane before use.

[0150] 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 calculated as A1 / A2, respectively. 10 / A 11 / A 00 , the pancreatic lipase inhibition rate was calculated according to formula (4).

[0151] Pancreatic lipase production rate (%) = [1-(A1-A11) / (A10-A00)]×100% (4)

[0152] (1) 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 (5).

[0153] Cholesterol esterase efficiency (%) = [1-(A1-A11) / (A10-A00)]×100% (5)

[0154] 4. In vitro simulated digestion-lipid absorption determination

[0155] 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, the pH was adjusted to 3, and the total volume was made up to 5 mL with deionized water, and then incubated at 37°C for 2 h with shaking (120 r / min). In the intestinal stage, 4 mL of trypsin solution (2.5 mg / mL dissolved in cholesterol micelles) and 10 μL of CaCl2(H2O)2 (0.3 mol / L) were added, and the total volume was made up to 10 mL with deionized water, and the pH was adjusted to 7 and incubated at 37°C for 2 h. Finally, the mixture was cooled in ice water and centrifuged (10000 g, 10 min, 4°C) to collect the supernatant. A portion of the supernatant was diluted 100 times and then the total bile acid content was determined using a bile acid determination kit. The other portion of the supernatant was extracted three times with methanol (1:1, v / v) and the cholesterol content was determined using the OPA method. The blank group was a reaction system without adding a sample. The bile acid or cholesterol content of the blank group and the sample group was calculated as B10 and B1, respectively. The bile salt adsorption rate and the cholesterol micelle dissolution inhibition rate were calculated according to formula (6)

[0156] Bile salt adsorption rate / cholesterol micelle dissolution inhibition rate (%) = [1-B1 / B10] × 100% (6)

[0157] For the convenience of analysis and description, in the analysis process, Dancong summer tea is recorded as OT, Fuzhuan broken tea is recorded as DT, the process used in the embodiment is enzymatic hydrolysis-physical field assisted extraction method recorded as EP, and the process used in the comparative example is high temperature water extraction method recorded as H. After measurement, the following analysis was made.

[0158] A. Comparative analysis of the active ingredients of the composite tea extracts prepared by enzymatic hydrolysis-physical field assisted extraction method and high temperature water extraction method.

[0159] Different proportions of Fuzhuan tea (DT) and Dancong summer tea (OT) were used as mixed raw materials for co-extraction under high temperature water extraction (H) and enzymatic hydrolysis-physical field assisted extraction (EP) to prepare the composite tea extracts. The yield, active substance content and extraction efficiency were as follows: Figure 1 As shown (including comparative examples 1 to 4 and embodiments 1, 4 to 6).

[0160] Tea polyphenols and tea polysaccharides are the most important active substances in tea, which have multiple functions such as anti-oxidation, cardiovascular protection, anti-hyperglycemia, anti-obesity and improvement of intestinal health. Due to the high temperature in summer, the polyphenols of single-bush summer tea accumulate more, which belongs to the series with higher polyphenol content in the oolong tea series. Fuzhuan tea, as the most representative series of Anhua dark tea, contains active substances such as tea polysaccharides, which have been widely reported to have significant glucose control and lipid-lowering activities. The two show complementary characteristics in chemical composition, which lays a material foundation for the development of composite tea extracts with single-bush summer tea as the main ingredient and Anhua dark tea as the auxiliary ingredient. Therefore, the difference in the proportion of Anhua dark tea and single-bush summer tea in the extraction process is one of the decisive factors for the composition and content of active substances in the composite tea extract. The ratio of the two should be optimized, and the total phenol and total sugar content can be used as indicators for optimizing the process of preparing composite tea extracts. 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 composite tea extracts.

