CN116217747A - Moringa oleifera leaf polysaccharide with high purity and high activity, and preparation method and application thereof - Google Patents
Moringa oleifera leaf polysaccharide with high purity and high activity, and preparation method and application thereof Download PDFInfo
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- CN116217747A CN116217747A CN202310145439.2A CN202310145439A CN116217747A CN 116217747 A CN116217747 A CN 116217747A CN 202310145439 A CN202310145439 A CN 202310145439A CN 116217747 A CN116217747 A CN 116217747A
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- polysaccharide
- moringa oleifera
- oleifera leaf
- polysaccharide extract
- extract
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention discloses a high-purity and high-activity moringa oleifera leaf polysaccharide, and a preparation method and application thereof. The method is characterized in that based on a moringa oleifera leaf polysaccharide extract (total sugar content is more than 50 percent, yield is more than 12 percent) with the function of adjusting the absorption of glycolipid, and a preparation method and application thereof (CN 202210833825.6), non-organic reagents, non-strong acid and non-chromatographic process flows such as double protease enzymolysis, double resin adsorption, ultrafiltration and dialysis are adopted to obtain the moringa oleifera leaf polysaccharide with high purity and high activity. The moringa oleifera leaf polysaccharide has the total sugar content of more than 80%, the purity of more than 80% and the yield of more than 2.5%, can effectively block the diffusion of glucose, adsorb cholate, inhibit the solubility of cholesterol in micelles, has good effect of regulating the absorption of glycolipid, has light color, and is suitable for various food systems. The invention has simple purification process, the whole process flow can meet the food-grade requirement, and the invention is suitable for the fields of common foods, health care products and the like.
Description
Technical Field
The invention belongs to the field of finish machining and high-value processing of moringa leaves, and particularly relates to a moringa leaf polysaccharide with high purity and high activity, and a preparation method and application thereof.
Background
With the development of modern society, people suffering from obesity and hyperglycemia increase year by year, and the demands of people for plant source nutrient substances and blood glucose and lipid reducing functional factors are increasing. The moringa leaves contain rich saccharides, proteins and other nutrients, and the polysaccharides are main active ingredients for playing roles of reducing blood sugar and controlling lipid, but the research on a green efficient preparation method capable of improving the purity and activity of the moringa leaf polysaccharides is less at present.
Unlike most plants, the protein content in the moringa leaves is close to 30%, and the protein content in the extract is always higher, so that the purification of moringa leaf polysaccharide is difficult, multiple methods are often needed to be combined, and the technical bottleneck exists. The patent application of the team discloses a moringa oleifera leaf polysaccharide extract with the effect of regulating the absorption of glycolipid, and a preparation method and application thereof, wherein the moringa oleifera leaf polysaccharide extract with the effect of regulating the absorption of glycolipid is obtained by adopting processes of liquid nitrogen crushing, cellulase enzymolysis, high-temperature extraction, yeast fermentation, ethanol precipitation, re-dissolution and the like, and has the advantages of having the total sugar content of 50 percent, having good activity of controlling the glucose and reducing the lipid, but still having a lifting space for further purification. The patent application of the team discloses a moringa oleifera leaf polyphenol-polysaccharide composition for reducing sugar and lipid, a preparation method and application thereof, wherein the moringa oleifera leaf polyphenol and polysaccharide are extracted and refined in sections by taking dry moringa oleifera leaves as raw materials, and the moringa oleifera leaf polysaccharide with the total sugar content of more than 65% and stronger sugar and lipid-reducing activity is obtained; the method adopts the methods of dephenolizing and extracting polysaccharide, and then adopts various impurity removing methods such as proteinase enzymolysis, ultrafiltration, ethanol precipitation, low-temperature dialysis and the like to purify the polysaccharide, so that the purity of the polysaccharide can be improved by combining the methods, the purification process flow is complex, the purity of the polysaccharide is not high, and the polysaccharide needs to be further optimized. In addition, research groups prepare high-purity anti-inflammatory moringa oleifera leaf polysaccharide (patent application number: 201810415879.4) by separating and purifying through a sevage method, low-temperature dialysis and anion exchange column chromatography, but the high-purity polysaccharide extraction quality of each batch is less than 15mg due to the limitation of sample loading, and the method belongs to a conventional plant polysaccharide purification process, is complex in operation, relates to organic hazardous chemicals, has low purification efficiency and is not beneficial to the green large-scale preparation of the high-purity moringa oleifera leaf polysaccharide.
In summary, the existing purification method of moringa oleifera leaf polysaccharide still has the following problems: (1) The polysaccharide in the moringa leaves is a main active ingredient, but the purity of the polysaccharide is difficult to break through the upper limit due to the fact that the moringa leaves contain a large amount of proteins, polyphenols and other substances; (2) Although the traditional purification method can obtain high-purity polysaccharide, the purification efficiency is low, the yield is low, the method is difficult to popularize and apply in the food industry, and the technology for purifying the moringa oleifera leaf polysaccharide in a green scale is deficient; (3) The traditional purification method does not consider the purity of moringa oleifera leaf polysaccharide and the activity of controlling sugar and reducing lipid.
Disclosure of Invention
The invention aims to provide a high-purity and high-activity moringa oleifera leaf polysaccharide, and a preparation method and application thereof. The total sugar content in the moringa oleifera leaf polysaccharide is more than 80%, the purity is more than 80%, the yield is more than 2.5%, and the polysaccharide has good effects of reducing blood sugar and controlling lipid and is light in color and suitable for various food systems through verification of an INFOGEST static digestion model. The purification process is simple, the whole process flow can meet the food-grade requirement, and the obtained moringa oleifera leaf polysaccharide can be applied to the fields of common foods, health care products and the like.
Based on the simplified technological process and the technical aim of comprehensively improving the purity of the moringa oleifera leaf polysaccharide and controlling the glucose and lipid-lowering activity, the invention obtains the moringa oleifera leaf polysaccharide with high purity and high activity by adopting non-organic reagents, non-strong acid and non-chromatographic technological processes such as double protease enzymolysis, double resin adsorption, ultrafiltration and dialysis on the basis of a moringa oleifera leaf polysaccharide extract (total sugar content >50 percent and yield >12 percent) with the function of adjusting the glucose and lipid absorption, a preparation method and application (CN 202210833825.6) thereof. Firstly, considering that polyphenol remains in the moringa oleifera leaf polysaccharide extract, the polyphenol can be oxidized into black quinone compounds under alkaline conditions (protease optimal action pH), so that the polysaccharide changes color and is difficult to remove in the later period, and the application of the polysaccharide in a light-colored food system is influenced; the moringa oleifera leaf protein is rich in hydrophobic amino acid and is difficult to hydrolyze into small molecular peptides by single protease, and the invention utilizes a method of 2 protease complex long-time enzymolysis to degrade proteins into small molecular peptides; the principle that the ionic resin can be combined with small molecular peptide, pigment and salt is utilized to carry out small molecular peptide removal, decoloration and desalination treatment on the zymolyte; further, polysaccharide is further purified by an ultrafiltration method, micromolecular substances are further removed by a dialysis method, original pigment in the moringa oleifera leaf polysaccharide extract and pigment introduced by biochemical reaction in the purification process are skillfully removed, and the purity of the moringa oleifera leaf polysaccharide and the activity of controlling glucose and reducing lipid are greatly improved. The method not only gives consideration to three latitudes of high purity, high activity and light color of the moringa oleifera leaf polysaccharide, breaks through the technical bottlenecks of high toxicity, complex operation, high polysaccharide loss of the traditional organic reagent method, low purity and high loss of the traditional strong acid method polysaccharide, and low operation, low yield and low separation efficiency of the traditional chromatographic method in the polysaccharide purification process, and the whole process flow can meet the food-grade requirements, accords with the green processing concept, has low requirements on the equipment processing performance, is a novel technology for purifying the moringa oleifera leaf polysaccharide in a green large-scale manner, and has potential of industrial popularization and application.
