Patent Application
20220323554
This application relates to nutritional functional foods and biological fermentation, and more particularly to a polysaccharide-peptide composite for lowering blood sugar, blood lipid, and glycated hemoglobin and a method of preparing the same.
In recent years, human life has been improved gradually, accompanied by the ascending incidence of diseases, such as diabetes, hyperlipidemia, and obesity, in elder people as well as younger people. Although in recent years, traditional Chinese medicine, western medicine, and health food aiming to prevent the above diseases have been available on the market, there are still many difficult problems needed to be solved. As a consequence, there is still a need for researchers to constantly develop new drugs and healthy foods for the prevention and treatment of diabetes, hyperlipidemia, and obesity.
Blood sugar and glycated hemoglobin are important indicators for evaluating the degree of diabetes, hyperlipidemia, and obesity. The blood sugar is a monosaccharide in the blood that is derived from carbohydrates in food, and usually merely refers to glucose. The blood sugar test results reveal the real-time blood sugar level. Glycated hemoglobin (HbA1c) is formed through non-enzymatic reaction between blood glucose and the N-terminal valine of hemoglobin in red blood cells via the cell membrane. The synthesis rate of HbA1c is proportional to the sugar concentration in the environment where the red blood cells are located. HbA1c is a product of the combination of hemoglobin in red blood cells and blood sugar. The combination of hemoglobin and blood sugar is irreversible, and will remain for about 120 days. Therefore, compared with blood sugar detection, the HbA1c detection is more clinically significant, which is known as the “gold standard” for diabetes monitoring, and thus often used as an indicator of the daily blood glucose control in clinical testing.
Hence, it is particularly important to provide a kind of food and drug that can more effectively lower the level of blood sugar and glycated hemoglobin.
To overcome the above deficiencies in the prior art, the present disclosure provides a polysaccharide-peptide composite for lowering blood sugar, blood lipid, and glycated hemoglobin and a method of preparing same. The method provided herein adopts a special extraction process for increasing the content of bitter melon polypeptide, combined with the use of other natural plant components to better lowering blood sugar and glycated hemoglobin.
Technical solutions of the present disclosure are described as follows.
In a first aspect, this application provides a polysaccharide-peptide composite, which is prepared from 20-25 parts by weight of oat dietary fiber powder, 10-15 parts by weight of konjac powder, 10-15 parts by weight of a corn silk, 20-30 parts by weight of bitter melon peptide (BMP) powder, 10-12 parts by weight of soybean polypeptide powder, 5-10 parts by weight of a mulberry leaf extract, 5-10 parts by weight of a gardenia fruit oil, 5-10 parts by weight of cocoa powder, 5-10 parts by weight of L-arabinose, 3-5 parts by weight of a Poria cocos extract, 5-10 parts by weight of a hawthorn extract, 1-2 parts by weight of nutritional yeast, 2-5 parts by weight of a pancreatin, and 5-8 parts by weight of xylitol.
In some embodiments, the nutritional yeast comprises selenium-rich yeast with a selenium concentration of 3000 ppm and chromium-rich yeast with a chromium concentration of 2000 ppm, with a weight ratio of 1:2.
In some embodiments, the pancreatin comprises trypsin, amylopsin and pancreatic lipase with a weight ratio of 1:2:2.
In some embodiments, the BMP powder is prepared through steps of:
In some embodiments, the buffer solution is a phosphate buffer solution prepared from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate.
In some embodiments, the gardenia fruit oil is extracted through steps of:
In a second aspect, this application also provides a method of preparing the polysaccharide-peptide composite, comprising:
This application has at least the following beneficial effects.
Through the extraction process including periodic warming, repeated digestion and multiple filtering, the content of BMP-protein in the BMP is equal to or greater than 30%. Moreover, the BMP is used in conjunction with other components, showing a significant effect in view of lowering blood sugar and glycated hemoglobin, and allowing users to get rid of side effects brought by the use of chemical hypoglycemic drugs.
Other advantages, objectives, and characteristics of the present application will be partly revealed by the following description and partly understood by those skilled in the art through the study and practice of the present application.
The present application will be further described in detail below to allow those skilled in the art to implement it according to the specification.
It should be understood that the terms used herein, such as “has”, “include” and “comprise”, do not match the existence or addition of one or more other components or the combination thereof.