[0161] Pectinase can specifically decompose the pectin component in the plant cell wall, making the cell wall structure loose, and cellulase can decompose cellulose into smaller units and destroy the plant cell wall. Both can promote the release of effective ingredients in the cell. The material that has been enzymatically hydrolyzed and is in a suspended state flows at high speed through a cavity with a special internal structure (high-pressure homogenization cavity) under high pressure, which can fully destroy the material, promote further dissolution of the substances in the material and achieve a homogenization effect. Compared with the traditional water extraction method for preparing tea extracts, which has the disadvantages of high loss and low sample yield, the enzymatic hydrolysis-physical field assisted extraction method is a more thorough extraction method and also improves the stability of the product. By comparing DOH1, DOH2, DOH3 and DOH4 (Comparative Examples 1-4), it was found that the composite tea extracts prepared by extracting DT and OT as mixed raw materials in different proportions under the H process, except for DOH2 and DOH3, had significant differences in total phenol and total sugar content, and the overall rule was that with the increase of OT ratio, the total phenol content increased, and the total sugar content showed a downward trend, but there was no significant difference in the extract yield; by comparing DOEP1, DOEP4, DOEP5 and DOEP6 (Examples 1, 4-6), it was found that the total phenol content and total sugar content of the composite tea extracts prepared by extracting DT and OT as mixed raw materials in different proportions under the EP process were consistent with those under the H process, but the change in yield was more significant, that is, with the increase of OT ratio, the yield of the composite tea extract also showed an upward trend. This confirms that DT and OT have more advantages in the content of tea polysaccharides and tea polyphenols, that is, the raw materials for preparing the composite tea extract have a material basis for active complementarity, and on the other hand, it shows that the EP process is more significant in promoting the release of effective substances in the two teas. Further simultaneous comparison of DOH1, DOH2, DOH3, DOH4, DOEP1, DOEP4, DOEP5 and DOEP6 revealed that one of the total phenol content, total sugar content or yield of DOH1, DOH2, DOEP4 and DOEP6 was relatively low, while when DT:OT=1:2 or 1:3, the chemical composition content of the samples prepared by the two processes was more balanced, and there was no significant difference in the total phenol content of the composite tea extracts prepared by the two processes, namely DOH2, DOH3, DOEP1 and DOEP5, but DOEP 1 Among them, the yield and total sugar content are significantly higher, which means that the composite tea extract prepared by the EP process with a tea raw material ratio of DT:OT=1:3 not only significantly improves the yield of tea extract compared with the traditional water extraction method H, but also significantly increases the total sugar content while ensuring that the total phenol content does not change significantly. This proves that the cellulase and pectinase enzymatic hydrolysis and high-pressure homogenization selected by the EP method can fully destroy tea cells, thereby more effectively promoting the release of the two most important active substances, tea polyphenols and tea polysaccharides, in OT and DT than the traditional water extraction method H.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 the composite tea extract, which can simultaneously promote the dissolution and release of the two most important active substances (tea polyphenols and tea polysaccharides) in the two types of tea (black tea and oolong tea). Compared with the traditional water extraction method, not only the yield of the prepared tea extract is significantly increased, but also the release of active substances in the two types of tea is more effectively promoted. In addition, from. Figure 6 It can be seen that when the tea raw material ratio of DT:OT=1:3 is selected, the tea extracts prepared by the EP method within the process conditions specified in the present invention can meet the requirements of total phenol content ≥28%, total sugar content ≥22% and yield ≥30%.

[0162] B. Comparative analysis of the glucose-controlling and lipid-lowering activities of the composite tea extracts prepared by enzymatic hydrolysis-physical field-assisted extraction method and high-temperature water extraction method.

[0163] The same proportion of Fuzhuan tea (DT) and Dancong summer tea (OT) were used as mixed raw materials and co-extracted by high temperature water extraction (H) and enzymatic hydrolysis-physical field assisted extraction (EP) to prepare the composite tea extracts. The inhibition rates of 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 2-4 (Different letters indicate significant differences among samples (p < 0.05)).

