Mulberry leaf extract rich in sugar inhibition component as well as preparation method and application of mulberry leaf extract

Abstract

The invention provides a mulberry leaf extract rich in sugar inhibition components and a preparation method and application thereof, and the method comprises the following steps: S1, after mulberry leaves are subjected to water extraction, collecting a first water extract and water extraction residues; s2, fermenting the water extraction residues, performing water extraction, and collecting a second water extraction solution; s3, mixing the first water extract and the second water extract, and then performing flocculation, microfiltration, ultrafiltration, membrane concentration and drying; a flocculating agent in the flocculation process is modified chitosan; the modified chitosan is prepared from the following raw materials in parts by weight: 10 parts of chitosan, 1-2 parts of aldehyde sucrose and 3-4 parts of amino polyethylene glycol. According to the preparation method, the steps of the preparation process are controlled, so that the mulberry leaf extract with high sugar inhibition component content is prepared.

Description

Technical Field

[0001] The present application relates to the technical field of plant extracts, and in particular to a mulberry leaf extract rich in sugar-suppressing components, and a preparation method and application thereof. Background Art

[0002] Mulberry leaves are rich in flavonoids, polysaccharides, polyphenols, alkaloids and other active ingredients, which have antioxidant, antibacterial, anti-inflammatory, hypoglycemic and hypolipidemic functions. Mulberry leaves are widely used in pharmaceutical, food, feed and cosmetic industries.

[0003] In the related art, the protein in mulberry leaves is extracted by alkali dissolution and acid precipitation, while other components are discarded; the polysaccharide in mulberry leaves is extracted by water boiling and alcohol precipitation, which can extract most of the polysaccharide, while other components are not used; the flavonoids in mulberry leaves are often extracted by ethanol extraction, which obtains relatively coarse flavonoids, while other components are also discarded, etc. That is, the related art extracts and separates one or a class of substances in mulberry leaves, while other components are often discarded or destroyed and are not fully utilized. Summary of the invention

[0004] The present application is made in view of the above-mentioned problems, and its purpose is to provide a method for preparing a mulberry leaf extract rich in anti-sugar components, by which the anti-sugar components in the mulberry leaves can be fully extracted and fully utilized.

[0005] Specifically, the first aspect of the present application provides a method for preparing a mulberry leaf extract rich in glucose-suppressing components, comprising the following steps:

[0006] S1, extracting mulberry leaves with water, and collecting the first water extract and water extraction residue;

[0007] S2, extracting the water-extracted residue by water after fermentation, and collecting the second water extract;

[0008] S3, mixing the first water extract and the second water extract, and then flocculating, microfiltering, ultrafiltering, membrane concentrating and drying;

[0009] The flocculant in the flocculation process is modified chitosan;

[0010] The modified chitosan comprises the following raw materials in parts by weight:

[0011] 10 parts of chitosan, 1 to 2 parts of aldehyde sucrose and 3 to 4 parts of amino polyethylene glycol.

[0012] According to one of the technical solutions of this application, at least the following beneficial effects are achieved:

[0013] The present application adopts water as the extraction medium of mulberry leaves to achieve the preliminary enrichment of the effective ingredients in the mulberry leaves; the present application also ferments the water extract residue to further destroy the plant cell walls in the water extract residue through the fermentation treatment, further promoting the dissolution of the effective ingredients in the mulberry leaves, and the present application also mixes the first water extract and the second water extract and flocculates to achieve the preliminary removal of the pigments in the water extract; the present application also intercepts macromolecular substances and impurities through ultrafiltration treatment, thereby obtaining a mulberry leaf extract with high sugar inhibition components.

[0014] In the flocculation process of the present application, modified chitosan is added, and chitosan undergoes adsorption bridging and electrical neutralization with impurity molecules such as proteins and tannins, so that impurity molecules such as proteins and tannins are flocculated and precipitated in large quantities, thereby increasing the content of total flavonoids and total polysaccharides in the extract; aminopolyethylene glycol has a molecule with a hydrophobic end and a hydrophilic center, so it can be introduced into chitosan, which can increase the hydrophilicity of chitosan, thereby increasing its water solubility, and further improving the flocculation effect of chitosan. The present application also uses aldehyde sucrose to cross-link chitosan and aminopolyethylene glycol, so that the whole forms a network structure, further improving the flocculation effect.

