Water extraction method sesame residue treatment method and application thereof

Abstract

The invention relates to the technical field of treatment and comprehensive utilization of sesame processing waste, in particular to a water extraction method sesame residue treatment method and application thereof. According to the method, protease Pronase E and alkaline protease Alcalase3.0 T which can effectively destroy the binding effect of protein and melanoidins are screened out, and melanoidins and polypeptides with high purity are obtained from the water extraction method sesame residues by adopting a double-enzyme auxiliary membrane separation technology at the same time. In addition, the functional activity of melanoidins is verified by using an animal model, and the nutritional value of polypeptides is evaluated on the basis of amino acid composition.

Description

Technical Field

[0001] The invention relates to the technical field of treatment and comprehensive utilization of sesame processing waste, and in particular to a method for treating sesame residues by a water replacement method and application thereof. Background Art

[0002] Sesame belongs to the herbaceous oil crop of the genus Sesame in the family Pedulaceae and is widely planted in Asia and Africa. According to statistics, more than half of the world's sesame is processed into sesame oil every year. About 1 million tons of sesame are used for oil production in my country every year, among which small-milled sesame oil is well-known both at home and abroad. The production process of small-milled sesame oil is mainly based on the water substitution method, which is a unique sesame oil production method in my country with a history of more than 600 years. The by-product of this production process, sesame residue, is dark brown and contains about 40% protein. Because it is rich in water, has a high temperature, is sticky, and is difficult to dry, it is very easy to mold and deteriorate, and emits a foul odor. According to statistics, about 1 ton of wet sesame residue is produced for every ton of sesame processed. Currently, more than 70% of the wet sesame residue will be simply used as animal feed or agricultural fertilizer along with corruption, which is a serious waste. Therefore, in view of the current treatment method of sesame residue by small-milled sesame oil enterprises, it is a problem that needs to be solved urgently to break the trouble of sesame residue characteristics and carry out its high-value utilization.

[0003] The research team found that the dark brown substance in the sesame residue produced by the water-replacement method mainly comes from the melanoidins produced by the Maillard reaction. The Maillard reaction starts in the sesame roasting stage, but in the water-extraction stage, due to the addition of hot water, the entire reaction continues but changes, giving the sesame oil a special flavor (Huang X., Xia X., Han J., Su C., Qi P., Wang X., Shi F., Han C., & Zhang F. (2024). Dynamic changes in the sensory properties, composition and antioxidant activity of sesame residues and oil during the preparation of sesame oil by aqueous extraction. LWT-Food Science and Technology, 199: 116060.). Moreover, these melanoidins are combined with proteins in sesame, have low water solubility, and have good antioxidant activity. At the same time, this combination also promotes the thermal aggregation of proteins (Mao Zhaojie, Lin Bingjie, Yu Yuan, Wang Xia, Xian Li, Zhang Yu, Yang Zhongxin, Zhang Fengxiang. Extraction and antioxidant activity analysis of melanoidins in sesame residues. Modern Food Science and Technology, 2021, 37(07): 221-227.). Therefore, in order to recover the protein substances in sesame residues, the two must be separated. At the same time, modern research believes that the melanoidins produced during the heat treatment of food are a type of potential food functional factor. Its functional characteristics are related to the source of food and the processing process. There are more research reports on coffee melanoidins, bread melanoidins, black garlic melanoidins, etc. The reported extraction methods of melanoidins include water extraction, organic solvent extraction, macroporous adsorption resin, exclusion chromatography, etc. These methods are mainly aimed at the separation and extraction of water-soluble melanoidins, while the extraction rate of melanoidins bound to other substances is low, and the extraction process only takes into account melanoidins, but does not consider the extraction of bound substances such as proteins. At present, there are very few research reports on the extraction and functional activity of melanoidins from sesame residues. Our research group has previously published on the extraction of melanoidins from sesame residues by water extraction, but it uses water extraction and compares it with alkali extraction and alcohol extraction. The extraction rate and purity of melanoidins are low, and the protein content is high (Mao Zhaojie, Lin Bingjie, Yu Yuan, Wang Xia, Xian Li, Zhang Yu, Yang Zhongxin, Zhang Fengxiang. Extraction and antioxidant activity analysis of melanoidins from sesame residues. Modern Food Science and Technology, 2021, 37(07): 221-227.).

[0004] At present, there are many studies on the recovery of protein from sesame residue by water substitution, most of which use enzyme and fermentation methods to hydrolyze the protein in sesame residue into small molecular proteins or polypeptides, among which alkaline protease, neutral protease, papain, pepsin, and composite protease have been reported. However, most of the obtained protein substances are dark in color and contain a certain amount of melanoidin components, which limits the further utilization of these products. Summary of the invention

[0005] The technical problem to be solved by the present invention is that about 1 ton of wet sesame residue is produced for every ton of sesame residue processed. Currently, more than 70% of the wet sesame residue is simply used as animal feed or agricultural fertilizer along with corruption, which is a serious waste. How to overcome the problem of the characteristics of sesame residue and develop its high-value utilization is a problem that needs to be solved urgently.

[0006] In order to solve the above problems, the present invention provides a method for treating sesame residue by water replacement method and its application, screens out protease Pronase E and alkaline protease Alcalase3.0T that effectively destroy the binding effect between protein and melanoidin, and adopts double enzyme-assisted membrane separation technology to obtain high-purity melanoidin and polypeptide from sesame residue by water replacement method at the same time. In addition, the functional activity of melanoidin is verified by using cell models and animal models, and the nutritional value of polypeptide is evaluated based on amino acid composition.

[0007] To achieve the above object, the present invention specifically implements a method for treating sesame residue by water substitution method through the following technical scheme, comprising the following steps:

[0008] (1) Mixing fresh sesame residue obtained by the water-substitution sesame oil process with water and stirring evenly;

[0009] (2) The pH value of the sesame residue aqueous solution was adjusted to 9-10 and ultrasonically treated at 100 kHz for 30 min; a pH value between 9-10 can allow more protein to dissolve into the solution, facilitating subsequent enzymatic hydrolysis.