The technical scheme of the invention is as follows:
the invention provides a preparation method of high-purity and high-activity moringa oleifera leaf polysaccharide, which is characterized in that based on a moringa oleifera leaf polysaccharide extract (total sugar content is more than 50 percent, yield is more than 12 percent) with the function of adjusting glycolipid absorption, and a preparation method and application thereof (CN 202210833825.6), non-organic reagents such as double protease enzymolysis, double resin adsorption, ultrafiltration, dialysis and the like, non-strong acid and non-chromatographic process flows are adopted to obtain the high-purity and high-activity moringa oleifera leaf polysaccharide. The moringa oleifera leaf polysaccharide has the total sugar content of more than 80%, the purity of more than 80%, the yield of more than 2.5%, and good glycolipid absorption regulation effect, and compared with the total sugar content of moringa oleifera leaf polysaccharide extract MEB (CN 202210833825.6) before purification, the total sugar content is improved by 52.50%, the purity is improved by 49.90%, the glucose dialysis delay index is improved by 20.88%, the cholate adsorption rate is improved by 17.39%, and the cholesterol micelle dissolution inhibition rate is improved by 3.07%. The extraction process is simple, the whole process flow can meet the food-grade requirement, and the method can be applied to the fields of common foods, health care products and the like. The preparation method comprises the following steps:
(1) Preparing a moringa oleifera leaf polysaccharide extract: preparing an extracting solution 2 in the Chinese patent application claim 1 with the application number of CN202210833825.6, namely, the polysaccharide extracting solution 1;
(2) Resin adsorption and filtration: adding nonionic macroporous resin into the polysaccharide extract 1 obtained in the step (1), stirring at a constant speed, and filtering to obtain a polysaccharide extract 2;
(3) Protease enzymolysis: regulating the pH value of the polysaccharide extract 2 obtained in the step (2), adding Alcalase enzyme and pancreatin, stirring at constant temperature for enzymolysis, and obtaining a polysaccharide extract 3;
(4) Resin adsorption, filtration and centrifugation: adding ion macroporous resin into the polysaccharide extract 3 obtained in the step (3), uniformly stirring, filtering, and centrifuging to obtain polysaccharide extract 4;
(5) Ultrafiltration and concentration: carrying out ultrafiltration separation on the polysaccharide extract 4 obtained in the step (4) by adopting an ultrafiltration membrane, and concentrating under reduced pressure to obtain a polysaccharide extract 5;
(6) And (3) dialysis: dialyzing the polysaccharide extract 5 obtained in the step (5) at low temperature to obtain a polysaccharide extract 6;
(7) And (3) freeze drying: and (3) freeze-drying the polysaccharide extract 6 obtained in the step (6) to obtain the moringa oleifera leaf polysaccharide with high purity and high activity.
Further, in the step (1), the preparation method of the polysaccharide extract 1 specifically comprises the following steps:
1) Crushing by liquid nitrogen: crushing the dried moringa leaves into dry powder by a liquid nitrogen crushing device to obtain moringa leaf dry powder;
2) Enzymatic hydrolysis of cellulase: uniformly mixing the moringa leaf dry powder obtained in the step 1) with water, regulating the pH, adding cellulase after fully and uniformly mixing, and stirring at constant temperature for enzymolysis to obtain a suspension 1;
3) Heating and extracting: heating and extracting the suspension 1 to obtain a suspension 2;
4) And (3) yeast fermentation: adding active dry yeast into the suspension 2, and fermenting at constant temperature to obtain a suspension 3;
5) Centrifuging and concentrating: centrifuging the suspension 3, and concentrating the supernatant under reduced pressure to obtain an extract A;
6) Ethanol precipitation: adding ethanol solution into the extract A, mixing to obtain mixed solution, standing, centrifuging, collecting precipitate, adding water, heating for dissolving, and concentrating under reduced pressure to obtain polysaccharide extract 1.
Further, in the step 1), the condition of liquid nitrogen pulverization is that pulverization is carried out at-150 to-120 ℃.
Further, in the step 2), the feed liquid ratio of the moringa oleifera leaf dry powder to the water is 1:8-1:12 g/mL; adjusting the pH value to 4.0-5.0 by using hydrochloric acid, adding cellulose with the mass of 0.25-0.75% of the moringa oleifera leaf dry powder obtained in the step 1), stirring at the speed of 120-180 r/min, and performing enzymolysis at the temperature of 45-55 ℃ for 1-3 h.
Further, in the step 3), the temperature of the heating extraction is 90-120 ℃, and the time of the heating extraction is 20-40 min.
Further, in the step 4), the mass ratio of the added active dry yeast to the water in the step 2) is 1g (200-1000) mL, the fermentation temperature is 26-30 ℃, the fermentation time is 1-3 h, and the rotating speed is 110-130 rpm.
Further, in the step 5), the temperature of the centrifugation is 4-8 ℃, the centrifugal force of the centrifugation is 6000-8000 g, and the time of the centrifugation is 15-25 min; the temperature of the reduced pressure concentration is 55-65 ℃, and the solid content in the extracting solution A is 4-10 wt%.
Further, in the step 6), the volume of the added ethanol solution accounts for 70-90% of the total volume of the mixed solution, the standing temperature is 4-8 ℃, the standing time is 2-4 h, the centrifuging temperature is 4-8 ℃, the centrifuging force is 4000-8000 g, and the centrifuging time is 15-25 min.
Further, in the step 6), the mass of the added water is 100-150 times of the mass of the sediment, the heating and dissolving temperature is 50-60 ℃, the heating and dissolving time is 1-3 hours, the decompressing and concentrating temperature is 55-65 ℃, and the solid content in the polysaccharide extract 1 is 2-4 wt%.
Further, in the step (2), the nonionic macroporous resin is XAD-16 macroporous resin, the ratio of the mass of the nonionic macroporous resin to the volume of the polysaccharide extract 1 in the step (1) is 40-60 g/L, the stirring speed is 100-120 r/min, the stirring temperature is 50-60 ℃, the stirring time is 1-2 h, and the mesh number of the filtering cloth is 80-120 meshes.