[0164] Diseases such as diabetes, metabolic syndrome and hyperlipidemia often occur together, which are usually caused by abnormal glucose and lipid metabolism. The most important enzymes related to glucose metabolism are α-glucosidase and α-amylase. The former mainly acts on the non-reducing ends of polysaccharides and oligosaccharides, hydrolyzes α-1,4-glucosidic bonds, and releases glucose. The latter randomly acts on the α-1,4-glycosidic bonds inside the starch chain. In addition to glucose, the hydrolysis products are mainly dextrin and maltose. The most important enzymes related to lipid metabolism are cholesterol esterase and pancreatic lipase. The two together hydrolyze a variety of lipid substrates, including phospholipids, triglycerides, fat-soluble vitamins and dietary cholesterol esters. Therefore, by inhibiting these four enzymes, the above-mentioned series of complications can be effectively prevented or alleviated by regulating glucose and lipid metabolism. The compound tea extract can show a good inhibitory effect on the above four enzymes at a certain concentration, and the inhibition rate at high concentration is greater than or equal to the inhibition rate at low concentration, such as Figure 2-3 As shown. Different proportions of Fuzhuan tea (DT) and Dancong summer tea (OT) were used as mixed raw materials and prepared by high temperature water extraction (H) and enzymatic hydrolysis-physical field assisted extraction (EP) respectively. All the extracts obtained had an inhibitory activity against α-glucosidase of μg level ( Figure 2). 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 DOH1 and DOEP2 prepared when DT:OT=1:1. For the samples at the other three ratios, the inhibition rate of the composite tea extract prepared by the H process was higher than that of the composite tea extract prepared by the EP method and showed significance. However, when the ratio of DT to OT was the same, the inhibition effect of the H method was only 4%-10% higher than that of the EP method. When the sample concentration was 10mg / mL and 14mg / mL, the inhibition rate of α-amylase by the composite tea extract prepared by the EP process was significantly higher than that of the composite tea extract prepared by the H method, and when the ratio of DT to OT was the same, the inhibition effect of the EP method was improved by 23%-200% compared with the H method ( Figure 2), therefore, combined with the inhibition rates of α-glucosidase and α-amylase, the compound tea extract prepared by the EP method has more advantages in exerting functional activity by regulating sugar metabolism. Similarly, at the set sample concentration, we compared the inhibition rates of the compound tea extracts under the two processes on pancreatic lipase and cholesterol esterase. In terms of inhibiting pancreatic lipase, the inhibition rate of the compound tea extract prepared by the H process is significantly higher than that of the compound tea extract prepared by the EP method. When the ratio of DT to OT is the same, the inhibition effect of the H method is improved by 9%-21% compared with the EP method. For cholesterol esterase, when the ratio of DT to OT is 1:2 and 1:4, the inhibition rate of the compound tea extract prepared by the H process is significantly higher than that of the compound tea extract prepared by the EP method, and when the ratio of DT to OT is 1:1 and 1:3, the inhibition rate of the compound tea extract prepared by the EP process on cholesterol esterase is significantly higher than that of the compound tea extract prepared by the H method. It is worth mentioning that DOEP1 showed the highest cholesterol esterase inhibition activity among all samples. Therefore, combined with the inhibition rates of pancreatic lipase and cholesterol esterase, the compound tea extracts prepared by the EP method and the H method have their own advantages in different targets by regulating lipid metabolism to exert functional activity. In addition, we found that under the H process, with the increase of the proportion of OT, although the inhibitory ability of the compound tea extracts on glycolipid digestive enzymes was mostly on the rise, when the OT ratio was increased to DT:OT=1:3, and further increased to DT:OT=1:4, this change was no longer significant. However, under the EP process, with the increase of the proportion of OT, the inhibitory ability of the compound tea extracts on glycolipid digestive enzymes was not stable and showed an upward trend. Among them, the activity of the samples prepared by DT:OT=1:4 was the most significant on α-amylase and pancreatic lipase; the activity of the samples prepared by DT:OT=1:3 was the most significant on α-glucosidase and cholesterol esterase. Therefore, the samples DOEP1 and DOEP5 prepared by the EP method have the best comprehensive glycolipid digestive enzyme inhibition ability among all compound tea extracts. They delay the absorption and metabolism of glycolipids by inhibiting the above glycolipid digestive enzymes, thereby achieving the purpose of controlling blood sugar and reducing blood lipids.

[0165] Bile salt adsorption and cholesterol micelle dissolution play an important role in regulating cholesterol levels in the body. This is another important way to exert glucose-control and lipid-lowering activity through lipid absorption in addition to sugar and lipid digestion. The final system concentration of the composite tea extract prepared by the H method and the EP method with different proportions of DT and OT as mixed raw materials in the in vitro simulated digestion was 10 mg / mL. Figure 4As shown. All compound tea extract samples have the ability to inhibit bile salt adsorption and cholesterol micelle dissolution, and the inhibition rate is distributed in 15%-35%, which is generally low. This shows that although the compound tea extract can exert glucose-controlling and lipid-lowering activity by inhibiting the adsorption of bile salts and reducing the solubility of cholesterol micelles, the effect is limited. And unlike digestive enzyme inhibition, this activity has no obvious correlation with the ratio of DT and OT used in the preparation of compound tea extracts. This may be because there are more types of active substances involved in lipid absorption and more diverse pathways of action, such as binding to micelles to directly destroy their stability, or absorbing bile acids during digestion to inhibit the formation of micelles. It is worth mentioning that only when DT:OT=1:3, there is no significant difference in bile salt adsorption between the compound tea extracts prepared by EP method and H method, and the rest show that H method is slightly better than EP method.