[0010] (3) The pH value of the sesame residue solution is adjusted to 7-8, and 35,000-45,000 U / g of Pronase E based on the dry weight of the sesame residue is added, and the pH value is adjusted to 7-8. Enzymatic hydrolysis is carried out at 40-50° C. for 30-48 h. This temperature and pH range is the optimal temperature range and optimal pH range of the enzyme, which can best exert the activity of the protease.

[0011] (4) The pH value of the above solution is adjusted to 7.5-8.5, and 2500U / g-3500U / g of protease Alcalase3.0T based on the dry weight of sesame residue is added, and the pH value is adjusted to 7.5-8.5. The solution is hydrolyzed at 55-65°C for 3-5h, and then the temperature is raised to 90°C to inactivate the enzyme for 15min. This temperature and pH range is the optimal temperature range and optimal pH range of the enzyme, which can best exert the activity of the protease.

[0012] (5) placing the enzymatic hydrolysis solution at a temperature below 10° C. for 24 h to allow the oil to precipitate and the insoluble matter to settle, which is convenient for subsequent centrifugation. Centrifuging is then performed to remove the oil from the upper lipid layer, collecting the supernatant aqueous solution, and filtering to obtain a filtrate;

[0013] (6) diluting the filtrate, performing ultrafiltration separation using an ultrafiltration device, selecting a membrane component with a molecular weight cutoff of 5 KDa, collecting the filtrate ≥ 5 KDa, evaporating and concentrating under reduced pressure, and freeze-drying to obtain sesame residue melanoidin; based on the dry weight of the sesame residue, the extraction rate of melanoidin is measured by a weight method, and the melanoidin content reaches 75.6%. Melanoidin is a macromolecular substance, and selecting a 5 KDa membrane component can just separate the peptide and melanoidin.

[0014] (7) The filtrate with a content of ≤5 KDa was collected and desalted by nanofiltration through a 200 Da nanofiltration membrane. The polypeptide solution was evaporated and concentrated under reduced pressure, and freeze-dried to obtain sesame residue polypeptide. Based on the protein content in the dry weight of sesame residue, the polypeptide extraction rate under this process was calculated to be 76.8%, and the polypeptide content was measured by the Lowry method to reach 87.6%.

[0015] Furthermore, in step (1), sesame residue is mixed with water in a ratio of w:v1:10. Deionized water is preferred. The concentration of the membrane filtration solution cannot be too high, which will lead to clogging and increased resistance of the membrane, affecting the filtration effect; if the concentration is too low, the filtration rate will be too slow.

[0016] Furthermore, in step (2) and step (4), the pH value is adjusted using a 0.5 mol / L NaOH solution; and in step (3), the pH value is adjusted using a 0.5 mol / L HCl solution.

[0017] Furthermore, the centrifugal parameters of step (5) are 5000 r / min for 15 min and filtering with four layers of gauze, which can achieve good centrifugal separation of oil and water.

[0018] Furthermore, in step (6), the filtrate is diluted 4 times and then ultrafiltered.

[0019] Application of melanoidins in the preparation of hypoglycemic products.

[0020] Application of melanoidin in the preparation of products for regulating intestinal flora.

[0021] Beneficial effects of the present invention:

[0022] (1) No organic solvents were used during the entire treatment process.

[0023] (2) The process is suitable for industrial production. The existing technology is difficult to implement in large-scale processing because fresh sesame residue is sticky, hot and humid, rich in protein, and very easy to spoil. In this process, the sesame residue raw material can be directly diluted with water without drying and defatting, which greatly reduces energy consumption. At the same time, the use of centrifugation and filtration methods instead of organic solvent degreasing also greatly increases the safety of the polypeptide.

[0024] (3) High-purity sesame residue melanin and polypeptide can be obtained simultaneously.

[0025] (4) The sesame residue polypeptide obtained by this process is light yellow and does not require further decolorization. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . The sesame residue melanoidin and polypeptide obtained by the present invention, in the figure, the left side is melanoidin and the right side is polypeptide.

[0027] Figure 2 .SDS-PAGE images of sesame protein (R: raw sesame protein; B: baked sesame protein; M: stone-ground sesame protein; W: sesame protein after water-extraction).

[0028] Figure 3 .TEM (A) and XRD spectrum (B) of the sesame residue melanin obtained in the present invention.

[0029] Figure 4 .The UV-visible absorption spectrum (A), fluorescence spectrum (B) and Fourier transform infrared spectrum (C) of the sesame residue melanin obtained by the present invention.

[0030] Figure 5 :In vitro hypoglycemic activity of MELs A. α-glucosidase inhibition rate B. α-amylase inhibition rate C. Pancreatic lipase inhibition rate (compared with acarbose / or orlistat, *, P<0.05; **, P<0.01; n=3)

[0031] Figure 6:Effects of MELs on glucose resistance and insulin resistance in T2DM mice. A, body weight; B, fasting blood glucose; C, glycosylated serum protein level; D, glucose tolerance test results; E, area under the curve of glucose tolerance test; F, insulin resistance test results; G, area under the curve of insulin resistance test; H, serum insulin content; I, insulin resistance index. (*p<0.05, **p<0.01 compared with the control group; #p<0.05, ##p<0.01 compared with the model; n=8).

[0032] Figure 7 :Effects of MELs on glycogen synthesis and metabolism-related indices in T2DM mice.A. Liver glycogen contentB. Skeletal muscle glycogen contentC. FBPase and PEPCK activitiesD. HK activityE. G6Pase activityF. PK activity (compared with the normal group, *, P < 0.05, **, P < 0.01; compared with the model group, #, P < 0.05, ##, P < 0.01; n = 6)

[0033] Figure 8 :Effects of MELs on liver function in T2DM mice. A. Changes in serum lipid indexes TC, TG, LDL-C, and HDL-C B. Serum free fatty acid content C. Changes in serum liver function indexes ALT, AST, and ALP D. HE staining of mouse liver tissue sections (compared with the normal group, **, P<0.01; compared with the model group, ##, P<0.01; n=6)

[0034] Fig. 9 :Effects of MELs on the expression of proteins related to blood glucose regulation pathways in the liver of T2DM mice. A, protein expression band of IRS1 / PI3K / Akt signaling pathway; B, protein expression level of IRS1 / PI3K / Akt signaling pathway; C, protein expression band of AMPK signaling pathway; D, protein expression level of AMPK signaling pathway; E, protein expression band of GLUT4; F, protein expression level of GLUT4. (Compared with the control group, *P<0.05, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; n=3).