Further, in the step (3), a sodium hydroxide solution is used for adjusting the pH value of the polysaccharide extract 2 to 7.0-8.0, the mass of Alcalase enzyme added is 1-3% (w/w) of the mass of protein in the polysaccharide extract 2 obtained in the step (2), the mass of pancreatin added is 1-3% (w/w) of the mass of protein in the polysaccharide extract 2 obtained in the step (2), the enzymolysis temperature is 50-60 ℃, the stirring speed is 120-180 r/min, and the enzymolysis time is 24-36 h.
Further, the amount of protein in the polysaccharide extract 2 was measured by the Kjeldahl method.
Further, in the step (4), the ionic macroporous resin is D-280 macroporous resin, the ratio of the mass of the added ionic macroporous resin to the volume of the polysaccharide extract 3 in the step (3) is 40-60 g/L, the stirring speed is 100-120 r/min, the stirring temperature is 50-60 ℃, the stirring time is 1-2 h, the mesh number of the filtering cloth is 80-120 meshes, the centrifugation temperature is 4-8 ℃, the centrifugal force of centrifugation is 6000-8000 g, and the centrifugation time is 15-25 min.
Further, in the step (5), the molecular weight of the ultrafiltration membrane is 8000-12000 Da, the times of ultrafiltration are 5-7 times, the polysaccharide extract 5 is ultrafiltration interception liquid with the molecular weight of more than 10000Da, the temperature of reduced pressure concentration is 50-60 ℃, and the solid content in the polysaccharide extract 5 is 2-4wt%.
In the step (6), the polysaccharide extract 5 is put into a dialysis bag with the molecular weight cut-off of 8000-14000 Da, dialyzed for 24-48 hours in deionized water with the temperature of 4-10 ℃ and changed for every 8-12 hours, and the polysaccharide extract 6 is the liquid in the dialysis bag.
The invention provides a high-purity and high-activity moringa oleifera leaf polysaccharide prepared by the preparation method.
The invention also provides application of the moringa oleifera leaf polysaccharide with high purity and high activity in foods, health products or medicines for reducing blood sugar and controlling lipid.
The invention has the following advantages and effects:
(1) The invention adopts nonionic macroporous resin to adsorb small molecular substances such as pigment, so that the color of the polysaccharide extract can not be deepened after the pH value of the polysaccharide extract is adjusted to be neutral and alkaline, then the horseradish tree leaf protein is fully degraded into small molecular peptide by two protease enzymolysis polysaccharide extracts so as to be beneficial to separation, and the method of coupling ion macroporous resin adsorption adsorbs the small molecular peptide, dark enzymolysis products and salt after enzymolysis, thereby playing roles in removing the small molecular peptide, decoloring and desalting, and the small molecular substances can be further removed for purification by ultrafiltration and dialysis, so that the obtained horseradish tree leaf polysaccharide has the capacity of adjusting the absorption of glycolipid. The double enzymolysis-double resin combined method not only gives consideration to two latitudes of high purity and high activity, but also breaks through the technical bottlenecks of high toxicity, complex operation, high polysaccharide loss, low purity and high loss of the conventional organic reagent method, complex operation, low yield and low separation efficiency of the conventional strong acid method, and has a certain common application prospect in the field of plant polysaccharide purification in the polysaccharide purification process.
(2) The whole preparation process flow of the invention meets the food-grade requirements and meets the green processing concept.
(3) The moringa oleifera leaf polysaccharide obtained by the invention has the total sugar content of more than 80%, the purity of more than 80% and the yield of more than 2.5%, can effectively block the diffusion of glucose, adsorb cholate, inhibit the solubility of cholesterol in micelles, has good effect of regulating the absorption of glycolipid, has light color, and is suitable for various food systems. Compared with the total sugar content of the moringa oleifera leaf polysaccharide extract MEB (CN 202210833825.6) before purification, the purity is improved by 52.50%, the glucose dialysis delay index is improved by 49.90%, the cholate adsorption rate is improved by 17.39%, the cholesterol micelle dissolution inhibition rate is improved by 3.07%, the separation and purification process is simple, the safety is high, and the moringa oleifera leaf polysaccharide extract MEB can be used for purifying moringa oleifera leaf polysaccharide in a large scale and is suitable for the fields of common foods, health-care products and the like.
Drawings
FIG. 1 is a bar graph of total sugar content of the Moringa oleifera leaf polysaccharides prepared in comparative examples 1-8 and examples 1-3.
FIG. 2 is a bar graph of protein content of Moringa oleifera leaf polysaccharide extracts MEB, P5, P6 of comparative examples 1, 6-7, and Moringa oleifera leaf polysaccharides PA, PB, PC of examples 1-3.
FIG. 3 is a bar graph of yields and polysaccharide retention of the Moringa oleifera leaf polysaccharide extracts MEB, P5, P6 of comparative examples 1, 6-7 and the Moringa oleifera leaf polysaccharides PA, PB, PC of examples 1-3.
FIG. 4 is a bar graph showing the glucose blocking capacity of the Moringa oleifera leaf polysaccharide extracts MEB, P5, P6 of comparative examples 1, 6-7 and the Moringa oleifera leaf polysaccharides PA, PB, PC of examples 1-3.
FIG. 5 is a bar graph showing the bile acid adsorption capacity of the Moringa oleifera leaf polysaccharide extracts MEB, P5, P6 of comparative examples 1, 6-7 and the Moringa oleifera leaf polysaccharides PA, PB, PC of examples 1-3.
FIG. 6 is a bar graph showing the cholesterol micelle solubility inhibition capacity of the Moringa oleifera leaf polysaccharide extracts MEB, P5, P6 of comparative examples 1, 6-7 and the Moringa oleifera leaf polysaccharides PA, PB, PC of examples 1-3.
Detailed description of the preferred embodiments
For a better understanding of the present invention, the operation of the InFOGEST static digestion model will be specifically elucidated below and further described in connection with specific examples, but embodiments of the present invention are not limited thereto.
1. The INFOGEST static digestion model evaluates glucose control lipid lowering activity:
(1) Simulated oral liquid: comprises 15.1mmol/LKCl, 3.7mmol/LKH 2 PO 4 、13.6mmol/LNaHCO 3 、0.15mmol/LMgCl 2 、0.06mmol/L(NH 4 ) 2 CO 3 1.1mmol/LHCl and 1.5mmol/L CaCl 2 The solvents were all water.
(2) Simulating gastric juice: comprises 6.9mmol/L KCl and 0.9mmol/L KH 2 PO 4 、25mmol/LNaHCO 3 、47.2mmol/LNaCl、0.12mmol/L MgCl 2 、0.5mmol/L(NH 4 ) 2 CO 3 15.6mmol/L HCl and 0.15mmol/LCaCl 2 The solvents were all water.
(3) Simulation of intestinal juice: comprises 6.8mmol/L KCl and 0.8mmol/L KH 2 PO 4 、85mmol/LNaHCO 3 、38.4mmol/LNaCl、0.33mmol/LMgCl 2 8.4mmol/LHCl and 0.6mmol/LCaCl 2 The solvents were all water.