[0015] According to some embodiments of the present application, the aldehyde sucrose is prepared by periodate oxidation method.

[0016] According to some embodiments of the present application, the periodate includes at least one of sodium periodate and potassium periodate.

[0017] According to some embodiments of the present application, the molar ratio of sucrose to sodium periodate in the periodate oxidation method is 1:1-3.

[0018] According to some embodiments of the present application, the reaction temperature of the periodate oxidation method is 25°C to 40°C.

[0019] According to some embodiments of the present application, the reaction time of the periodate oxidation method is 1 h to 6 h.

[0020] According to some embodiments of the present application, the method for preparing the modified chitosan comprises the following steps:

[0021] Adding chitosan and amino polyethylene glycol to an acetic acid aqueous solution to prepare a first mixture;

[0022] The aldehyde sucrose and the first mixture are mixed and reacted, and after the reaction is completed, the solid phase is collected and dried.

[0023] According to some embodiments of the present application, the Mp of the aminopolyethylene glycol is 4000-6000.

[0024] According to some embodiments of the present application, the Mp of the aminopolyethylene glycol is 5000-6000.

[0025] According to some embodiments of the present application, the amino polyethylene glycol is O-(2-aminoethyl) polyethylene glycol (CAS No.: 32130-27-1).

[0026] According to some embodiments of the present application, the reaction temperature in the method for preparing modified chitosan is 40°C to 60°C.

[0027] According to some embodiments of the present application, the reaction time in the method for preparing modified chitosan is 0.5 h to 2 h.

[0028] According to some embodiments of the present application, the temperature of the water extraction in step S1 is 60°C to 80°C.

[0029] According to some embodiments of the present application, the water extraction time in step S1 is 1 h to 3 h.

[0030] According to some embodiments of the present application, the mass volume ratio of mulberry leaves and water during the water extraction process is 1 g: 10 mL to 20 mL.

[0031] According to some embodiments of the present application, ultrasonic treatment is performed during the water extraction process, and the power of the ultrasonic treatment is 100W to 200W.

[0032] According to some embodiments of the present application, the ultrasonic treatment time is 30 min to 120 min.

[0033] According to some embodiments of the present application, cellulase, pectinase and tea polyphenols are added during the fermentation process.

[0034] According to some embodiments of the present application, the mass ratio of the cellulase to the pectinase is 1:1 to 1.5.

[0035] According to some embodiments of the present application, the mass ratio of the cellulase to the tea polyphenols is 1:1.3-1.7.

[0036] According to some embodiments of the present application, the mass ratio of the water-extracted residue to the cellulase is 100:0.5-1.

[0037] Cellulase helps to decompose plant cell walls. It can enzymatically hydrolyze and destroy the surface structure of the cell walls of natural mulberry leaves, making the cellulose network structure loose, which is not only beneficial to the release of cell contents, but also beneficial to the contact of substances involved in the mulberry leaf fermentation reaction, thereby promoting the orderly progress of the fermentation reaction.

[0038] Pectinase acts on the water-extracted residue, causing the degradation of insoluble pectin, cellulose and other components, destroying the cell wall and causing the contents to overflow, thereby promoting the orderly progress of the fermentation reaction.

[0039] According to some embodiments of the present application, the fermentation temperature is 30°C to 40°C.

[0040] According to some embodiments of the present application, the fermentation time is 5 h to 10 h.

[0041] According to some embodiments of the present application, the mass volume ratio of the water-extracted residue and water is 1 g: 20 mL to 30 mL.

[0042] According to some embodiments of the present application, the temperature of the water extraction in step S2 is 60°C to 80°C.

[0043] According to some embodiments of the present application, the water extraction time in step S2 is 1 h to 3 h.

[0044] According to some embodiments of the present application, the mass volume ratio of the modified chitosan and the mixed water extract is 1 to 2:1000.