[0035] Fig.10:Effects of MELs on the intestinal microbiota at the phylum level in T2DM mice. A, ASV / OUT Venn diagram (n=5); B, alpha diversity (n=4); C, principal coordinate analysis (PCoA) (n=5); D, phylum-level relative species abundance histogram (n=5); E, UPGMA clustering tree and community structure histogram (n=5); F, relative abundance of Firmicutes (n=5); G, relative abundance of Bacteroidetes (n=5); H, Firmicutes / Bacteroidetes ratio (n=5). (Different letters a, b, and c are marked between groups, indicating statistically significant differences, P<0.05).

[0036] Fig.11 :Effects of MELs on the intestinal microbiota of T2DM mice at the genus level. A, genus-level relative species abundance bar graph; B, UPGMA clustering tree and genus-level community structure bar graph; C, genus-level composition of the intestinal microbiota of T2DM mice. (Different letters a, b, c between groups indicate statistically significant differences, P<0.05; n=5). DETAILED DESCRIPTION

[0037] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the art without making creative work are within the scope of protection of the present invention. The following raw materials, unless otherwise specified, are commercially available.

[0038] Embodiment 1:

[0039] A method for treating sesame residue by water substitution method comprises the following steps:

[0040] (1) Mixing fresh sesame residue obtained by the water-replacement sesame oil process (raw sesame, roasted sesame, slurry from the stone grinding stage, and oil and residue from the final water extraction stage) with deionized water in a ratio of 1:10 and stirring evenly;

[0041] (2) The pH value of the sesame residue aqueous solution was adjusted to 10 using a 0.5 mol / L NaOH solution and ultrasonically treated at 100 kHz for 30 min;

[0042] (3) Using 0.5 mol / L HCl solution, the pH value of the sesame residue solution was adjusted to 7.5, and 40,000 U / g (dry weight of sesame residue) of Pronase E (Cat. No.: 1.074333.0001, MERCK) was added, and enzymatic hydrolysis was performed at 40°C for 36 h;

[0043] (4) The pH value of the above solution was adjusted to 8.0 with 0.5 mol / L NaOH solution, and 3000 U / g (dry weight of sesame residue) Alcalase 3.0 T (Alcalase 3.0 T, Novonesis) was added, and enzymatic hydrolysis was performed at 60° C. for 4 h, and then the temperature was raised to 90° C. to inactivate the enzyme for 15 min;

[0044] (5) The enzymatic hydrolysis solution was placed at 4°C for 12 h, and then centrifuged at 5000 r / min for 15 min to aspirate the upper lipid layer, collect the supernatant aqueous solution, and filter it through four layers of gauze to obtain a filtrate;

[0045] (6) diluting the filtrate 4 times, performing ultrafiltration separation using an ultrafiltration device, selecting a membrane component with a molecular weight cutoff of 5 KDa, collecting the filtrate ≥ 5 KDa, concentrating it by evaporation under reduced pressure, and freeze-drying it to obtain sesame residue melanin ( Figure 1 The product on the left side), based on the dry weight of sesame residue, the extraction rate of melanoidin is measured by weight method, and the melanoidin content reaches 75.6%.

[0046] (7) The filtrate with a concentration of ≤5 KDa was collected and desalted by nanofiltration through a 200 Da nanofiltration membrane. The polypeptide solution was evaporated and concentrated under reduced pressure and freeze-dried to obtain sesame residue polypeptide ( Figure 1 The product on the right side) was extracted by taking the protein content in the dry weight of sesame residue as the benchmark, and the polypeptide extraction rate under this process was calculated to be 76.8%. The polypeptide content was measured by the Lowry method to be 87.6%.

[0047] In the early stage, our research group sampled the traditional water-replacement production process (Shandong Ruifu Oil Co., Ltd.) (raw sesame, sesame in the roasting stage, pulp in the stone grinding stage, and oil and residue in the final water extraction stage). The study found that the dark brown substance in the sesame residue of the water-replacement method mainly comes from the melanoidins produced by the Maillard reaction. The Maillard reaction starts in the sesame roasting stage, but in the water extraction stage, due to the addition of hot water, the entire reaction continues but changes, forming more brown substances, making the sesame residue and sesame oil further darker, and giving the small-milled sesame oil a special flavor (Huang X., Xia X., Han J., Su C., Qi P., Wang X., Shi F., Han C., & Zhang F. (2024). Dynamic changes in the sensory properties, composition and antioxidant activity of sesame residues and oil during the preparation of sesame oil byaqueous extraction. LWT-Food Science and Technology, 199: 116060.). Modern research believes that melanoidins produced during the heat treatment of food are a type of potential food functional factor, and their functional properties are related to the food source and processing process (Mesias M, Cristina DA. (2017). Melanoidins as a potential functional food ingredient. Current Opinion in Food Science, 14: 37-42). Our research group tried to extract the melanoidins from sesame residues using deionized water, pH 10 NaOH solution, and 70% ethanol solution, and found that these melanoidins are combined with proteins in sesame, have low water solubility, and have good antioxidant activity. In addition, the research group used alkali extraction and acid precipitation to extract proteins from sesame at various stages of the water replacement process, and used SDS-PAGE to separate and identify them. It was found that the Maillard reaction caused the protein to aggregate severely, and almost no bands appeared on SDS-PAGE ( Figure 2), the binding of melanoidins to proteins enhances protein aggregation and reduces protein solubility (Brudzynski K, Miotto D. Honey melanoidins: Analysis of the compositions of the high molecular weight melanoidins exhibiting radical-scavenging activity. Food Chem. 2011; 127(3): 1023-1030.). These research findings provide important information for the extraction and separation of melanoidins or protein peptides from sesame residues by water substitution, that is, this binding property must be destroyed in order to separate the two.