The digestion process of the samples in the info GEST model is as follows:
(1) Oral digestion: sampling, dissolving in 2mL simulated oral liquid, adjusting pH to 7.0 with deionized water and 5mol/LNaOH aqueous solution to make the final volume of oral digest be 2.5mL, and oscillating at 37deg.C for 2min.
(2) Gastric digestion: 2mL simulated gastric fluid and 7.50. Mu.L 0.3mmol/L CaCl were added to the oral digest 2 The aqueous solution was dissolved in 40mg of pepsin (final concentration 2000U/mL) with 0.17mL of simulated gastric fluid, added to the digest, and the pH was adjusted to 3.0 with deionized water and 5mol/LHCl aqueous solution to give a final volume of 5mL of gastric digest, and the gastric digest was shaken at constant temperature of 37℃for 2h.
(3) Small intestine digestion: 4mL simulated intestinal fluid and 10. Mu.L 0.3mmol/L CaCl were added to the stomach digest 2 In the aqueous solution, 5mg of pancreatin (final concentration 100U/mL) was dissolved in 1.25mL of simulated intestinal fluid, and then added to the digest, 66.4mg of pig bile salt was added to 0.75mL of simulated intestinal fluid, and after dissolution, the digest was added to the pH of the mixture, and deionized water and 5mol/LNaOH aqueous solution were used to adjust the pH to 7.0, so that the final volume of the digest was 10mL, and the mixture was subjected to constant temperature shaking at 37℃for 2 hours.
1.1 glucose blocking Capacity determination step:
180mg glucose and 0.1g of sample (concentration 10mg/mL in final 10mL digestion volume) were taken, simulated oral digestion and simulated gastric digestion were performed as described above, all of the digestion solution was transferred to a dialysis bag with a cut-off molecular weight of 3000Da before the last step of simulated intestinal digestion was started with constant temperature shaking, the dialysis bag was placed in a container containing 100mL of simulated intestinal fluid, after shaking dialysis at 37℃for 2 hours, 2mL of dialysis bag external fluid was collected, and the glucose content of the dialysis bag external fluid was determined using a glucose kit. The blank was treated in the same way as the sample group without the addition of sample. The delay index (%) of glucose diffusion from the dialysis bag into the dialysate is calculated as follows:
Wherein the unit of glucose amount is mmol/L.
1.2 procedure for determining adsorption Capacity of bile acid salt:
preparation of cholesterol micelle solution: 3.125g of pig bile salt, 386.65mg of cholesterol and 706.15mg of oleic acid are dissolved by using 150mL of simulated intestinal fluid, ultrasonic emulsification is carried out for 2 hours until the cholesterol is completely dissolved, deionized water is used for fixing the volume to 200mL, the gel bundles are filled in a closed container, and the gel bundles are stored at 37 ℃ for 24 hours for use.
A sample of 0.05g (5 mg/mL in the final 10mL digestion volume) was taken and simulated oral digestion and simulated gastric digestion were performed as described above. Dissolving 5mg of pancreatin in 4mL of cholesterol micelle solution before digestion of simulated small intestine, and adding the mixed solution into simulated intestinal juice after uniform mixing; then 0.01mL of 0.3mmol/L CaCl is added 2 An aqueous solution; adjusting pH to 7.0 with deionized water and 5mol/LNaOH aqueous solution to give digestion volume of 10mL, oscillating at 37deg.C for 2h, ice-bathing for 15min, centrifuging at 8000g at 4deg.C for 15min, collecting supernatant, and measuring cholate content. The blank was treated in the same way as the sample group without the addition of sample. The cholate adsorption (%) was calculated according to the following formula:
wherein, C blank is the concentration of cholate in blank control group, and the unit is mmol/L; c the sample is the concentration of cholate in the sample group, and the unit is mmol/L.
1.3 cholesterol micelle dissolution inhibition ability measurement step:
a sample of 0.05g was taken (at a final volume of digestion of the final 10mLMedium concentration of 5 mg/mL) simulated oral digestion and simulated gastric digestion were performed as described above. Dissolving 5mg of pancreatin in 4mL of cholesterol micelle solution before digestion of simulated small intestine, and adding the mixed solution into simulated intestinal juice after uniform mixing; then 0.01mL of 0.3mmol/L CaCl is added 2 An aqueous solution; adjusting pH to 7.0 with deionized water and 5mol/LNaOH aqueous solution to obtain a digestion final volume of 10mL, oscillating at 37deg.C for 2h, ice-bathing for 15min, centrifuging at 8000g and 4deg.C for 15min, collecting the digestion supernatant, adding anhydrous methanol at a ratio of 1:1 (v/v), repeatedly extracting the supernatant twice, and measuring cholesterol content by OPA method. The blank was treated in the same way as the sample group without the addition of sample. Cholesterol solubility inhibition (%) was calculated according to the following formula:
wherein, C blank is cholesterol concentration in μg/mL in blank control group; c the concentration of cholesterol in the sample group in μg/mL.
The glucose blocking ability, cholate adsorption ability and cholesterol micelle solubility inhibition ability of the moringa oleifera leaf extracts obtained in examples and comparative examples were measured using the above methods.
1. The method for measuring the yield and the polysaccharide retention rate comprises the following steps:
the yield (%) and polysaccharide retention (%) were calculated according to the following formulas:
3. molecular weight distribution and purity determination method:
the sample was dissolved in 0.1mol/LNaNO 3 (containing 0.05% g/mL, (w/v) sodium azide) to prepare 5mg/mL polysaccharide solution, passing through 0.22 μm microporous filter membrane, and adopting molecular exclusion gel chromatographyThe molecular weight distribution and purity were measured using a multi-angle laser light scatterometer and a differential detector (SEC-MALLS-RI). Chromatographic conditions: ultraHydrogel guard column (40 mm. Times.6 mm), ultraHydrogel 2000SEC column (7.8 mm. Times.300 mm) and UltraHydrogel 1000SEC column (7.8 mm. Times.300 mm) were used in series with a column temperature of 35℃and a mobile phase of 0.1mol/LNaNO 3 (containing 0.05% g/mL, (w/v) sodium azide) solution, the flow rate is 0.6mL/min, the sample injection amount is 100 mu L, the data collection is carried out for 60min, the refractive index increment (dn/dc) is 0.138mL/g, the data collection and analysis are carried out by adopting ASTRA 6.1 software, the molecular weight (Mw) is the molecular weight of the polysaccharide, and the mass fraction (mass fraction) is defined as the ratio of the polysaccharide in the whole substance, namely the purity of the polysaccharide.
Preparing a moringa oleifera leaf polysaccharide extract:
(1) Crushing by liquid nitrogen: 15kg of dry moringa leaves are put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain moringa leaf dry powder M.
(2) Enzymatic hydrolysis of cellulase: mixing 200g of the moringa oleifera leaf dry powder M obtained in the step (1) with deionized water at a ratio of 1:10g/mL, adding hydrochloric acid to adjust the pH to 4.5, adding cellulase after fully mixing, wherein the mass of the cellulase is 0.50% (w/w, g/g) of the moringa oleifera leaf dry powder M obtained in the step (1), and stirring at a constant temperature of 50 ℃ at a rotating speed of 150r/min for enzymolysis for 2 hours to obtain a suspension S1.