[0045] According to some embodiments of the present application, the pore size of the microfiltration membrane is 0.2 μm to 0.3 μm.

[0046] According to some embodiments of the present application, the molecular weight cutoff of the ultrafiltration is 2000Da to 3000Da.

[0047] According to some embodiments of the present application, the operating pressure of the ultrafiltration is 0.05 MPa to 1 MPa.

[0048] According to some embodiments of the present application, the molecular weight cutoff of the membrane concentration is 200Da to 300Da.

[0049] According to some embodiments of the present application, the operating pressure of the membrane concentration is 1 MPa to 2 MPa.

[0050] According to some embodiments of the present application, after the membrane is concentrated, the concentrated liquid is collected and dried.

[0051] According to some embodiments of the present application, the drying is spray drying.

[0052] According to some embodiments of the present application, the spray drying is followed by sieving. The second aspect of the present application provides a mulberry leaf extract prepared by the method described in the first aspect of the present application.

[0053] The third aspect of the present application provides the use of the mulberry leaf extract of the second aspect of the present application in the preparation of a glucose-suppressing product BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present drawings or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present drawings. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0055] Figure 1 This is the blood glucose response curve of the sucrose group and sucrose + mulberry leaf group of this application.

[0056] Figure 2 This is the blood insulin content curve of the sucrose group and the sucrose + mulberry leaf group of this application.

[0057] The purpose, features and advantages of this figure will be further described in conjunction with the embodiments with reference to the accompanying drawings. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application is described and illustrated below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. Based on the embodiments provided in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present application.

[0059] Obviously, the following descriptions are only some examples or embodiments of the present application. For ordinary technicians in this field, the present application can also be applied to other similar scenarios without creative work. In addition, it can also be understood that although the efforts made in this development process may be complicated and lengthy, for ordinary technicians in the field related to the content disclosed in this application, some changes in design, manufacturing or production based on the technical content disclosed in this application are just conventional technical means, and should not be understood as insufficient content disclosed in this application.

[0060] However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of substantially the same structures may be omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art. In addition, the following description is provided for those skilled in the art to fully understand the present application and is not intended to limit the subject matter described in the claims.

[0061] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values ​​or not including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following scope can be all expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0062] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0063] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0064] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0065] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0066] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0067] In this application, the phenol-sulfuric acid method was used to determine the content of total polysaccharides in mulberry leaf extract.

[0068] The present application adopts the aluminum nitrate-sodium nitrite method to determine the content of total flavonoids in mulberry leaf extract.

[0069] The content of total alkaloids in mulberry leaf extract was determined by Reinecke's salt colorimetry.

[0070] The preparation method of aldehyde sucrose in the examples and comparative examples of the present application consists of the following steps:

[0071] Sodium periodate (1 mol) and water are mixed to form a sodium periodate solution; sucrose is then added to the sodium periodate solution (sucrose is 0.4 mol) and reacted at 35°C in the dark for 1 hour. After the reaction, the mixture is filtered, the filtrate is collected, and the filtrate is concentrated by heating to obtain a solid, which is then dried.

[0072] Example 1

[0073] This embodiment is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which comprises the following steps:

[0074] S1, extracting mulberry leaf powder (1 kg, mesh size 325 mesh) with water, separating the solid and liquid, and collecting the first water extract and the water extraction residue;

[0075] The parameters of water extraction in this step are as follows:

[0076] The temperature was 70℃, the time was 2h, reflux treatment was used during the water extraction process, the mass volume ratio of mulberry leaf powder and water was 1g:20mL, ultrasonic treatment was used during the water extraction process, and the ultrasonic power was 200W;

[0077] S2, extracting the water-extracted residue by water after fermentation, and collecting the second water extract;

[0078] The fermentation parameters were as follows:

[0079] Cellulase, pectinase and tea polyphenols were added during the fermentation process with a mass ratio of 1:1.4:1.5.

[0080] The mass ratio of water-extracted residue to cellulase was 100:0.8, the fermentation temperature was 35°C, and the fermentation time was 8 h.