[0166] Based on the above in vitro models, the compound tea extract can regulate sugar and lipid metabolism, and has a significant effect on inhibiting sugar and lipid digestion enzymes. It can also play a certain role in the pathway of lipid absorption, thereby exerting glucose control and lipid-lowering activity. Therefore, combined with the content of active substances, samples DOEP1 and DOEP6 prepared by the EP method have the greatest comprehensive glucose control and lipid-lowering potential. Since there is no significant difference in the yield and total phenol content of DOEP1 compared with DOEP6, and the total sugar content is significantly higher (such as Figure 1 As shown in the figure, the homogenization pressure is lower and the enzymatic hydrolysis time is shorter, so the process cost is lower, so DOEP1 is the optimal compound tea extract.

[0167] C. Comparative analysis of sensory evaluation of composite tea extracts prepared by enzymatic hydrolysis-physical field assisted extraction method and high temperature water extraction method.

[0168] Different proportions of Fuzhuan tea (DT) and Dancong summer tea (OT) were used as mixed raw materials and co-extracted by high temperature water extraction (H) and enzymatic hydrolysis-physical field assisted extraction (EP) to prepare the composite tea extracts. The results are shown in the following table. Figure 5 As shown in Figure 2, the quantitative descriptive analysis (QDA) experimental results of the Fuzhuan Tea extract (DTH) and the single-bush summer tea extract (OTH) prepared by the room temperature water extraction method are shown in Figure 2. Figure 5 shown.

[0169] QDA is a sensory analysis method used to describe and evaluate the sensory characteristics of products. It is widely used in the food industry for product quality control, new product development and product quality improvement. On the premise that DOEP1 has been confirmed to have good glucose control and lipid-lowering activity, the QDA experiment simultaneously compares DOH1, DOH2, DOH3, DOH4, DOEP1, DOEP4, DOEP5 and DOEP6, which is conducive to quantifying the sensory characteristics of compound tea extracts, including bitterness. That is, the more prominent the radar, the higher the score, and the more obvious the corresponding sensory characteristics. Figure 5 In the experiment, the sensory differences between DTH and OTH were obvious. DTH had obvious warehouse smell (warehouse smell was a kind of not so refreshing, low-pitched smell of old wood, which was different from moldy smell. Warehouse smell did not mean that tea was spoiled) and thick feeling, and the soup color was brown-yellow, while OTH had the characteristics of strong freshness and heavy bitterness, and the soup color was clear and yellow. Further analysis showed that from the comparison between H method and EP method, when the ratio of DT to OT was the same during the preparation of composite tea extract, the bitterness of composite tea extract under EP method was relatively weaker, which may be related to the higher content of tea polysaccharides in composite tea extract prepared by EP method than that by H method. At the same time, except when DT:OT=1:2, the composite tea extract prepared by EP method was more refreshing than that prepared by H method, which showed that the flavor of DT and OT under EP process was coordinated, and the flavor improvement effect on OT was relatively more obvious than that of H process. In addition, with the increase of OT ratio, the overall bitterness continued to increase, and the change law between the ratio of the two and the sensory characteristics was basically consistent with the characteristic flavor of OT and DT reported in the literature, indicating that the experimental results were reliable. It is worth mentioning that when DT:OT changes from 1:2 to 1:3, the bitterness of OT does not increase significantly, and even decreases. Compared with the single OT extract, the bitterness decreases by 25%, which may be affected by the flavor of DT. The sample prepared by the EP method (DT:OT=1:3) is the composite tea extract DOEP1 with the best comprehensive glucose control and lipid-lowering potential. Therefore, under the premise of ensuring functional activity, the process adopted by the present invention has improved the shortcomings of the flavor of single-bush summer tea to a certain extent.

[0170] In summary, by comparing the embodiments of the present invention with the comparative examples, it can be seen that: considering the sample yield, active substance content, process cost, functional activity and sensory properties, the composite tea extract prepared by the cellulase and pectinase enzymatic hydrolysis method combined with the physical field assisted method is optimal when DT:OT=1:3, and can improve glucose and lipid metabolism by inhibiting multiple pathways of sugar digestion, fat digestion and fat absorption. The specific target is that it can inhibit the activity of α-glucosidase, α-amylase, pancreatic lipase and cholesterol esterase, and inhibit bile salt adsorption and cholesterol micelle dissolution to a certain extent to exert glucose control and lipid-lowering activity. By introducing Fuzhuan tea and Dancong summer tea for compounding, and further combining cellulose enzymatic hydrolysis-assisted extraction method and physical field-assisted extraction method for innovative application in the preparation of composite tea extract, a way of thinking is provided to change the current situation of serious waste of Dancong summer tea resources, and at the same time, the application field of Anhua black tea is further broadened, and the flavor coordination of the two teas is achieved to a certain extent under the premise of ensuring functional activity, which caters to the current situation of people's increasingly high requirements for the health quality of food, and explores the blood sugar control and lipid-lowering potential of the two teas in a different way, expanding the market demand for the two teas. This shows that the technical solution of the present invention can obtain a class of composite tea extracts with blood sugar control and lipid-lowering activity through a better preparation process.