(3) High-temperature extraction: extracting suspension S1 at 105deg.C for 30min to obtain suspension S2.
(4) And (3) yeast fermentation: cooling the suspension S2 to room temperature, adding active dry yeast into the suspension S2, wherein the volume ratio of the mass of the added active dry yeast to the deionized water in the step (2) is 1g:333mL, and fermenting at the constant temperature of 28 ℃ for 2h at the rotating speed of 120rpm to obtain the suspension S3.
(5) Centrifuging and concentrating: after the suspension S3 was centrifuged at 7000g at 6℃for 20min at high speed, the supernatant was concentrated under reduced pressure at 60℃until the solid content in the final concentrate became 7wt%, giving extract E1.
(6) Ethanol precipitation: adding absolute ethyl alcohol into the extracting solution E1 to obtain a mixed solution, adding absolute ethyl alcohol with the volume of 80% (v/v) of the total volume of the mixed solution, fully mixing at 6 ℃ and standing for 3h, centrifuging at 6000g speed for 20min at 6 ℃, taking out the lower layer of sediment, adding deionized water with the mass being 125 times of that of the sediment, heating at 55 ℃ for fully dissolving for 2h, and concentrating under reduced pressure at 60 ℃ until the solid content in the final concentrated solution is 3wt%, wherein the polysaccharide extracting solution P1A.
Example 1
A method for improving purity and activity of moringa oleifera leaf polysaccharide comprises the following steps:
(1) Resin adsorption and filtration: adding XAD-16 nonionic macroporous resin into 1L of polysaccharide extract P1A, mixing the mass of the added XAD-16 nonionic macroporous resin and the volume ratio of the polysaccharide extract P1A at 40g/L, stirring at a constant speed of 100r/min for 1h at 50 ℃, and filtering by a 80-mesh filter cloth to obtain polysaccharide extract P2A.
(2) Protease enzymolysis: adding sodium hydroxide aqueous solution to adjust pH value of polysaccharide extract P2A to 7.0, adding Alcalase enzyme and pancreatin, wherein the addition mass of Alcalase enzyme and pancreatin is 1% (w/w) of protein in polysaccharide extract P2A, and stirring at constant temperature of 50deg.C at 120r/min for enzymolysis for 24 hr to obtain polysaccharide extract P3A.
(3) Resin adsorption, filtration and centrifugation: d-280 ion macroporous resin is added into polysaccharide extract P3A, the volume ratio of the mass of the added D-280 ion macroporous resin to the polysaccharide extract P3A is 40g/L, the mixture is stirred at a constant speed of 100r/min for 1h at 50 ℃, the mixture is filtered by a filter cloth with 80 meshes, the filtrate is centrifuged, and the filtrate is centrifuged at 6000g for 15min at 4 ℃ to obtain polysaccharide extract P4A.
(4) Ultrafiltration and concentration: and (3) carrying out ultrafiltration separation on the polysaccharide extract P4A for 5 times by adopting an ultrafiltration membrane with the molecular weight of 8000Da, collecting ultrafiltration trapped fluid with the molecular weight of more than 10000Da, and concentrating under reduced pressure at 50 ℃ until the solid content in the final concentrated solution is 2wt% to obtain polysaccharide extract P5A.
(5) And (3) dialysis: the polysaccharide extract P5A is put into a dialysis bag with the molecular weight cut-off of 8000Da, dialyzed for 24 hours at the temperature of 4 ℃ in deionized water, water is changed every 8 hours, and the polysaccharide extract P6A is obtained by taking the liquid in the dialysis bag.
(6) And (3) freeze drying: and freeze-drying the polysaccharide extract P6A to obtain the moringa oleifera leaf polysaccharide PA with high purity and high activity.
Example 2
A method for improving purity and activity of moringa oleifera leaf polysaccharide comprises the following steps:
(1) Resin adsorption and filtration: adding XAD-16 nonionic macroporous resin into 1L polysaccharide extract P1A, adding 50g/L of the mass of the XAD-16 nonionic macroporous resin and the volume ratio of the polysaccharide extract P1A, stirring at a constant speed of 110r/min for 1.5h at 55 ℃, and filtering the polysaccharide extract P2B by a 100-mesh filter cloth.
(2) Protease enzymolysis: adding sodium hydroxide aqueous solution to adjust pH value of polysaccharide extract P2B to 7.5, adding Alcalase enzyme and pancreatin, wherein the addition mass of Alcalase enzyme and pancreatin is 2% (w/w) of protein in polysaccharide extract P2B, stirring at 55deg.C at 150r/min, and performing enzymolysis for 30 hr.
(3) Resin adsorption, filtration and centrifugation: d-280 ion macroporous resin is added into polysaccharide extract P3B, the volume ratio of the mass of the added D-280 ion macroporous resin to the polysaccharide extract P3B is 50g/L, the mixture is stirred at a constant speed of 100r/min for 1.5h at 55 ℃, the filtrate is centrifuged through a 100-mesh filter cloth after being filtered, and the filtrate is centrifuged at 7000g for 20min at 6 ℃ to obtain polysaccharide extract P4B.
(4) Ultrafiltration and concentration: and (3) carrying out ultrafiltration separation on the polysaccharide extract P4B for 6 times by adopting an ultrafiltration membrane with the molecular weight of 10000Da, collecting ultrafiltration trapped fluid with the molecular weight of more than 10000Da, and concentrating under reduced pressure at 55 ℃ until the solid content in the final concentrated solution is 3wt% to obtain polysaccharide extract P5B.
(5) And (3) dialysis: the polysaccharide extract P5B is put into a dialysis bag with the molecular weight cut-off of 11000Da, dialyzed for 36 hours at 7 ℃ in deionized water, water is changed every 10 hours, and the polysaccharide extract P6B is obtained from the liquid in the dialysis bag.
(6) And (3) freeze drying: and freeze-drying the polysaccharide extract P6B to obtain the moringa oleifera leaf polysaccharide PB with high purity and high activity.
Example 3
A method for improving purity and activity of moringa oleifera leaf polysaccharide comprises the following steps:
(1) Resin adsorption and filtration: adding XAD-16 nonionic macroporous resin into 1L polysaccharide extract P1A, adding 60g/L of the mass of the XAD-16 nonionic macroporous resin and the volume ratio of the polysaccharide extract P1A, stirring at a constant speed of 120r/min for 2h at 60 ℃, and filtering by a 120-mesh filter cloth to obtain polysaccharide extract P2C.
(2) Protease enzymolysis: adding sodium hydroxide aqueous solution to adjust pH value of polysaccharide extract P2C to 8.0, adding Alcalase enzyme and pancreatin, wherein the addition mass of Alcalase enzyme and pancreatin is 3% (w/w) of protein in polysaccharide extract P2C, and stirring at 60deg.C at 180r/min for enzymolysis for 36 hr to obtain polysaccharide extract P3C.