It should be noted that if there are no special instructions, the following test method described in the examples are conventional methods, and described reagents and materials can be available commercially.
Provided herein was a polysaccharide-peptide composite, which was prepared from 20 parts by weight of oat dietary fiber powder, 11 parts by weight of konjac powder, 12 parts by weight of a corn silk, 20 parts by weight of bitter melon polypeptide (BMP) powder, 10 parts by weight of soybean polypeptide powder, 6 parts by weight of a mulberry leaf extract, 6 parts by weight of a gardenia fruit oil, 5 parts by weight of cocoa powder, 5 parts by weight of L-arabinose, 3 parts by weight of a Poria cocos extract, 6 parts by weight of a hawthorn extract, 1 parts by weight of nutritional yeast, 2 parts by weight of a pancreatin, and 5 parts by weight of xylitol. The nutritional yeast included selenium-rich yeast with a selenium concentration of 3000 ppm and chromium-rich yeast with a chromium concentration of 2000 ppm. The weight ratio of the selenium-rich yeast to the chromium-rich yeast was 1:2. The pancreatin included trypsin, amylopsin and pancreatic lipase with a weight ratio of 1:2:2.
The BMP powder was prepared through the following steps.
The temperature treatment was performed through the following steps.
The mixed system was heated to 45-55° C. (preferably, 50° C.), kept for 45-60 min (preferably, 55 min), cooled to 20-25° C. (preferably, 22° C.), and kept for 25-30 min (preferably, 28 min), and a volume of the mixed system was recorded at this time as a first volume V1. During the aforementioned heating and insulation processes, the water and acids in the mixed system were evaporated, resulting in the change in the solubility of acid, base and inorganic ions in the mixed system. Therefore, after the aforementioned heating and insulation processes, a first mixed liquid was added to the mixed system for compensation such that the mixed system could constantly stay at a good extraction environment, where the first mixed liquid contained deionized water and the buffer solution with a weight ratio of 4:1; and a volume of the first mixed liquid was calculated by: (V0-V1)*0.6. After being added with the first mixed liquid, the mixed system was heated to 60-75° C. (preferably, 65° C.), kept at 60-75° C. for 60-75 min (preferably, 65 min), cooled to 45-55° C. (preferably, 50° C.), and kept at 45-55° C. for 30-35 min (preferably, 32 min), and a volume of the mixed system was recorded at this time as a second volume V2. A second mixed liquid was added to the mixed system, where the second mixed liquid contained deionized water and the buffer solution with a weight ratio of 3:1; and a volume of the second mixed liquid was calculated by: (Vt-V2)*0.75. In this case, the temperature of heating and insulation was higher, so the evaporation of water and acids in the mixed system was greater. As a consequence, the addition volume of the second mixed liquid was increased (to 75%), and the proportion of the buffer solution in the second mixed liquid was also increased. After being added with the second mixed liquid, the mixed system was heated to 80-90° C. (preferably, 85° C.), kept at 80-90° C. for 75-85 min (preferably, 80 min), cooled to 60-75° C. (preferably, 70° C.), and kept at 60-75° C. for 35-45 min (preferably, 40 min).
In this step, through periodic heating and insulation, cellular structure (such as cell wall) of components were repeatedly impacted and destroyed under different temperature environment. And at the same time, after heating and insulation, the reaction system was supplemented with corresponding proportion of water and the buffer solution for compensating the evaporation of water and acids, so that the reaction system was always in a great extraction environment to achieve the best extraction effect.
Primary Enzymatic Hydrolysis
The bitter melon extract was regulated to pH 7.5-8.5 (preferably, 7.0). Trypsin was added to the extract followed by heating to 35-40° C. (preferably, 37° C.) under stirring at 80-100 r/min and keeping at 35-40° C. for 45-60 min (preferably, 55 min) to obtain a first enzymatic hydrolysis system, where the trypsin was 5% by weight of the bitter melon extract.
Secondary Enzymatic Hydrolysis
After the first enzymatic hydrolysis system was cooled to 20-25° C., the first enzymatic hydrolysis system was regulated to pH 3.0-4.0 (preferably, 3.5). Pectinase was added to the first enzymatic hydrolysis system followed by heating to 45-55° C. (preferably, 50° C.) under stirring at 80-100 r/min and keeping at 45-55° C. for 40-60 min (preferably, 50 min) to obtain a second enzymatic hydrolysis system, where the pectinase was 3% by weight of the first enzymatic hydrolysis system.