[0171] 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 a composite tea extract, characterized in that: The enzymatic hydrolysis-physical field assisted combined method includes the following steps: S1: preparing materials, taking oolong summer tea and black tea pieces, and putting them into a grinder for grinding respectively, to obtain oolong tea powder and black tea powder for later use; S2 enzymatic hydrolysis, taking oolong tea powder and dark tea powder in proportion, mixing them, adding deionized water according to the material-liquid ratio, and adding cellulase and pectinase to perform enzymatic hydrolysis in a shaking process, and obtaining an enzymatic hydrolyzate after the enzymatic hydrolysis is completed; S3: inactivating the enzyme, inactivating the enzyme in the enzymatic solution by a constant temperature water bath method, and obtaining a suspension after cooling; S4 shearing, shearing the suspension using a shearing machine to obtain a sheared liquid; S5 homogenization, placing the shearing liquid into a homogenizer for homogenization to obtain a homogenized liquid; S6 Centrifugation: Place the homogenized liquid in a centrifuge for centrifugation and take the supernatant after centrifugation; S7: filtration, placing the supernatant in a Buchner funnel for filtration to obtain a filtrate; S8: Concentrating, placing the filtrate in a rotary evaporator for concentration under reduced pressure to obtain a concentrated solution; S9 freeze drying, transferring the concentrated solution to a freeze dryer for freeze drying, and recovering the concentrated solution after drying, and the obtained powder is the composite tea extract; Among them, the oolong summer tea is single bush summer tea, and the broken black tea is broken Fuzhuan tea.

2. The method for preparing the composite tea extract according to claim 1, characterized in that: In the step S1 of preparing materials, the oolong tea powder and the dark tea powder are crushed by a grinder and then passed through a 60-mesh sieve to obtain the powder.

3. The method for preparing the composite tea extract according to claim 1, characterized in that: In the S2 enzymatic hydrolysis step, the weight ratio of oolong tea powder to black tea powder is 1:1~1:4; the material-liquid ratio is 1:10~1:20 g / mL; the amount of cellulase added is 0.25%~0.75% of the total mass of the tea powder; the amount of pectinase added is 0.25%~0.75% of the total mass of the tea powder; the enzymatic hydrolysis temperature is 50~60°C; the enzymatic hydrolysis time is 2~6 h; the shaking process is shaking on a shaking table at a rotation speed of 100~160 r / min.

4. The method for preparing the composite tea extract according to claim 3, characterized in that: The total amount of the cellulase and the pectinase added is 0.8-1.2% of the total mass of the tea powder.

5. The method for preparing the composite tea extract according to any one of claims 1 to 4, characterized in that: In the S3 enzyme inactivation step, the constant temperature water bath method is carried out in an environment of 90-100° C., the reaction time is 10-20 min, and the cooling time after the reaction is 5-15 min.

6. The method for preparing the composite tea extract according to any one of claims 1 to 4, characterized in that: In the S4 shearing step, the rotation speed of the shearing machine is 1000-5000 r / min, and the shearing time is 5-10 min; in the S5 homogenizing step, the homogenizing pressure is 10 Mpa-30 Mpa, and the homogenization times are 1-3 times.

7. The method for preparing the composite tea extract according to any one of claims 1 to 4, characterized in that: In the S6 centrifugation step, the centrifugal force of the centrifuge is set to 8000 ~ 10000 g , the centrifugation time is 20~30 min; in the step S8, the reduced pressure concentration is carried out at a temperature of 50~60°C, and concentrated to 10%~20% of the original volume; in the step S9, the freeze-drying temperature of the freeze dryer is -10~-20°C, and the drying time is 20~30h.

8. A composite tea extract, characterized in that: The compound tea extract is prepared by the preparation method of any one of claims 1 to 8.

9. The composite tea extract according to claim 8, characterized in that: The active ingredient yield of the composite tea extract is ≥30%, the tea polyphenol content is ≥28%, and the tea polysaccharide content is ≥22%. The composite tea extract has the function of inhibiting α -glucosidase, α -Amylase, pancreatic lipase and cholesterol esterase capacity as well as bile salt adsorption and cholesterol micellar solubilization inhibition capacity.

10. An application of a composite tea extract, characterized in that: Use of the composite tea extract as claimed in claim 9 as an additive in food or health products.

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