(3) Resin adsorption, filtration and centrifugation: d-280 ion macroporous resin is added into polysaccharide extract P3C, the volume ratio of the mass of the added D-280 ion macroporous resin to the polysaccharide extract P3C is 60g/L, the mixture is stirred at a constant speed of 120r/min for 2h at 60 ℃, the mixture is filtered by a 120-mesh filter cloth, the filtrate is centrifuged, and the filtrate is centrifuged at 8000g for 25min at 8 ℃ to obtain polysaccharide extract P4C.
(4) Ultrafiltration and concentration: and (3) carrying out ultrafiltration separation on the polysaccharide extract P4C for 7 times by adopting an ultrafiltration membrane with the molecular weight of 12000Da, collecting ultrafiltration trapped fluid with the molecular weight of more than 10000Da, and concentrating under reduced pressure at 60 ℃ until the solid content in the final concentrated solution is 4wt% and obtaining polysaccharide extract P5C.
(5) And (3) dialysis: and (3) placing the polysaccharide extract P5C into a dialysis bag with the molecular weight cut-off of 14000Da, dialyzing in deionized water at 10 ℃ for 48 hours, changing water every 12 hours, and taking the liquid in the dialysis bag to obtain the polysaccharide extract P6C.
(6) And (3) freeze drying: and freeze-drying the polysaccharide extract P6C to obtain the high-purity and high-activity moringa oleifera leaf polysaccharide PC.
Comparative example 1
A preparation method of Moringa oleifera leaf polysaccharide extract with glycolipid absorption regulating effect comprises the following steps:
the preparation method of the moringa oleifera leaf polysaccharide extract MEB of example 2 in the specification of Chinese patent application No. CN202210833825.6 is as follows:
(1) Crushing by liquid nitrogen: 15kg of dry moringa leaves are put into a liquid nitrogen crushing device at the temperature of minus 135 ℃ and crushed into dry powder to obtain moringa leaf dry powder M1.
(2) Enzymatic hydrolysis of cellulase: mixing 200g of the moringa oleifera leaf dry powder M1 obtained in the step (1) with deionized water at a ratio of 1:10g/mL, adding hydrochloric acid to adjust the pH to 4.5, adding cellulase after fully mixing, wherein the mass of the cellulase is 0.50% (w/w, g/g) of the moringa oleifera leaf dry powder M1 obtained in the step (1), and stirring at a constant temperature at a rotating speed of 150r/min for enzymolysis for 2h at 50 ℃ to obtain a suspension S1B.
(3) High-temperature extraction: extracting suspension S1B at 105deg.C for 30min to obtain suspension S2B.
(4) And (3) yeast fermentation: cooling the suspension S2B to room temperature, adding active dry yeast into the suspension S2B, fermenting at constant temperature for 2h at 28 ℃ at a rotating speed of 120rpm, wherein the volume ratio of the mass of the added active dry yeast to the deionized water in the step (2) is 1g:333mL, and obtaining the suspension S3B.
(5) Centrifuging and concentrating: after the suspension S3B was centrifuged at 7000g for 20min at 6℃at high speed, the supernatant was concentrated under reduced pressure at 60℃until the solid content in the final concentrate became 7wt%, giving extract E1B.
(6) Ethanol precipitation: adding absolute ethyl alcohol into the extract E1B to obtain a mixed solution, adding absolute ethyl alcohol with the volume of 80% (v/v) of the total volume of the mixed solution, fully mixing at 6 ℃ and standing for 3h, centrifuging at 6000g speed for 20min at 6 ℃, taking out the lower precipitate, adding deionized water with the mass being 125 times of that of the precipitate, heating at 55 ℃ for fully dissolving for 2h, and concentrating under reduced pressure at 60 ℃ until the solid content in the final concentrated solution is 3wt%, thereby obtaining the extract E2B.
(7) And (3) freeze drying: and (3) freeze-drying the extracting solution E2B to obtain a moringa oleifera leaf polysaccharide extract MEB.
Comparative example 2
Adding sodium hydroxide aqueous solution into 1L of polysaccharide extract P1A to adjust the pH value of the polysaccharide extract P1A to 7.5, adding pancreatin, wherein the added mass of pancreatin is 2 percent (w/w) of the protein content in the polysaccharide extract P1A, stirring at constant temperature of 55 ℃ at a rotating speed of 150r/min for enzymolysis for 8 hours, adding XAD-16 nonionic macroporous resin, adding the ratio of the mass of the XAD-16 nonionic macroporous resin to the volume of the polysaccharide extract P1A to be 50g/L, stirring at constant speed of 110r/min for 1.5 hours at 55 ℃, filtering by a 100-mesh filter cloth, centrifuging at rotating speed of 7000g for 20 minutes at 6 ℃, concentrating the supernatant at reduced pressure at 55 ℃ until the solid content in the final concentrated solution is 3wt%, loading into a dialysis bag with a cut-off molecular weight of 11000Da, dialyzing at 7 ℃ for 36 hours, changing water every 10 hours, taking liquid in the dialysis bag, and drying to obtain the moringa polysaccharide P1.
Comparative example 3
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding sodium hydroxide aqueous solution into 1L of polysaccharide extract P1A to adjust the pH value of the polysaccharide extract P1A to 7.5, adding pancreatin, wherein the added mass of pancreatin is 2 percent (w/w) of the protein mass in the polysaccharide extract P1A, stirring at a constant temperature of 150r/min at 55 ℃ for enzymolysis for 8 hours, adding XAD-16 nonionic macroporous resin, adding the ratio of the mass of the XAD-16 nonionic macroporous resin to the volume of the polysaccharide extract P1A to be 50g/L, stirring at a constant speed of 110r/min for 1.5 hours at 55 ℃, filtering by a 100-mesh filter cloth, centrifuging at a rotating speed of 7000g for 20 minutes at 6 ℃, taking supernatant, performing ultrafiltration separation for 6 times by adopting an ultrafiltration membrane with a molecular weight of 10000Da, collecting ultrafiltration retentate with a molecular weight of 10000Da, concentrating under reduced pressure at 55 ℃ until the solid content in the final concentrate is 3wt%, loading into a dialysis bag with a retention molecular weight of 11000Da, changing water once every 10 hours at 7 ℃ in deionized water, and taking the dialysis bag for drying to obtain the moringa oleifera polysaccharide P2.
Comparative example 4
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding sodium hydroxide aqueous solution into 1L of polysaccharide extract P1A to adjust the pH value of the polysaccharide extract P1A to 7.5, adding pancreatin, wherein the added mass of pancreatin is 2 percent (w/w) of the protein mass in the polysaccharide extract P1A, stirring at a constant temperature of 150r/min at 55 ℃ for enzymolysis for 30h, adding XAD-16 nonionic macroporous resin, adding the ratio of the mass of the XAD-16 nonionic macroporous resin to the volume of the polysaccharide extract P1A to be 50g/L, stirring at a constant speed of 110r/min for 1.5h at 55 ℃, filtering by a 100-mesh filter cloth, centrifuging at a rotating speed of 7000g for 20min at 6 ℃, taking supernatant, performing ultrafiltration separation for 6 times by adopting an ultrafiltration membrane with a molecular weight of 10000Da, collecting ultrafiltration retentate with a molecular weight of 10000Da, concentrating under reduced pressure at 55 ℃ until the solid content in the final concentrate is 3wt%, loading into a dialysis bag with a retention molecular weight of 11000Da, changing water once every 10h at 7 ℃ in deionized water, and taking the dialysis bag for drying to obtain the moringa oleifera polysaccharide P3.