[0081] The parameters of water extraction in this step are as follows:

[0082] The temperature was 70°C, the time was 2h, reflux treatment was performed during the water extraction process, the mass volume ratio of water extraction residue and water was 1g:20mL, ultrasonic treatment was performed during the water extraction process, and the ultrasonic power was 200W;

[0083] S3, the first water extract and the second water extract are mixed and then flocculated, microfiltered (filter membrane pore size is 0.22 μm), ultrafiltered (molecular weight cutoff is 3000 Da, operating pressure is 0.2 MPa), membrane concentrated (nanofiltration membrane with molecular weight cutoff of 200 Da, operating pressure is 1 MPa) and dried (spray dried, and then passed through an 80-mesh sieve);

[0084] The flocculant in the flocculation process was modified chitosan (the mass volume ratio of modified chitosan and mixed water extract was 1.8:1000);

[0085] The modified chitosan comprises the following raw materials in parts by weight:

[0086] 10 parts of chitosan, 1.5 parts of aldehyde sucrose and 3.2 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0087] The preparation method of modified chitosan in this embodiment consists of the following steps:

[0088] Adding chitosan and aminopolyethylene glycol to an acetic acid aqueous solution (mass fraction is 2%, and the volume mass ratio of chitosan is 50mL:1g) to prepare a first mixture;

[0089] The aldehyde sucrose and the first mixture were mixed and reacted (temperature was 60° C., time was 1 h), and after the reaction was completed, the solid phase was collected and dried.

[0090] Example 2

[0091] This embodiment is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Embodiment 1 in that:

[0092] Cellulase, pectinase and tea polyphenols were added during the fermentation process with a mass ratio of 1:1:1.7;

[0093] The mass ratio of water-extracted residue to cellulase was 100:1;

[0094] The modified chitosan comprises the following raw materials in parts by weight:

[0095] 10 parts of chitosan, 1 part of aldehyde sucrose and 4 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0096] Example 3

[0097] This embodiment is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Embodiment 1 in that:

[0098] Cellulase, pectinase and tea polyphenols were added during the fermentation process in a mass ratio of 1:1.5:1.3;

[0099] The mass ratio of water-extracted residue to cellulase is 100:0.5;

[0100] The modified chitosan comprises the following raw materials in parts by weight:

[0101] 10 parts of chitosan, 2 parts of aldehyde sucrose and 3 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0102] Example 4

[0103] This embodiment is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Embodiment 1 in that:

[0104] Cellulase, pectinase and tea polyphenols were added during the fermentation process with a mass ratio of 1:1.5:1.7;

[0105] The mass ratio of water-extracted residue to cellulase is 100:0.5;

[0106] The modified chitosan comprises the following raw materials in parts by weight:

[0107] 10 parts of chitosan, 2 parts of aldehyde sucrose and 4 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0108] Example 5

[0109] This embodiment is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Embodiment 1 in that:

[0110] Cellulase, pectinase and tea polyphenols were added during the fermentation process with a mass ratio of 1:1:1.3;

[0111] The mass ratio of water-extracted residue to cellulase is 100:0.5;

[0112] The modified chitosan comprises the following raw materials in parts by weight:

[0113] 10 parts of chitosan, 1 part of aldehyde sucrose and 3 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0114] Comparative Example 1

[0115] This comparative example is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Example 5 in that:

[0116] The modified chitosan comprises the following raw materials in parts by weight:

[0117] 10 parts chitosan and 1 part aldehyde sucrose.

[0118] The preparation method of modified chitosan in this comparative example consists of the following steps:

[0119] Adding chitosan to an acetic acid aqueous solution (mass fraction of 2%, and volume mass ratio of chitosan of 50 mL:1 g) to prepare a first mixture;

[0120] The aldehyde sucrose and the first mixture were mixed and reacted (temperature was 60° C., time was 1 h), and after the reaction was completed, the solid phase was collected and dried.

[0121] Comparative Example 2

[0122] This comparative example is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Example 5 in that:

[0123] The modified chitosan comprises the following raw materials in parts by weight:

[0124] 10 parts of chitosan and 3 parts of amino polyethylene glycol (CAS No.: 32130-27-1, Mp is 5000).