[0048] Through the analysis of existing melanoidin extraction methods, it is found that melanoidin that is easily soluble in water is usually directly extracted with a certain proportion of water and then separated; for bound melanoidin, the water solubility is poor, and this binding needs to be broken first. Enzyme hydrolysis is an environmentally friendly and efficient method. As mentioned earlier, the melanoidin in sesame residue by the water replacement method is mainly bound to protein, so the best choice is to use protease for hydrolysis. Although the protease method is also commonly used to extract melanoidin from biscuits and bread, the extraction process of melanoidin in sesame residue cannot be completely copied. The reasons are as follows:

[0049] 1. The distribution of melanoidins is different. The melanoidins in biscuits and bread are mainly distributed in the epidermis, while the melanoidins in sesame residue are evenly distributed in the sesame residue.

[0050] 2. The composition of the samples is different. The main components of biscuits and bread are carbohydrates, while most of the oil in the sesame residue produced by the water replacement method is extracted, and its main component is protein, accounting for about 50% of the dry weight of the sesame residue. The residual oil content is also relatively high, accounting for about 20% of the dry weight of the sesame residue.

[0051] 3. The samples have different shapes. The biscuit and bread samples have less moisture and are easy to crush and disperse, while the sesame residue obtained by the water replacement process is rich in moisture, sticky and difficult to dry.

[0052] Therefore, the extraction of melanoidins from sesame residue by water substitution method requires not only attention to its protein binding properties, but also full consideration of the effects of its properties, oils and high content of aggregated proteins.

[0053] In the early stage, our research group also studied the changes in amino acid composition during the water-substitution sesame oil extraction process and found that the main amino acid composition of sesame protein includes glutamic acid (8.37%), followed by arginine (5.35%), aspartic acid (3.19%), leucine (2.99%), glycine (2.10%), and serine (2.07%). The amino acids involved in the Maillard reaction include: serine, threonine, cysteine, arginine, histidine, and lysine. When amino acids participate in the Maillard reaction, they become part of melanoidin, and their structure changes. The peptide bonds connected to them are not easily recognized and catalyzed by the corresponding proteases. Therefore, enzymes that catalyze the peptide bonds of amino acids that do not participate in the Maillard reaction are selected as hydrolases. For melanoidins bound to sesame residue protein, the best separation site is the peptide bond on the side of the amino acid not involved in the reaction connected to the amino acid residues involved in the reaction, such as selecting enzymes that catalyze peptide bonds connected to glutamic acid, aspartic acid, etc., and these amino acids are high in sesame protein, while Pronase E tends to hydrolyze the peptide bonds on the carboxyl side of glutamic acid or aspartic acid. In addition, considering the aggregation of proteins, even the amino acids involved in the Maillard reaction mentioned above are only a small part of them. Therefore, alkaline protease Alcalase3.0T (the main catalytic site is the serine side chain peptide bond, Alcalase3.0T product manual) is further selected to complement Pronase E, releasing the bound melanoidins as much as possible, and further converting the macromolecular protein into small molecular polypeptides, effectively achieving the acquisition of high-purity melanoidins and polypeptides from sesame residues by water substitution.

[0054] Verification results:

[0055] 1. The structure of sesame residue melanin

[0056] (1) Apparent structure of sesame residue melanin

[0057] The sesame residue melanin is in an amorphous state under an electron microscope, with some showing a nano-sized spherical shape, but there is also a part of flocculent disordered form, which is speculated to be unaggregated linear molecular substances. Figure 3 As shown, the X-ray diffraction (XRD) pattern shows diffuse diffraction characteristics, with a diffuse broad peak near 20°, indicating that MEL exists in a non-crystalline form with weak internal crystallinity and belongs to an amorphous structure.

[0058] (2) Spectroscopic properties of sesame residue melanin

[0059] like Figure 4As shown, the full wavelength scan of UV-vis absorbance found strong absorption in the range of 200-230nm, indicating the existence of structures connected with unsaturated aldehydes, unsaturated ketones or conjugated systems; there is a weak peak at 280nm, representing heterocyclic compounds such as furan or pyrrole or heterocyclic and conjugated substructures of Maillard reaction-derived chromophores. When the excitation wavelength is about 350nm, the corresponding emission wavelength has a peak in the range of 420-440nm, which is the fluorescence spectrum characteristic of the Maillard reaction system. Sesame residue melanin has a maximum absorption peak at 420nm, which is consistent with the fluorescence characteristics of the Maillard reaction product. Fourier infrared spectroscopy shows that sesame residue melanin contains aromatic amines and heteroaromatic structures such as furan and pyrrole.

[0060] In summary, the sesame residue melanin prepared by the method is a type of chain-like macromolecular amorphous brown substance rich in carbonyl and nitrogen-oxygen heterocyclic structures, and is easily soluble in water.

[0061] 2. Evaluation of the nutritional value of peptides

[0062] The amino acid composition of the polypeptide was determined by an amino acid analyzer. The essential amino acid model standard recommended by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) was used. Through the essential amino acid model (Table 1), amino acid score (AAS) (Table 2), amino acid ratio coefficient (RC) and amino acid ratio coefficient score (SRC) (Table 3), and the comparative analysis of the closeness to the amino acid composition of egg protein (closeness is 0.986), it was found that the main limiting amino acid is lysine, and the other essential amino acids are very rich, which is close to or even better than egg protein and soy protein isolate. Therefore, the sesame residue polypeptide should be used in combination with lysine-rich protein, or lysine should be added to enhance its nutritional value.

[0063] Table 1 Essential amino acids and amino acid patterns of sesame residue polypeptides obtained in the present invention (mg / g pro)

[0064]

[0065] Table 2 Essential amino acid scores of sesame residue polypeptides obtained in the present invention

[0066]

[0067]

[0068] Table 3 RC and SRC of sesame residue polypeptide EAA obtained in the present invention

[0069]

[0070] Embodiment 2:

[0071] Use of the melanoidin (hereinafter referred to as MEL) prepared in Example 1 in the preparation of hypoglycemic products.

[0072] Animal Experimentation:

[0073] (1) Establishment and grouping of T2DM mouse model

[0074] The T2DM mouse model was established using C57BL / 6J mice, and the modeling method was based on the method of Han Caijing et al.