Third Enzymatic Hydrolysis
After the second enzymatic hydrolysis system was cooled to 20-25° C., the second enzymatic hydrolysis system was regulated to pH 4.5-5.0 (preferably, 4.7). Cellulase was added to the second enzymatic hydrolysis system followed by heating to 55-60° C. (preferably, 58° C.) under stirring at 80-100 r/min and keeping at 55-60° C. for 30-45 min (preferably, 35 min) to obtain the enzymatic hydrolysis product, where the cellulase was 2% by weight of the second enzymatic hydrolysis system.
As the components used herein were plant components, of which cellular structure contained cell walls. Therefore, in this step, through the use of different enzymes and enzymatic conditions under different stages for complete enzymatic hydrolysis of cell walls, cellulose and pectin of the cell walls were destroyed, which facilitated active ingredients (such as BMP protein) in the cell walls to release fully to enhance the extraction efficiency.
The first supernatant was filtered with diatomite at 0.2-0.3 MPa (preferably, 0.25 MPa) to obtain a first filtrate. The first filtrate was added with activated carbon followed by standing for 45-50 min, centrifugation, and residue removal to obtain a second supernatant, where the activated carbon was 4-5% by weight of the first filtrate.
Through the adsorption treatment by activated carbon and diatomite, the pigment, suspended particles and colloid in the enzymatic hydrolysate of BMP were removed to offer a final product with high purity.
The second filtrate was filtered with a spiral wound ultrafiltration membrane having a cut-off molecular weight of 100-200 kDa at 45-50° C. (preferably, 48° C.) to remove macromolecular impurities to obtain a third filtrate, where the spiral wound ultrafiltration membrane was used in parallel with two membranes.
The third filtrate was concentrated with a spiral reverse-osmosis membrane with having a cut-off molecular weight of 150-1000 kDa below 40° C. to remove water, residual inorganic salts and small molecule impurities to obtain a BMP concentrate, where the solid content of the BMP concentrate was equal to or more than 40%; and the spiral reverse-osmosis membrane was a high-pressure concentration membrane, made of polysulfone (PS), polyethersulfone (PFS), or other composite material film, and used in parallel with four membranes.
In this step, the multilayer membrane separation and purification technology was used to separate and purify the BMP protein. The low concentration temperature used herein effectively guaranteed the natural activity and high content of the BMP protein.
Moreover, gardenia fruit was abundant, cheap, and had a variety of functions, and has played a more and more important role in the modern food industry. Specifically, gardenia fruit mainly contained flavonoids, iridoids, crocin, pectin, tannins, polysaccharides, crocetin, and volatile oil. Among them, flavonoids had an auxiliary therapeutic effect on hypertension and other diseases, and could reduce blood pressure and blood sugar. Hence, this example also provided a method for extracting the gardenia fruit oil, which included the following steps.
Primary Enzymatic Hydrolysis
The mixture was added with trypsin and a permeability-regulating fluid, where the trypsin was 5% by weight of the gardenia fruit powder; and the permeability-regulating fluid was 45-55% (preferably, 50%) by weight of the gardenia fruit powder. The mixture was regulated to pH 6.5-7.5 (preferably, 7.0), followed by heating to 42-45° C. (preferably, 43.5° C.) under stirring, and keeping at 42-45° C. for 30-45 min (preferably, 35 min) to obtain a first enzymatic hydrolysis system, where the permeability-regulating fluid consisted of an acid solution, glycerol, sodium chloride, and lysozyme in a weight ratio of 1:(0.7-1.0):(0.02-0.05):(0.03-0.06), preferably, 1:0.8:0.03:0.04; and the acid solution was a citric acid-sodium citrate buffer solution with a pH of 6-7.
Secondary Enzymatic Hydrolysis
After the first enzymatic hydrolysis system was cooled to 20-25° C., the first enzymatic hydrolysis system was regulated to pH 3.5-4.5 (preferably, 4.0). Pectinase was added to the first enzymatic hydrolysis system, followed by heating to 50-60° C. (preferably, 55° C.) under stirring, and keeping at 50-60° C. for 30-35 min (preferably, 32 min) to obtain a second enzymatic hydrolysis system, where the pectinase was 4% by weight of the first enzymatic hydrolysis system.