[0125] The preparation method of modified chitosan in this comparative example consists of the following steps:

[0126] Adding chitosan and aminopolyethylene glycol to an acetic acid aqueous solution (mass fraction is 2%, and the volume mass ratio of chitosan is 50mL:1g) to prepare a first mixture;

[0127] The first mixture was concentrated to dryness.

[0128] Comparative Example 3

[0129] This comparative example is a method for preparing a mulberry leaf extract rich in glucose-suppressing components, which differs from Example 5 in that:

[0130] The modified chitosan was replaced by chitosan.

[0131] Comparative Example 4

[0132] This comparative example is a method for preparing a mulberry leaf extract rich in glucose-inhibiting components, which differs from Example 5 in that:

[0133] This comparative example did not undergo flocculation treatment.

[0134] Comparative Example 5

[0135] This comparative example is a method for preparing a mulberry leaf extract rich in glucose-inhibiting components, which differs from Example 5 in that:

[0136] In this comparative example, the water residue was not subjected to fermentation treatment, that is, the water-extracted residue was water-extracted again to collect the second water extract (the water extraction parameters refer to Example 5).

[0137] Table 1

[0138]

[0139] It can be seen from Table 1 that the extract prepared in the examples of the present application has high contents of total alkaloids, total polysaccharides and total flavonoids.

[0140] The mulberry leaf extracts prepared in the examples and comparative examples of the present application were used in the glucose inhibition test, and the specific test operation was as follows:

[0141] The principle of using zebrafish for glucose suppression experiments is as follows:

[0142] Long-term high blood sugar levels in the human body can cause obesity, diabetes and other unhealthy conditions. Therefore, effectively controlling blood sugar levels and stabilizing blood sugar levels are of great significance to human health. Zebrafish, like humans, will also experience high sugar levels when induced by high-sugar foods, and the free sugar level can be measured by a blood glucose meter. Therefore, the high-sugar food-induced hyperglycemia model was used for the experiment. By comparing the difference in zebrafish free sugar levels between the test group and the model control group, it can be used to evaluate the sugar control effect of the test substance.

[0143] The instruments and equipment used for the test are as follows:

[0144] Zebrafish breeding equipment:

[0145] The equipment must be equipped with temperature control devices, water circulation and filtration devices, and the breeding container is made of glass. Use universal zebrafish breeding equipment equipped with fish tanks (length, width and height 45cm×45cm×30cm).

[0146] Egg laying box / tank:

[0147] Made of glass, equipped with a mesh screen (mesh size 2mm x 2mm, made of stainless steel to protect fish eggs).

[0148] Water quality monitoring equipment:

[0149] pH meter, dissolved oxygen meter, salinity meter (conductivity meter).

[0150] Analytical balance: precision 0.1 mg.

[0151] Microscope: A stereo microscope equipped with a camera system and a maximum magnification greater than 80 times.

[0152] Test container: glass (such as 6-well plate, culture dish).

[0153] Thermostat: Accuracy ±1°C.

[0154] Brine Shrimp Hatchery:

[0155] The equipment must be equipped with an incubation bucket, an air pipe, an air volume regulating valve, an air pump cut-off switch and a check valve device.

[0156] Blood glucose meter: used to test blood glucose.

[0157] Other testing instruments and equipment:

[0158] Pipettes, centrifuge tubes, glass containers (beakers, volumetric flasks, etc.), vortex mixers, ultrasonic water baths, centrifuges, low-temperature refrigerators, adjustable pipettes and tips.

[0159] Main reagents and consumables:

[0160] Water: in accordance with GB / T 6682.

[0161] Cosolvents such as methanol, dimethyl sulfoxide, ethanol, etc.

[0162] Water for broodstock breeding: weigh 4g of sea salt and dissolve it in 10L of water, with a salinity of 0.25‰~0.50‰, a conductivity of 500μS / cm~800μS / cm, dissolved oxygen ≥80% saturation, a pH of 6.5~8.5, and a hardness of 30mg / L~300mg / L (calculated as calcium carbonate).