[0075] After 48 C57BL / 6J mice were adapted to feeding in an environment with a humidity of 50±10% and a temperature of 25℃ for 1 week, 8 mice were randomly selected as the control group (Control) and given normal feed. The other mice were fed with a high-sugar-high-fat feed (basic feed 58.5%, cholesterol 1%, egg yolk powder 10%, sucrose 20%, lard 10%, sodium cholate 0.5%). After 4 weeks, fasting but not water for 12 hours, each mouse was intraperitoneally injected with 1% STZ working solution (dissolved in 0.1mol / L sodiumcitrate buffer with pH=4.2-4.5) at a dose of 110mg / kg body weight, and the control group was given the same dose of sodium citrate buffer. After 3 days of STZ induction, blood samples were collected from the tail vein of mice that had fasted overnight to measure their fasting blood glucose (FBG). When the FBG level within 2 weeks was greater than 11.2mmol / L, it represented that the model was successfully established.

[0076] The mice with successful modeling were randomly divided into model group (Model), low-dose group (MELL, 50 mg / kg / d), medium-dose group (MELM, 250 mg / kg / d), high-dose group (MELH, 500 mg / kg / d) and metformin group (Met, 200 mg / kg / d), with 8 mice in each group. They were gavaged with MEL and Met at 14:00-15:30 PM every day. The normal group and model group were given the same amount of normal saline for 6 weeks.

[0077] (2) Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)

[0078] After 5 weeks of MEL gavage treatment, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed.

[0079] After fasting for 12 hours, mice were gavaged with 50% glucose solution at a dose of 1g / kg. Blood glucose levels of mice were measured at 0, 30, 60 and 120min after gavage, and OGTT curves were drawn. ITT and OGTT were separated by three days. Mice in each group fasted and deprived of water for 4h, and then injected with 0.5U / kg insulin on an empty stomach. Blood glucose levels were measured at 0, 30, 60 and 120min, respectively. Time was used as the horizontal axis, and the blood glucose value at 0min was converted to 100% as the vertical axis. The ITT curve was drawn using percentage data. The area under the curve (AUC) values ​​of OGTT and ITT experiments were calculated using formula (1), and the insulin resistance index (HOMA-IR) was calculated using formula (2).

[0080]

[0081] Wherein Bg in formula (1) refers to the blood glucose value at each time point; FBG in formula (2) refers to the fasting blood glucose value (mmol / L), and FINS refers to the fasting insulin level (mIU / L).

[0082] (3) Detection of biochemical indicators

[0083] After the experiment, mice were anesthetized by intraperitoneal injection of sodium pentobarbital 90 mg / kg, and blood was collected from the orbital vein. Serum samples were allowed to stand for 30 min and then centrifuged at 1000×g for 20 min at 4°C. The corresponding kits were used to measure the contents of serum glycosylated serum protein (GSP), free fatty acid content (FFA), blood lipid metabolism (TG, TC, HDL-C, LDL-C) and liver function (ALT, ALP and AST) related indicators in mice.

[0084] (4) Histochemical staining of mouse liver

[0085] Fresh liver tissues of mice in each group were placed in a pre-prepared 4% polyformaldehyde solution and fixed for more than 24 hours, embedded in paraffin, sliced, and stained with hematoxylin and eosin. The slices were dehydrated and sealed, and then placed under an Olympus IX73 inverted binocular optical microscope (Olympus, Japan) for 200 times photography observation.

[0086] (5) Detection of glycogen content and enzyme activities related to its synthesis and metabolism in mice

[0087] The activities of G6Pase, FBPase, PEPCK, HK, and PK in mouse serum and the glycogen content in liver and skeletal muscle were detected according to the operating procedures of the corresponding detection kits.

[0088] (6) Determination of the expression of proteins related to sugar synthesis and metabolism in the liver

[0089] 10% liver homogenate was prepared with normal saline, and the protein expression levels of p-IRS1, p-PI3K, p-AKT, GSK-3β, p-AMPK, FOXO1, G6Pase, PEPCK and β-actin in liver tissue were determined by Western Blot method.

[0090] data:

[0091] (1) In vitro hypoglycemic activity of MEL

[0092] Using acarbose and orlistat as controls, the effects of MEL on α-glucosidase, α-amylase and pancreatic

[0093] The in vitro inhibition ability of lipase activity was as follows Figure 5 As shown in A-5C. At low concentrations, the inhibition level of MEL on the three enzyme activities increased with the increase of MEL concentration, but when the concentration reached a certain level, the inhibition rate tended to stabilize or decrease. When the MEL concentration was 10 mg / mL, the highest inhibition rate for α-amylase was 58.08% ( Figure 5 B), and the inhibition rate of pancreatic lipase reached 66.85% ( Figure 5 A), and then the inhibition rate did not increase significantly when the concentration was increased. When the MEL concentration was 20 mg / mL, the inhibition rate of α-glucosidase reached 82.57% ( Figure 5 C). α-amylase and α-glucosidase can hydrolyze dietary carbohydrates into monosaccharides and promote their absorption and utilization. For diabetic patients, MEL can inhibit the activity of these enzymes to help reduce glucose absorption in patients, thereby lowering blood sugar. Inhibiting the activity of pancreatic lipase can effectively inhibit the hydrolysis and absorption of fat, thereby achieving the effect of lowering blood lipids and preventing the occurrence of obese diabetes. The results of in vitro inhibition experiments show that MEL has potential anti-diabetic effects. In order to better study its hypoglycemic effect, this study used a T2DM mouse model experiment.

[0094] (2) Effects of MEL on body weight, blood glucose and insulin in T2DM mice

[0095] T2DM mice were gavaged with Met and MEL, and the weight changes of the mice were recorded every week. The specific results are shown in Figure 6A. The weight of mice in the control group remained in a stable range, while the weight of T2DM mice in the model group continued to decrease, and after 6 weeks, the weight decreased by 8.99% compared with the initial weight. Diabetic mice have a disorder of glucose metabolism in the body, which leads to compensatory consumption of protein and fat for energy supply, which will cause the weight of mice in the late stage of diabetes to decrease. After 6 weeks of intervention with Met and MELL, MELM, and MELH, the weight of T2DM mice decreased by -5.31%, 4.38%, 3.44%, and 3.34% respectively compared with their initial weight, and the degree of weight loss was lower than that of the model group.