Comparative example 5
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding sodium hydroxide aqueous solution into 1L of polysaccharide extract P1A to adjust the pH value of the polysaccharide extract P1A to 7.5, adding Alcalase enzyme and pancreatin, stirring at constant temperature of 150r/min for enzymolysis for 30h at 55 ℃, adding XAD-16 nonionic macroporous resin, adding the ratio of the mass of the XAD-16 nonionic macroporous resin to the volume of the polysaccharide extract P1A to be 50g/L, stirring at constant speed of 110r/min for 1.5h at 55 ℃, filtering by a 100-mesh filter cloth, centrifuging at 7000g at 6 ℃ for 6 times, taking supernatant, carrying out ultrafiltration separation by adopting an ultrafiltration membrane with molecular weight of 10000Da for 6 times, collecting ultrafiltration liquid with molecular weight of 10000Da, concentrating under reduced pressure at 55 ℃ until the solid content in the final concentrate is 3wt%, and drying to obtain moringa leaf polysaccharide P4.
Comparative example 6
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding trichloroacetic acid solution (water as solvent) with 10% (v/v) of polysaccharide extract P1A into 1L of polysaccharide extract P1A, standing at 4deg.C for 12h, adding XAD-16 nonionic macroporous resin, adding 50g/L of the ratio of the mass of XAD-16 nonionic macroporous resin to the volume of polysaccharide extract P1A, stirring at 55deg.C at uniform speed of 110r/min for 1.5h, filtering with 100 mesh filter cloth, centrifuging at 6deg.C at 7000g for 20min, separating supernatant by ultrafiltration membrane with molecular weight of 10000Da for 6 times, collecting ultrafiltration retentate with molecular weight of >10000Da, concentrating under reduced pressure at 55deg.C until the solid content in the final concentrate is 3wt%, loading into dialysis bag with molecular weight of 11000Da, dialyzing at 7deg.C for 36h, changing water every 10h, and lyophilizing to obtain Moringa leaf polysaccharide P5.
Comparative example 7
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding sevage solution (volume ratio of chloroform to n-butanol is=4:1) with 25% (v/v) of polysaccharide extract P1A volume into 1L polysaccharide extract P1A, stirring at constant speed of 150r/min for 5min, standing for 30min for layering, collecting supernatant, adding sevage solution (volume ratio of chloroform to n-butanol is=4:1) with 25% (v/v) of supernatant volume, stirring at constant speed of 150r/min for 5min, standing for 30min for layering, collecting supernatant, repeating the above process (adding sevage solution for removing protein) for 14 times until no precipitate is generated, adding XAD-16 nonionic macroporous resin with a volume ratio of 50g/L to polysaccharide extract P1A, stirring at constant speed of 110r/min for 1.5h at 55 ℃, ultrafiltering with ultrafiltration membrane with molecular weight of 10000Da for 6 times, collecting cut-off molecular weight of 10000Da at constant speed of 55 ℃ for 6h, concentrating into dialysis solution at a dry concentration of 6h under dialysis condition of 6 Da, and concentrating into water of dialysis solution under dialysis condition of 6h, and concentrating under dialysis condition of water for 6h under dialysis condition of concentrating into a dialysis bag.
Comparative example 8
A preparation method of moringa oleifera leaf polysaccharide specifically comprises the following steps:
adding sevage solution (volume ratio of chloroform to n-butanol is=4:1) with 25% (v/v) of polysaccharide extract P1A volume into 1L polysaccharide extract P1A, stirring at constant speed of 150r/min for 5min, standing for 30min for layering, collecting supernatant, adding sevage solution (volume ratio of chloroform to n-butanol is=4:1) with 25% (v/v) of supernatant volume, stirring at constant speed of 150r/min for 5min, standing for 30min for layering, collecting supernatant, repeating the above steps (adding sevage solution for removing protein) for 14 times until no precipitate is generated, adding XAD-16 nonionic macroporous resin with a mass to polysaccharide extract P1A volume ratio of 50g/L, stirring at constant speed of 110r/min for 1.5h at 55deg.C, ultrafiltering with ultrafiltration membrane with molecular weight of 10000Da for 6 times, collecting ultrafiltration retentate with molecular weight of >10000Da, concentrating under reduced pressure at 55deg.C until solid content in final concentrate is 3wt%, loading into dialysis bag with molecular weight cutoff of 11000Da, dialyzing at 7deg.C for 36 hr in deionized water, changing water every 10 hr, lyophilizing the liquid in the dialysis bag, dissolving with ultrapure water to obtain 5mg/mL solution, loading into pre-packed and balanced DEAE-Sepharose Fast Flow chromatographic column (2.6X40 cm), collecting eluate subjected to gradient elution with 500mL of 0.1mol/LNaCl aqueous solution, loading into dialysis bag with molecular weight cutoff of 3000Da, dialyzing at 4deg.C for 24 hr in deionized water, changing water every 8 hr, lyophilizing the liquid in the dialysis bag, obtaining the moringa oleifera leaf polysaccharide P7.
Analysis of results
1. Comparison of total sugar content in Moringa oleifera leaf polysaccharide
As can be seen from FIG. 1 (the different letters in FIG. 1 indicate that there is a significant difference between groups, P < 0.05), the methods of comparative examples 2-8 and examples 1-3 according to the present invention can further increase the total sugar content based on the Moringa oleifera leaf polysaccharide extract MEB, however, because comparative example 2 is not ultrafiltered, comparative example 3 is not enough in enzymolysis time, comparative example 4 only uses a single protease for enzymolysis, comparative example 5 is not dialyzed, and all of the five comparative examples only use a single resin for decolorization, the total sugar content of the obtained Moringa oleifera leaf polysaccharides P1, P2, P3, P4 is significantly lower, the effect of increasing the total sugar content of comparative examples 6, comparative example 7 and three examples cannot be achieved, and the total sugar content of Moringa oleifera leaf polysaccharides PA, PB, PC and P6 in comparative example 7 is greater than 80%.
2. Protein content comparison of Moringa leaf polysaccharide
The protein content in the moringa leaves is higher, but the polysaccharide is a main active substance component, and the effective reduction of the protein content in the polysaccharide extraction and purification process is very critical. The methods of examples 1-3 and comparative examples 2-8 are all purification methods of moringa oleifera leaf polysaccharide extract MEB (protein content exceeds 15%), and examples 1-3 are further optimized in the processes (comprising the addition sequence of resin, enzymolysis condition, ultrafiltration treatment, dialysis treatment and resin combined treatment) of comparative examples 2, 3, 4 and 5, and after optimization, the total sugar content in moringa oleifera leaf polysaccharide is obviously improved, so that the protein contents in moringa oleifera leaf polysaccharides P5, P6, PA, PB and PC prepared by the other five preparation methods are compared. As shown in figure 2 (different letters in figure 2 show that significant differences exist among groups, and p is less than 0.05), as the invention adopts a double protease deep enzymolysis method, a double resin combined adsorption impurity removal method and an ultrafiltration combined dialysis further purification method, proteins are efficiently converted into small molecular peptides, the small molecular peptides are further removed by using a resin, membrane separation method and a dialysis method, proteins are removed to a great extent, and the protein content in moringa oleifera leaf polysaccharides PA, PB and PC is reduced to 2%; the protein content in P5 prepared by the traditional strong acid (TCA) method and P6 prepared by the traditional organic reagent (sevage) method still exceeds 5 percent.