[0163] Brine shrimp (Artemia salina) eggs: The dormant eggs of brine shrimp need to be kept dry and dark and refrigerated.

[0164] Weigh about 15g of sea salt and dissolve it in 1L of water to prepare brine shrimp egg hatching water. The density should not exceed 4g / L brine shrimp eggs.

[0165] Fish embryo culture medium: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride were dissolved in 10 L of water. The pH value was 6.5-8.5. All chemicals were of analytical grade.

[0166] 4% rice mixture: The working solution is prepared with fish embryo culture medium to prepare a 4% rice homogenate mixture, which should be prepared fresh before use.

[0167] Test organisms:

[0168] Spawning tests should be conducted using wild-type AB strain zebrafish (Daniorerio) from a reliable source (e.g., China National Zebrafish Resource Center). Broodstock have good reproductive capacity – the fish are aged 6 to 12 months. Test crosses are used for subculture to maintain genetic diversity, and a new batch of broodstock should be replaced after 5 generations of pure strain broodstock. Broodstock have no obvious visible signs of infection and disease, and have not been treated with drugs within 2 months. Broodstock should be acclimated for more than 14 days after being introduced into the laboratory before spawning tests.

[0169] Breeding requirements:

[0170] The breeding water temperature is controlled at 26℃~28.5℃, and the indoor temperature is controlled at 20℃~25℃.

[0171] The stocking density is controlled at 1 to 2 fish per liter of water, with a fixed daily light intensity of 12 to 16 hours, and a good filtration system must be maintained.

[0172] Feed at least twice a day, including at least 1 serving of Artemia salina larvae, with an interval of more than 3 hours between feedings to avoid overfeeding that could affect water quality and cleanliness.

[0173] Spawning Requirements:

[0174] Use a spawning tank to collect fish eggs. Place male and female fish in a 2:1 ratio into the spawning tank 1 to 2 hours before turning off the lights the day before spawning. Since zebrafish occasionally fail to spawn, it is recommended to prepare multiple spawning tanks for backup.

[0175] To avoid genetic bias, fish eggs collected from at least three spawning tanks are mixed and selected for use. If fish eggs are collected in a breeding fish tank, the egg collection box is placed in the breeding fish tank before turning off the lights the day before spawning or before turning on the lights on the day of spawning.

[0176] To prevent the eggs from being eaten by adult fish, the egg collection box is covered with an inert net.

[0177] Mating, spawning and fertilization are completed within about 30 minutes after turning on the lights, at which time the egg box can be removed from the fish tank.

[0178] After the fish eggs are taken out of the egg collection box, it is recommended to clean the fish embryos with fish embryo culture medium.

[0179] Healthy zebrafish embryos were selected and cultured at 28°C ± 1°C at a density of no more than 1 fish embryo in 200 μL fish embryo culture medium until 6 days after fertilization.

[0180] Blank control group settings:

[0181] 60 zebrafish were randomly selected and placed in a 6-well plate, with 20 zebrafish in each well and 5 mL of zebrafish embryo culture medium.

[0182] Model control group settings:

[0183] 60 zebrafish were randomly selected and placed in a 6-well plate, with 20 zebrafish in each well and 5 mL of 4% rice mixture.

[0184] Test substance treatment:

[0185] 60 zebrafish were randomly selected from each group and placed in a 6-well plate, with 20 zebrafish per well, 5 mL of 4% rice (mass fraction) mixed solution and 0.2 g of the test substance, and placed in a 28°C±1°C incubator for 16h±1h.

[0186] Free Sugar Measurement:

[0187] The zebrafish from each test well were collected into a test tube, anesthetized in ice, washed once with fish embryo culture medium, homogenized, and then centrifuged at room temperature for 5 minutes and 15,000 rpm for 5 minutes. 1.5 μL of the supernatant was taken on a blood glucose meter test strip to measure the glucose level.