[0096] After intragastric administration of MEL and Met, the fasting blood glucose of mice in each group was measured once a week. Figure 6 As shown in B. The blood glucose of each MEL group decreased significantly in the first week of oral administration. After six weeks of continuous observation, the fasting blood glucose values ​​of mice in each MEL group decreased to 72.0% (MELL), 62.88% (MELM) and 54.73% (MELH) of the initial blood glucose value, respectively, and there was a dose-dependent relationship. GSP can reflect the blood glucose level of the body in the past 1 to 2 weeks and is not affected by temporary blood glucose concentration fluctuations. It provides a good indicator for the diagnosis of diabetes and the study of blood glucose control levels over a long period of time. Figure 6 In C, the GSP value of the model group was significantly higher than that of the normal group, while the GSP levels of the mice in the MEL groups were significantly lower than those in the model group, and there was no statistical difference compared with the control group (P>0.05). It can be seen that MEL has a certain control effect on the blood sugar of T2DM mice.

[0097] The oral glucose tolerance test (OGTT) simulates postprandial hyperglycemia and is used to evaluate the function of pancreatic β cells and the body's ability to regulate blood sugar. Within 30 minutes after oral administration of glucose, the blood sugar levels of mice in all groups increased rapidly, and then gradually decreased ( Figure 6 D). The AUC of OGTT in the model group was significantly higher than that in the normal group ( Figure 6 E), showing abnormal glucose tolerance. The AUC of each MEL group was significantly lower than that of the model group, indicating that it can improve the glucose tolerance of T2DM mice, and MELH has the best effect (P<0.01).

[0098] The decrease in insulin sensitivity is mainly manifested in the decrease in the efficiency of insulin in promoting glucose absorption and utilization, that is, blood sugar decreases relatively slowly, but increases rapidly. The effect of MEL on insulin resistance in T2DM mice can be observed through the insulin tolerance test (ITT). After insulin injection, the blood sugar of T2DM mice in the model group decreased slowly, and at 60 minutes, the blood sugar only dropped to 75.59% of the initial blood sugar, while the other groups dropped to less than 50% ( Figure 6 F); The AUC of ITT in the model group was significantly higher than that in the control group, while the AUC values ​​in the MEL group and the Met group were significantly lower than those in the model group ( Figure 6 G). The results showed that the T2DM mice in the model group showed significant IR or insulin insensitivity, while MEL improved the insulin tolerance of T2DM mice and alleviated the insulin resistance. At the same time, the serum insulin content of T2DM mice after 6 weeks of MEL intervention was measured, and it was found that their insulin content and insulin resistance index (HOMA-IR) were significantly lower than those in the model group ( Figure 6 H and 6I), further indicating that MEL can improve the IR phenomenon in T2DM mice.

[0099] (4) Effects of MEL on glycogen synthesis and glucose metabolism in T2DM mice

[0100] Glycogen is a storage form of glucose, mainly found in tissues and organs such as liver and muscle. The glycogen levels in the liver and muscle of each group of mice were as follows: Figure 7 A and Figure 7 As shown in B. The glycogen contents of the liver and skeletal muscle in the model group were 71.25 mg / gprot and 9.21 mg / gprot, respectively, which were 58.36% and 26.78% lower than those in the normal group; while the contents of both types of glycogen in the MELM group and the MELH group were higher than those in the model group, 107.47, 12.65 mg / gprot and 105.42, 13.83 mg / gprot, respectively, and the difference in muscle glycogen content was no longer significant compared with the control group (P>0.05), indicating that MEL can promote the synthesis of glycogen.

[0101] Glucose metabolism disorder is the root cause of diabetes. G6Pase, FBPase and PEPCK are three key enzymes in the gluconeogenesis pathway, which convert non-sugar substances into glucose, while HK and PK are key rate-limiting enzymes in the glucose catabolism pathway. Together, they play an important role in maintaining blood glucose homeostasis. By detecting the activity of these enzymes in mouse serum, the effect of MEL on glucose metabolism in T2DM mice was evaluated. The results are as follows: Figure 7 As shown in C-7F. Compared with the control group, the activities of PEPCK, FBPase, and G6Pase in the model group mice were significantly increased, while the activities of HK and PK were significantly decreased; while compared with the model group, the activities of PEPCK, FBPase, and G6Pase in the MEL group were significantly decreased, approaching normal levels, while the activities of HK and PK were significantly increased. This shows that MEL can improve the glucose metabolism disorder of T2DM mice to a certain extent, inhibit gluconeogenesis, and promote glycolysis, thereby reducing the blood sugar of diabetic mice.

[0102] (5) Effects of MEL on blood lipids and liver function in T2DM mice

[0103] Dyslipidemia is a risk factor for diabetes. Obvious symptoms of lipid metabolism disorders exist in the early stages of diabetes and become more severe as the disease progresses. The results of the effects of MEL on blood lipid indicators in T2DM mice are as follows: Figure 8 As shown in A. The mice in the model group showed obvious lipid metabolism disorders, which were manifested in the significant increase of TC, TG, and LDL-C content, and the significant decrease of HDL-C content. However, the use of MEL significantly reversed the changes of these indicators, and there was a certain dose-dependent relationship. When the lipid metabolism in the body is disturbed, the concentration of free fatty acids (FFA) will increase. The concentration of free fatty acids in the model group increased to 1.64mmol / L ( Figure 8 B), which was 1.6 times that of the control group, while that of the MELH group was 0.80mmol / L, which was nearly 50% lower than that of the model group. These results indicate that MEL can improve the abnormal lipid metabolism in diabetic mice.

[0104] The liver is an important organ involved in sugar and lipid metabolism in the body. Abnormal sugar and lipid metabolism in diabetic patients will increase the burden on the liver and affect its function. The activity of AST, ALT and ALP in serum is a characteristic indicator to determine whether liver function is abnormal. Abnormal liver function will lead to increased activity of ALT, AST and ALP in serum. Figure 8 As shown in C, the ALT, AST and ALP activities in the serum of the mice in the model group were significantly higher than those in the control group, which were 11.41, 10.59, and 368.53 U / L, respectively, increasing by 1.65 times, 1.67 times, and 5.47 times, respectively; while the AST and ALP activities in the serum of the mice in the MEL group were significantly lower than those in the model group, and the effects were almost at the same level as those in the Met group, and there was no statistical difference compared with the control group (P>0.05). The HE staining results of the liver group were observed ( Figure 8 D), it can be found that the liver tissue of the control group mice is intact, and the liver lobules are clearly visible; the cytoplasm of the model group is full of round fat droplets of different sizes, the hepatic cords are arranged in disorder, and the hepatic sinusoids are obviously dilated; after feeding with MEL, the accumulation of fat droplets in the liver tissue of mice decreased with the increase of MEL intervention concentration, and the morphology of hepatocytes was close to that of the control group. These results indicate that MEL has a certain protective effect on the liver function of T2DM mice.