Tertiary Enzymatic Hydrolysis
After the second enzymatic hydrolysis system was cooled to 20-25° C., the second enzymatic hydrolysis system was regulated to pH 4.0-5.5 (preferably, 5.0). Cellulase was added to the second enzymatic hydrolysis system, followed by heating to 50-65° C. (preferably, 60° C.) under stirring, and keeping at 50-65° C. for 25-35 min (preferably, 30 min) to obtain the enzymatic hydrolysis product, where the cellulase was 3.5% by weight of the second enzymatic hydrolysis system.
Example 2 was different from Example 1 in the composition of the polysaccharide-peptide composite. In Example 2, the polysaccharide-peptide composite was prepared from 25 parts by weight of oat dietary fiber powder, 15 parts by weight of konjac powder, 14 parts by weight of a corn silk, 28 parts by weight of BMP powder, 11 parts by weight of soybean polypeptide powder, 9 parts by weight of a mulberry leaf extract, 8 parts by weight of a gardenia fruit oil, 9 parts by weight of cocoa powder, 10 parts by weight of L-arabinose, 5 parts by weight of a Poria cocos extract, 8 parts by weight of a hawthorn extract, 2 parts by weight of nutritional yeast, 4.5 parts by weight of pancreatin, 7.5 parts by weight of xylitol, and 200 parts by weight of water.
Example 3 was different from Example 1 in the composition of the polysaccharide-peptide composite. In Example 3, the polysaccharide-peptide composite was prepared from 22 parts by weight of oat dietary fiber powder, 13 parts by weight of konjac powder, 12.5 parts by weight of a corn silk, 25 parts by weight of a BMP powder, 11 parts by weight of soybean polypeptide powder, 8 parts by weight of a mulberry leaf extract, 8 parts by weight of a gardenia fruit oil, 8 parts by weight of cocoa powder, 7 parts by weight of L-arabinose, 4 parts by weight of a Poria cocos extract, 8 parts by weight of a hawthorn extract, 1.5 parts by weight of a nutritional yeast, 3.5 parts by weight of a pancreatin, 6.5 parts by weight of xylitol, and 200 parts by weight of water.
Molecular weight detection results of BMP were as follows.
The BMP of Comparative Example 1 was prepared according to Example 1 of Chinese Patent Application No. 201710832199.8. The BMP samples of Examples 1-3 and Comparative Example 1 were detected by using a high-performance gel filtration chromatography to acquire the molecular weight of BMP and distribution range thereof. The results were shown in Table 1.
In the method for preparing the BMP provided herein, through periodic heating and insulation, cellular structure (such as cell wall) of components were repeatedly impacted and destroyed under different temperature environments. And at the same time, after heating and insulation, the reaction system was supplemented with a corresponding proportion of water and the buffer solution for compensating the evaporation of water and acids, so that the reaction system was always in a great extraction environment. Moreover, through further periodic heating, repeated enzymatic hydrolysis, and multilayer membrane separation and purification technology, the BMP protein in the obtained BMP powder was greater than 30%. In addition, there was no soybean polypeptide protein and other non-BMP proteins in the obtained extracts. As can be seen from Table 1, the BMP fragment within the range of 5000-7000 Da prepared herein was close to 30%, which was exactly the fragment with the function of regulating blood sugar and the fragment size closest to the molecular weight of insulin. Therefore, the obtained BMP extract had a good effect on regulating blood sugar metabolism, especially could greatly improve the binding ability of insulin receptors and lowering blood sugar.
Detection results of gardenia fruit oil were as follows.
The gardenia fruit oil of Comparative Example 2 was prepared according to Example 1 of Chinese Patent Application No. 201110321487.X. The gardenia fruit oil samples of Examples 1-3 and Comparative Example 2 were detected to acquire the content of crocin, chlorogenic acid, flavone and geniposide. Those components were mainly effective components for lowering blood sugar and blood lipid. The results were shown in Table 2.
In the method provided herein, through the use of different enzymes and enzymatic conditions under different stages for complete enzymatic hydrolysis of cell walls of the gardenia fruit, cellulose and pectin of the cell walls were destroyed. And at the same time, acid solutions, glycerol, sodium chloride, and lysozyme could alter the permeability of cell wall or cell membrane by altering their structure. Therefore, by employing a permeability regulatory solution to regulate the permeability of cell membrane and/or cell wall, the structure of cell membrane and/or cell wall could be destroyed, which facilitated active ingredients (such as crocin, and flavone) to release fully to fulfil the hypoglycemic effect.