[0188] Calculate the inhibition rate of free sugar production:

[0189] Inhibition rate = (MS) / (MC) × 100%;

[0190] Where:

[0191] S—mean value of free sugar level in zebrafish in the test substance treatment group;

[0192] M—mean value of free sugar level in zebrafish of model control group;

[0193] C—Average free sugar levels in zebrafish of the blank control group.

[0194] Table 2

[0195] - Free sugar level (mM) Inhibition rate (%) Example 1 7.8 54.3 Example 2 8.2 50.0 Example 3 8.1 51.1 Example 4 8.4 47.9 Example 5 8.7 44.7 Comparative Example 1 9.3 38.3 Comparative Example 2 9.5 36.2 Comparative Example 3 9.6 35.1 Comparative Example 4 9.8 33.0 Comparative Example 5 10.0 30.9 Model Group 12.9 - Blank control group 3.5 -

[0196] It can be seen from Table 2 that the extract prepared in the examples of the present application has an excellent inhibitory effect.

[0197] The present application also uses the mulberry leaf extract in Example 1 in a human glucose suppression test, and the specific test process is as follows:

[0198] Fourteen participants with normal blood sugar were randomly divided into 2 groups according to age and gender.

[0199] Sucrose group: 5 g sucrose was consumed.

[0200] Sucrose + mulberry leaf group: 5 g sucrose and 1.5 g mulberry leaf extract prepared according to Example 1 were taken.

[0201] The average blood glucose and insulin levels were tested before intake, and the average blood glucose (15min, 30min, 45min, 60min, 90min and 120min) and insulin levels (15min, 30min, 45min, 60min, 90min, 120min and 180min) were tested after intake, and the changes were calculated.

[0202] Test results see Figures 1-2 ,from Figures 1-2 It is known that the mulberry leaf extract in Example 1 has an excellent effect of inhibiting the increase of blood sugar.

[0203] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing a mulberry leaf extract rich in glucose-suppressing ingredients, characterized in that: The following steps are involved: S1, extracting mulberry leaves with water, and collecting the first water extract and water extraction residue; S2, extracting the water-extracted residue by water after fermentation, and collecting the second water extract; S3, mixing the first water extract and the second water extract, and then flocculating, microfiltering, ultrafiltering, membrane concentrating and drying; The flocculant in the flocculation process is modified chitosan; The modified chitosan comprises the following raw materials in parts by weight: 10 parts of chitosan, 1 to 2 parts of aldehyde sucrose and 3 to 4 parts of amino polyethylene glycol.

2. The method according to claim 1, characterized in that The aldehyde sucrose is prepared by periodate oxidation method.

3. The method according to claim 1, characterized in that The preparation method of the modified chitosan, The following steps are involved: Adding chitosan and amino polyethylene glycol to an acetic acid aqueous solution to prepare a first mixture; The aldehyde sucrose and the first mixture are mixed and reacted, and after the reaction is completed, the solid phase is collected and dried.

4. The method according to claim 3, characterized in that The reaction temperature in the preparation method of the modified chitosan is 40°C to 60°C.

5. The method according to claim 1, characterized in that The parameters of the water extraction in step S1 include at least one of the following (1) to (3): (1) The water extraction temperature is 60°C to 80°C; (2) The mass volume ratio of mulberry leaves to water during the water extraction process is 1 g: 10 mL to 20 mL; (3) Ultrasonic treatment is performed during the water extraction process, and the power of the ultrasonic treatment is 100W to 200W.

6. The method according to claim 1, characterized in that Cellulase, pectinase and tea polyphenols are added during the fermentation process.

7. The method according to claim 6, characterized in that The mass ratio of the cellulase to the pectinase is 1:1 to 1.5; The mass ratio of the cellulase to the tea polyphenols is 1:1.3-1.

7.

8. The method according to claim 6, characterized in that The pore size of the microfiltration membrane is 0.2 μm to 0.3 μm; The ultrafiltration molecular weight cut-off is 2000Da to 3000Da.

9. A mulberry leaf extract, characterized in that Prepared by the method according to any one of claims 1 to 8.

10. Use of the mulberry leaf extract according to claim 9 in the preparation of a glucose-suppressing product.

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