[0105] (6) Effects of MEL on the expression of proteins related to glycogen synthesis and metabolism in the liver of T2DM mice

[0106] The phosphorylation levels of Tyr632 in IRS-1, Tyr458 in PI3K, and Ser473 in AKT and the expression level of GSK-3β protein were detected by western blot. Fig. 9AB). The p-IRS-1 / IRS-1 value in the liver of mice in the model group was 0.77. Compared with the control group, the phosphorylation level of Tyr632 was reduced, which inhibited the IRS-1 / PI3K / Akt signaling pathway. The phosphorylation levels of Tyr458 in PI3K and Ser473 in Akt were significantly reduced, which promoted the expression of GSK-3β, inhibited the activity of glycogen synthase, and increased blood sugar. Compared with the model group, the p-IRS-1 / IRS-1 values ​​of the MELM and MELH groups were 0.81 and 0.93, respectively. The phosphorylation level of Tyr632 was significantly enhanced, which activated the IRS-1 / PI3K / Akt signaling pathway, inhibited the expression of GSK-3β, and promoted glycogen synthesis, thereby reducing blood sugar levels.

[0107] MEL significantly improved fasting blood glucose in T2DM mice ( Figure 6 B), it is speculated that the AMPK signaling pathway is also involved in the hypoglycemic effect of MEL. Compared with the control group, the AMPK phosphorylation level in the liver of the model group mice was 0.43, a decrease of 57%, while the protein expression levels of FOXO1, PEPCE, and G6Pase increased by 25%, 65%, and 33%, respectively ( Fig. 9 CD), indicating that its gluconeogenesis is enhanced, leading to increased fasting blood glucose, which is consistent with Figure 5 The activity results of these enzymes shown in A-5C correspond to each other. After MEL feeding, the phosphorylation level of AMPK increased significantly, and there was a dose-dependent relationship, reaching 0.88 in the MELH group. The phosphorylation activation of AMPK and Akt jointly promoted the expression of GLUT4 protein and induced FOXO1 to transfer from the nucleus to the cytoplasm through phosphorylation, thereby leading to the decrease of G6Pase and the inhibition of PEPCK expression, inhibiting gluconeogenesis. The expression levels of FOXO1, G6Pase, PEPCE and other proteins in the MELH group were 1.01, 0.92, and 0.97, respectively, approaching the control group and significantly lower than the model group. This shows that MEL affects the AMPK signaling pathway, slows down the metabolic efficiency of gluconeogenesis, inhibits glucose production, and ultimately reduces blood glucose content.

[0108] GLUT4 is an insulin-sensitive glucose transporter. The expression of GLUT4 protein in the liver of T2DM mice in the model group decreased by 35% compared with the control group ( Fig. 9 EF), indicating that the glucose transport function of diabetic mice was inhibited, resulting in a decrease in the body's glucose utilization rate. The protein expression of GLUT4 in the liver of mice fed with Met and MEL for 6 weeks increased by about 1 times compared with the model group, indicating that MEL increased the transport of glucose in the liver, promoted the body's liver to consume glucose, and thus lowered blood sugar.

[0109] Embodiment 3:

[0110] Use of the melanoidins (hereinafter referred to as MELs) prepared in Example 1 in the preparation of products for regulating intestinal flora.

[0111] Animal Experimentation:

[0112] (1) Analysis of mouse intestinal flora and detection of intestinal short-chain fatty acid content

[0113] Five mice were randomly selected from each group to collect their intestinal contents, which were quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for testing. First, the total DNA of the samples was extracted for quality inspection. After passing the quality inspection, PCR amplification was performed, and quantitative sampling was performed using the Illlumina HiSeq4000 pair-end 2×150bp platform for 16S rDNA sequencing. The data were checked and plotted using the Prozeno gene cloud platform (https: / / www.gene Cloud.cn).

[0114] The determination of short-chain fatty acids was carried out by GC-MS / MS. Weigh 50 mg of the sample and place it in 0.2 ml of 5% (V / V) phosphoric acid solution for homogenization. Add 500 μL of the solution containing the internal standard, swirl and low-temperature ultrasonic for 5 minutes, centrifuge at 12000 r / min, take the supernatant, and use an Agilent 7890B gas chromatograph equipped with a DB-5MS 30m×0.25mm×0.25μm chromatographic column and a 7000D mass spectrometer for determination. Determination conditions: injection volume 2 μL, flow rate 1.2 mL / min, carrier gas is helium, injection port temperature 200°C, transfer line temperature 230°C, temperature program: keep at 90°C for 1 minute, increase to 100°C at a rate of 25°C / min, increase to 150°C at a rate of 20°C / min, keep for 0.6 minutes, increase to 200°C at a rate of 25°C / min, and keep for 0.5 minutes.

[0115] Data processing:

[0116] SPSS Statistics25.0, GraphPad Prism 8.0 and Origin 85 software were used to analyze and plot routine data; Western blot (WB) image data were mainly acquired using Image J. Each group of experiments was set up at least 3 times in parallel, and the data were expressed as mean ± standard deviation. The independent sample t test was used to analyze the differences between the two groups, and the mean differences between multiple groups were analyzed by one-way ANOVA. P < 0.05 indicated that the difference was statistically significant.

[0117] data:

[0118] More and more studies have shown that intestinal flora can prevent and alleviate metabolic diseases, and melanoidin is considered a potential prebiotic. Therefore, this study explored the changes in the composition of the intestinal flora in T2DM mice. In order to facilitate the study of the composition of each sample species, the samples were homogenized and then OTUs were clustered. A total of 279 OTUs were obtained. The number of common and unique OTUs in each group of experimental samples at the OTU level was analyzed by Venn diagram. Fig.10 As shown in A, the number of OTUs shared by the control group, model group, Met group and MELM group was 7, and the number of unique OTUs was 106, 16, 14 and 64, respectively; compared with the control group, the intestinal flora species of mice in the model group were significantly reduced, and MEL intervention could increase the number of intestinal flora species in diabetic mice.