Provided was a method of preparing the polysaccharide-peptide composite, which included the following steps.
A raw material (Poria cocos, mulberry leaf or hawthorn) was soaked in water for 12-15 h (preferably, 13 h), heated to boiling for 1-2 h, and filtrated to obtain a first filtrate and a first filter residue, where a weight ratio of the raw material to the water was 1:(8-10).
The first filter residue was dried, soaked with a 60% (v/v) ethanol solution with for 1-2 h (preferably, 1.5 h) followed by heating to 65-75° C. (preferably, 70° C.), extraction for 1.5-2 h, standing at 8° C. for 24 h, and filtration to obtain a second filtrate and a second filter residue, where a weight ratio of the dried first filter residue to the ethanol solution was 1:(6-8) (preferably, 1:7). During the extraction, stirring was performed once every 10 min at a rate of 200-300 rpm.
The second filter residue was soaked with a 60% (v/v) ethanol solution for 6-7 h (preferably, 6.5 h) followed by heating to 65-75° C. (preferably, 70° C.), extraction for 2.5-3 h, standing at 8° C. for 24 h, and filtration to obtain a third filtrate and a third filter residue, where a weight ratio of the dried second filter residue to the ethanol solution was 1:(8-10) (preferably, 1:9). During the extraction, stirring was performed once every 10 min at a rate of 200-300 rpm. The first filtrate, the second filtrate, and the third filtrate were combined to obtain the Poria cocos extract, the mulberry leaf extract, or the hawthorn extract.
Therefore, through the repeated extraction of mulberry leaves and hawthorn raw materials, the active ingredients (such as mulberry leaf polysaccharides, mulberry leaf alkaloids, mulberry flavonoids, hawthorn polysaccharides, etc.) could be fully released. In addition, the filter residue was re-extracted after the first extraction, which maximized the utilization rate of raw materials, so as to obtain higher purity of raw materials, ensuring that active ingredients could effectively exert hypoglycemic effects.
Provided herein was a polysaccharide-peptide composite, which was prepared from 20 parts by weight of oat dietary fiber powder, 11 parts by weight of konjac powder, 12 parts by weight of a corn silk, 20 parts by weight of a BMP powder, 10 parts by weight of soybean polypeptide powder, 6 parts by weight of a mulberry leaf extract, 6 parts by weight of a gardenia fruit oil, 5 parts by weight of cocoa powder, 5 parts by weight of L-arabinose, 3 parts by weight of a Poria cocos extract, 6 parts by weight of a hawthorn extract, 1 parts by weight of nutritional yeast, 2 parts by weight of a pancreatin, and 5 parts by weight of xylitol. The nutritional yeast included a selenium-rich yeast. The pancreatin included trypsin, amylopsin and pancreatic lipase with a weight ratio of 1:2:2.
The bitter melon polypeptide powder was prepared through the following steps.
The temperature treatment was performed through the following steps.
The mixed system was subjected to heating to 50° C., keeping at 50° C. for 55 min, cooling to 22° C., and keeping at 22° C. for 28 min, and a volume of the mixed system was recorded at this time as a first volume V1. During the aforementioned heating and insulation processes, the water and acids in the mixed system were evaporated, resulting in the change in the solubility of acid, base and inorganic ions in the mixed system. Therefore, after the aforementioned heating and insulation processes, a first mixed liquid was added to the mixed system for compensation such that the mixed system could constantly stay at a good extraction environment, where the first mixed liquid contained deionized water and the buffer solution with a weight ratio of 4:1; and a volume of the first mixed liquid was calculated by: (Vt-V1)*0.6. After being added with the first mixed liquid, the mixed system was subjected to heating to 65° C., keeping at 65° C. for 65 min, cooling to 50° C., and keeping at 50° C. for 30 min, and a volume of the mixed system was recorded at this time as a second volume V2. A second mixed liquid was added to the mixed system, where the second mixed liquid contained deionized water and the buffer solution with a weight ratio of 3:1; and a volume of the second mixed liquid was calculated by: (Vt-V2)*0.75. In this case, the temperature of heating and insulation was higher, so the evaporation of water and acids in the mixed system was greater. As a consequence, the addition volume of the second mixed liquid was increased (to 75%), and the proportion of the buffer solution in the second mixed liquid was also increased. After being added with the second mixed liquid, the mixed system was subjected to heating to 85° C. for 80 min, and cooling to 70° C. for 40 min.