[0119] In ecology, α diversity is usually used to measure the diversity of microbiota within an individual. This study used Chao1, Faith_pd, Shannon, and Simpson indices to evaluate the α diversity of intestinal microbiota ( Fig.10 B), the higher the index value, the higher the species diversity within the community. Compared with the control group, the Faith_pd, Shannon and Simpson indices of the model group tended to decrease, while the Chaol, Faith_pd and Shannon indices of the MELM group tended to increase, indicating that the richness of the intestinal flora of the mice in the model group was destroyed and the diversity was reduced, while the MEL intervention significantly increased the abundance and diversity of the intestinal flora in T2DM mice, balancing the disordered intestinal flora to a certain extent. Compared with α diversity, β diversity analysis focuses on comparing the diversity between different communities or ecosystems. In this study, principal coordinate analysis (PCoA) was used to perform β diversity analysis on the intestinal flora of each group of mice, and the results are shown in Figure 2. Fig.10 As shown in C, the clusters of the model group and the control group are far apart, indicating that the microbial communities between the two groups are quite different; compared with the model, the microbial communities of the Met group and the MELM group shifted toward the control group, indicating that MEL and Met significantly changed the distribution of intestinal flora in T2DM mice.

[0120] The relative abundance of species at the phylum level is as follows Fig.10 As shown in D, among the first 10 phyla, Firmicutes and

[0121] Bacteroidetes is the most abundant microbial phylum in the mouse intestine, followed by Verrucomicrobia, Proteobacteria, and Actinobacteria. Fig.11As shown in B, through the abundance histogram combined with the UPGMA clustering tree, it can be observed that the model group is far away from the control group, while the control group and the MELM group are clustered together, indicating that the colony composition of the control group and the MELM group is similar. Compared with the control group, the relative abundance of Firmicutes in the model group increased significantly ( Fig.10 F), the relative abundance of Bacteroidetes decreased significantly ( Fig.10 G), while the ratio of Firmicutes to Bacteroidetes (F / B) increased significantly ( Fig.10 H). After intervention with MEL and Met, the F / B ratio was significantly reduced (p<0.05). It can be seen that MEL can reduce the F / B ratio and play a role in regulating the structural imbalance of intestinal flora.

[0122] The community composition and dominant genus of intestinal microorganisms in each group of mice were further analyzed at the genus level. The relative abundance bar graph of the top 30 species in each experimental group ( Fig.11 A) UPGMA clustering tree at the phylum level combined with abundance histogram ( Fig.11 B)

[0123] It can be seen that the samples in the model group are farthest from the control group, indicating that the species composition of the model group is quite different from that of the control group. The distance between the MELM group and the control group is relatively close, indicating that the species composition and abundance of the two groups of samples are more similar, which is consistent with the previous analysis results at the phylum level. This shows that diabetes has changed the composition and structure of the intestinal flora of mice at the genus level, and after MEL intervention, the dysbiosis at the genus level has been improved. The top 10 bacterial genera with high relative abundance at the genus level that are different between groups were analyzed, and the results are as follows: Fig.11 C. Compared with the control group, the relative abundance of beneficial bacteria such as Ruminococcus, Coprococcus, Allobaculum, Bacteroides, and Bifidobacterium in the model group decreased significantly. Compared with the model group, the relative abundance of beneficial bacteria such as Lactobacillus, Coprococcus, and Ruminococcus in the MELM group increased significantly, suggesting that MEL may reverse intestinal flora imbalance by proliferating these beneficial bacteria.

[0124] The above description is only the preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for treating sesame residue by water substitution, characterized in that The following steps are involved: (1) Mixing fresh sesame residue obtained by the water-substitution sesame oil process with water and stirring evenly; (2) adjusting the pH value of the sesame residue aqueous solution to 9-10 and subjecting it to ultrasonic treatment; (3) adjusting the pH value of the sesame residue solution to 7-8, adding 35000-45000 U / g of protease Pronase E based on the dry weight of the sesame residue, and performing enzymolysis at 40-50° C. for 30-48 hours; (4) adjusting the pH value of the above solution to 7.5-8.5, adding 2500U / g-3500U / g of protease Alcalase 3.0T based on the dry weight of sesame residue, performing enzymolysis at 55-65°C for 3-5h, and heating to 90°C to inactivate the enzyme; (5) placing the enzymatic hydrolysis solution at a temperature below 10° C., then centrifuging it, sucking out the upper lipid layer, collecting the supernatant aqueous solution, and filtering it to obtain a filtrate; (6) diluting the filtrate, performing ultrafiltration separation using an ultrafiltration device, selecting a membrane component with a molecular weight cutoff of 5 KDa, collecting the filtrate ≥5 KDa, concentrating it by evaporation under reduced pressure, and freeze-drying it to obtain sesame residue melanin; (7) The filtrate with a content of ≤5 KDa was collected and desalted by nanofiltration through a 200 Da nanofiltration membrane. The polypeptide solution was concentrated by evaporation under reduced pressure and freeze-dried to obtain the sesame residue polypeptide.

2. The processing method according to claim 1, characterized in that: Step (1) Mix sesame residue and water in a w:v ratio of 1:

10.

3. The processing method according to claim 1, characterized in that: Step (2) is ultrasonically treated at 100 kHz for 30 min.

4. The processing method according to claim 1, characterized in that: In step (2) and step (4), a 0.5 mol / L NaOH solution is used to adjust the pH value; in step (3), a 0.5 mol / L HCl solution is used to adjust the pH value.

5. The processing method according to claim 1, characterized in that: The centrifugal parameters of step (5) are 5000 r / min for 15 min; and filtering is performed using four layers of gauze.

6. The processing method according to claim 1, characterized in that: Step (6) The filtrate is diluted 4 times and then ultrafiltered.

7. Use of the melanoidin prepared according to claim 1 in the preparation of hypoglycemic products.

8. Use of the melanoidin prepared according to claim 1 in the preparation of products for regulating intestinal flora.

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