In this step, through periodic heating and insulation, cellular structure (such as cell wall) of components were repeatedly impacted and destroyed under different temperature environment. And at the same time, after heating and insulation, the reaction system was supplemented with corresponding proportion of water and the buffer solution for compensating the evaporation of water and acids, so that the reaction system was always in a great extraction environment to achieve the best extraction effect.
Primary Enzymatic Hydrolysis
The bitter melon extract was regulated to pH 7.0. Trypsin was added to the extract followed by heating to 37° C. under stirring at 80-100 r/min and keeping at 37° C. for 55 min to obtain a first enzymatic hydrolysis system, where the trypsin was 5% by weight of the extract.
Secondary Enzymatic Hydrolysis
After the first enzymatic hydrolysis system was cooled to 20-25° C., the first enzymatic hydrolysis system was regulated to pH 3.5. Pectinase was added to the first enzymatic hydrolysis system followed by heating to 50° C. under stirring at 80-100 r/min and keeping at 50° C. for 50 min to obtain a second enzymatic hydrolysis system, where the pectinase was 3% by weight of the first enzymatic hydrolysis system.
Tertiary Enzymatic Hydrolysis
After the second enzymatic hydrolysis system was cooled to 20-25° C., the second enzymatic hydrolysis system was regulated to pH 4.7. Cellulase was added to the second enzymatic hydrolysis system followed by heating to 58° C. under stirring at 80-100 r/min and keeping at 58° C. for 35 min to obtain an enzymatic hydrolysis product, where the cellulase was 2% by weight of the second enzymatic hydrolysis system.
As the components used herein were plant components, of which cellular structure contained cell walls. Therefore, in this step, through the use of different enzymes and enzymatic conditions under different stages for complete enzymatic hydrolysis of cell walls, cellulose and pectin of the cell walls were destroyed, which facilitated the full release of active ingredients (such as BMP protein) in the cell walls to enhance the extraction efficiency.
The first supernatant was filtered with diatomite at 0.25 MPa to obtain a first filtrate. The first filtrate was added with activated carbon followed by standing for 45-50 min, centrifugation, and residue removal to obtain a second supernatant, where the activated carbon was 4-5% by weight of the first filtrate.
Through the adsorption treatment by activated carbon and diatomite, the pigment, suspended particles and colloid in the enzymatic hydrolysate of BMP were removed to offer a final product with high purity.
The second filtrate was filtered with a spiral wound ultrafiltration membrane having a cut-off molecular weight of 100-200 kDa at 45-50° C. to remove macromolecular impurities to obtain a third filtrate, where the spiral wound ultrafiltration membrane was used in parallel with two membranes.
The third filtrate was concentrated with a spiral reverse-osmosis membrane with having a cut-off molecular weight of 150-1000 kDa below 40° C. to remove water, residual inorganic salts and small molecule impurities to obtain a BMP concentrate, where the solid content of the BMP concentrate was equal to or more than 40%; and the spiral reverse-osmosis membrane was a high-pressure concentration membrane, made of polysulfone (PS), polyethersulfone (PFS), or other composite material film, and used in parallel with four membranes.
In this step, the multilayer membrane separation and purification technology was used to separate and purify the BMP protein. The low concentration temperature used herein effectively guaranteed the natural activity and high content of the BMP protein.
Moreover, gardenia fruit was abundant, cheap, and had a variety of functions, and has played a more and more important role in the modern food industry. Specifically, gardenia fruit mainly contained flavonoids, iridoids, crocin, pectin, tannins, polysaccharides, crocetin, and volatile oil. Among them, flavonoids had an auxiliary therapeutic effect on hypertension and other diseases, and could reduce blood pressure and blood sugar. Hence, this example also provided a method for extracting the gardenia fruit oil, which included the following steps.
Primary Enzymatic Hydrolysis
The mixture was added with trypsin and a permeability-regulating fluid, where the trypsin was 5% by weight of the gardenia fruit powder; and the permeability-regulating fluid was 50% by weight of the gardenia fruit powder. The mixture was regulated to pH 7.0, followed by heating to 43.5° C. under stirring, and keeping at 43.5° C. for 30-45 min (preferably, 35 min) to obtain a first enzymatic hydrolysis system, where the permeability-regulating fluid consisted of an acid solution, glycerol, sodium chloride, and lysozyme in a weight ratio of 1:0.8:0.03:0.04; and the acid solution was a citric acid-sodium citrate buffer solution with a pH of 6-7.
Secondary Enzymatic Hydrolysis
After the first enzymatic hydrolysis system was cooled to 20-25° C., the first enzymatic hydrolysis system was regulated to pH 4.0. Pectinase was added to the first enzymatic hydrolysis system, followed by heating to 55° C. under stirring, and keeping at 55° C. for 32 min to obtain a second enzymatic hydrolysis system, where the pectinase was 4% by weight of the first enzymatic hydrolysis system.
Third Enzymatic Hydrolysis
After the second enzymatic hydrolysis system was cooled to 20-25° C., the second enzymatic hydrolysis system was regulated to pH 5.0. Cellulase was added to the second enzymatic hydrolysis system, followed by heating to 60° C. under stirring, and keeping at 50-65° C. for 30 min to obtain the enzymatic hydrolysis product, where the cellulase was 3.5% by weight of the second enzymatic hydrolysis system.
Evaluation tests for lowering glycated hemoglobin (HbA1c) were described below.
Healthy male Sprague-Dawley (SD) rats with a body weight of 185-225 g were selected and fed adaptively for one week. After adaptive feeding, the SD rats were randomly divided into six groups each for 20 rats. One of the six groups was selected as a blank control group, and fed with ordinary feed. The rest five groups were subjected to intragastric administration with high-lipid emulsion for 10 mL/kg per day for 1 month. After the last feeding, rats were fasted for 12 h expect for water. The rats fed with high-lipid emulsion were subjected to disposable intraperitoneal injection with streptozotocin STZ solution for 25 mg/kg to establish a diabetic rat model. After streptozotocin injection for 72 h, the rats were fasted for 12 h expect for water and was taken blood samples from the tail to detect fasting blood sugar. The rat with a fasting blood glucose>10.0 mmol/L was considered as a successful model. The blank control group was intraperitoneally injected with citric acid-sodium citrate buffer solution.
The rats that were successfully modeled were randomly divided into model group, positive control group, low-dose group, medium-dose group and high-dose group according to blood sugar and body weight, with 20 rats in each group. The polysaccharide-peptide composite having functions of lowering blood sugar and glycated hemoglobin (denoted as polysaccharide-peptide composite) prepared in Examples 1-3 were diluted with water into intragastric solution, which were fed to the low-dose group for 1.5 g/kg, the medium-dose group for 2.5 g/kg and the high-dose group for 4.0 g/kg, respectively. While the positive control group was given metformin hydrochloride solution. Each group was given intragastric administration once per day for 8 weeks. Before detection, rats were fasted for 12 h expect for water. After the last feeding for 2 h, the urine was collected and tested for HbA1c content and blood lipid content. The results were shown in Table 3. The above groups were fed with different feeds and not restricted for drinking water.
2.52 ± 0.24#*
#P < 0.05,
## P < 0.01.
As shown in Table 3, compared with the blank control group, the contents of blood lipid and HbA1c in the model group were significantly increased (both increased by 50%), indicating the successful modeling of diabetic rats. Compared with model group, the contents of blood lipid and HbA1c in the positive control group and drug groups (high, medium and low-dose groups) were decreased, and presented significant differences among them. Specifically, the contents of HbA1c and blood lipid in high-dose group declined more obvious (about 40% and 18%, respectively), indicating that the polysaccharide-peptide composite prepared in this application had good therapeutic effect on diabetes.
Evaluation tests for lowering blood sugar were described below.
100 of healthy male rats were selected, numbered and fed adaptively in normal environment for 2 weeks, and free to eat and drink.
After adaptive feeding, the rats were randomly divided into 5 groups with 20 rats in each group. Only one group was selected as the blank control group, the rest groups were subjected to disposable intraperitoneal injection with 5% streptozotocin STZ solution for 200 mg/kg. If there were coma among those rats within 2 h, glucose sugar solution was fed until awake. Two days later, the fasting blood sugar was detected. The rat with a blood sugar of more than 10 was selected as a diabetic animal model.
The rats that were successfully modeled were randomly divided into model group, positive control group, low-dose group, medium-dose group and high-dose group according to blood sugar and body weight, with 20 rats in each group. The polysaccharide-peptide composites prepared in Examples 1-3 were diluted with water into intragastric solutions, which were fed to the low-dose group for 1.0 g/kg, the medium-dose group for 2.0 g/kg and the high-dose group for 3.0 g/kg, respectively. While the positive control group was given metformin hydrochloride solution. Each group was given intragastric administration once per day for 15 days. After that, rats were fasted for 8 h to measure fasting blood sugar and urine sugar. The results were shown in Table 4. The above groups were fed with different feeds and not restricted for drinking water.
During the experiment, rats of each group were intraperitoneally injected every day for 15 consecutive days. After 45 days, rats were fasted for 8 h to measure fasting blood sugar. At the end of the experiment, the final blood sugar content was recorded.
Table 4 showed that compared with the model group, the contents of urine sugar and fasting blood sugar of the rats in the positive control group and drug groups (high, medium and low-dose group) declined, and presented significant differences among them. Specifically, compared with the model group, the contents of urine sugar in the high, medium and low-dose groups decreased by 9%, 32% and 56%, respectively, and the content of fasting blood sugar decreased by 21%, 39% and 52%, respectively, indicating that high-dose polysaccharide-peptide composite significantly reduced contents of urine sugar and fasting blood sugar.
Evaluation tests for lowering blood lipid were described below.
60 of healthy male rats were selected, numbered and fed adaptively in normal environment for 2 weeks, and free to eat and drink. After the adaptive feeding, the rats were randomly divided into 6 groups with 10 rats in each group, consisting of a blank control group, a high-lipid model group, a positive treatment group, a high-dose group, a medium-dose group and a low-dose group. Except the blank control group, the other groups were given high lipid diet. The polysaccharide-peptide composites prepared in this application and simvastatin were respectively diluted. The positive control group was given simvastatin solution by intragastric administration for 50 mg/kg bw d, and the high, medium and low-dose groups were given polysaccharide-peptide solutions for 100 mg/kg bw d, 50 mg/kg bw d and 25 mg/kg bw d, respectively. Other groups were intragastric with corresponding volume of distilled water. The treatment period lasted for 4 weeks. After continuous feeding for 10 weeks, and after the last feeding, rats were fasted excepted for water for 10 h, and total cholesterol (TC) and triglyceride (TG) were measured by aortic blood samples, and the results were shown in Table 5.
2.59 ± 0.24#*
#P < 0.05,
##P < 0.01.
As can be seen from Table 5, compared with the high-lipid group, the contents of TC and TG of rats in the drug groups (high-dose, medium-dose and low-dose groups) were significantly decreased, proving that the polysaccharide-peptide composite prepared in this application had a good effect on lowering blood lipid.
It should be noted that the technical solutions in the above Examples 1-5 can be combined arbitrarily, and the technical solutions obtained after combination shall fall within the protection scope of this application.
In summary, through the extraction process including periodic warming, repeated digestion and multiple filtering, the content of bitter melon peptide-protein in the bitter melon peptide is equal to or greater than 30%. Moreover, bitter melon peptide is used in conjunction with other components, showing a significant effect in view of lowering blood sugar and glycated hemoglobin, and allowing users to get rid of side effects brought by the use of chemical hypoglycemic drugs.
The number of devices and the scale of processing shown herein are intended to simplify the description of the present disclosure. The application, modification and variation of the present disclosure are obvious to those skilled in the art.
Described above are merely illustrative of the disclosure, and are not intended to limit the disclosure. It should be understood that any modifications, replacements and variations made by those skilled in the art without departing from the spirit and scope of the disclosure should fall within the scope of the disclosure defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911020502.X | Oct 2019 | CN | national |
This application is a continuation of International Patent Application No. PCT/CN2020/123563, filed on Oct. 26, 2020, which claims the benefit of priority from Chinese Patent Application No. 201911020502.X, filed on Oct. 25, 2019. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/123563 | Oct 2020 | US |
| Child | 17728965 | US |