3. Comparing the yield of Moringa oleifera leaf polysaccharide with the polysaccharide retention rate
The sevage method combined with anion exchange chromatography in comparative example 8 is the most traditional and most classical method for obtaining high-purity polysaccharide, and the method is generally used for preparing polysaccharide with higher purity, but has the advantages of less sample loading, extremely low yield, extremely complex operation and longer period, and limits the popularization and application of the polysaccharide in industry.
As can be seen from fig. 3 (the different letters in fig. 3 indicate that there is a significant difference between groups, P < 0.05), the yields and polysaccharide retention rates of five moringa oleifera leaf polysaccharides P5, P6, PA, PB, PC, which are higher than the rest of the total sugar content, are all significantly reduced (P < 0.05) compared to the moringa oleifera leaf polysaccharide extract MEB, because of the removal of impurities such as proteins and the loss of part of the polysaccharides. The yield of polysaccharide P6 prepared by the sevage method is the lowest, and the yields of polysaccharide P5, PA, PB and PC prepared by the TCA method and the double-enzymolysis double-resin combined method are not remarkably different (P is less than 0.05) and reach 2.5 percent. The loss of the PA, PB and PC after deproteinization is minimum, the retention rate can reach 90%, the risk of excessive acidolysis of polysaccharide exists in the TCA method, the sevage method needs to be repeatedly operated for 14 times, the operation requirement is high, the process is complex, the reaction environment is severe, the structure of the polysaccharide can be influenced, a large amount of polysaccharide can be lost, and the retention rate of the polysaccharide is less than 60%. The double-enzymolysis double-resin combined method can not only remarkably improve the total sugar content, but also remove impurities to the greatest extent and break through the traditional purification method to ensure that the yield reaches 2.5%, the polysaccharide retention rate reaches 90%, the method is convenient for further application of high-purity polysaccharide, and the method is more efficient, safer, milder and simpler to operate, and can be applied to various fields such as common food, functional food and the like.
4. Molecular weight distribution and purity comparison of moringa oleifera leaf polysaccharide
As shown in Table 1, as the total sugar content increases, the molecular weight ratio of the moringa oleifera leaf polysaccharide gradually increases, the main peak intensity in the molecular weight distribution diagram is higher, the impurity peak intensity is lower, the distribution ratio of the moringa oleifera leaf polysaccharide PA, PB and PC prepared by the double-enzymolysis double-resin combined method can reach 80.5%, namely the purity of the polysaccharide can reach more than 80%, and the purity of the polysaccharide P7 obtained by the traditional chromatography in comparative example 8 is higher. Compared with the moringa oleifera leaf polysaccharide in the comparative example, the dispersion coefficient Mw/Mn of the moringa oleifera leaf polysaccharide in the example is closer to 1, which indicates that the uniformity of the polysaccharide is better. Therefore, the double-enzymolysis double-resin combined method can break through the technical bottleneck of the traditional purification method, and the moringa oleifera leaf polysaccharide with higher purity is prepared.
TABLE 1 molecular weight distribution of Moringa leaf polysaccharides
The different letters in table 1 indicate significant differences between groups (p < 0.05).
5. Color value comparison of Moringa oleifera leaf polysaccharide solutions
The moringa oleifera leaf polysaccharide extract contains partial micromolecular phenols, and is oxidized into quinone substances under the conditions of medium alkalinity and heating, so that the polysaccharide extract turns dark brown when the pH value is regulated to medium alkalinity (the optimal pH value of protease) before enzymolysis of protease, the pH value is regulated after the nonionic macroporous resin is added before enzymolysis to fully adsorb the micromolecular phenols, but does not turn dark brown, a certain salinity is introduced after the pH is regulated, dark enzymolysis products are generated due to Maillard reaction during enzymolysis, the ionic macroporous resin is added after enzymolysis, the color of the micromolecular peptides, pigments and the salinity can be further fully adsorbed, the color of the light polysaccharide solution is further improved, the clarity and the brightness of the light brown are improved, and a pure white moringa oleifera leaf polysaccharide freeze-dried sample can be obtained after ultrafiltration and dialysis treatment.
As can be seen from table 2, when all samples were dissolved in deionized water to prepare a polysaccharide solution with a concentration of 2mg/mL, the aqueous solution of moringa oleifera leaf polysaccharide extract MEB still had a distinct tan color, the aqueous solutions of moringa oleifera leaf polysaccharide P5 and P6 in the comparative example had a lower brightness and a pale yellow color than that of deionized water, while the aqueous solutions of moringa oleifera leaf polysaccharide PA, PB, and PC in the examples had an overall color difference value of <0.4 as compared with deionized water, and the aqueous solutions of moringa oleifera leaf polysaccharide exhibited a clear, transparent colorless shape.
TABLE 2 color values of Moringa oleifera leaf polysaccharide extracts and polysaccharide solutions
The different letters in table 2 indicate significant differences between groups (p < 0.05).
6. Analysis of glucose blocking ability of moringa oleifera leaf polysaccharide
As can be seen from fig. 4 (the different letters in fig. 4 indicate significant differences between groups, p < 0.05), when all samples were 10mg/mL (concentration in the final volume of digestion), the samples had a certain glucose blocking effect, and glucose was effectively adsorbed so that the concentration of glucose diffused into the dialysis fluid was reduced. The glucose blocking capacity of five moringa oleifera leaf polysaccharides P5, P6, PA, PB and PC with higher total sugar content is obviously improved by 20.88% compared with that of moringa oleifera leaf polysaccharide extract MEB (P is less than 0.05), and along with the improvement of the total sugar content and the improvement of the polysaccharide purity, the glucose blocking capacity can be improved along with the improvement of the total sugar content, and the glucose dialysis delay index of the moringa oleifera leaf polysaccharide with the highest purity in the in-vitro simulated digestion process is highest and can reach 45%.
7. Analysis of cholate adsorption capacity and cholesterol micelle dissolution inhibition capacity of moringa oleifera leaf polysaccharide
From fig. 5 (the different letters in fig. 5 indicate significant differences between groups, p < 0.05), and fig. 6 (the different letters in fig. 6 indicate significant differences between groups, p < 0.05), it is known that when all samples are 5mg/mL (concentration in final volume of digestion), all samples have a certain adsorption effect on gastrointestinal cholate during simulated digestion, and have an inhibitory effect on solubility of cholesterol in gastrointestinal micelles. In the embodiment, the adsorption capacity of cholate of the moringa oleifera leaf polysaccharide PA, PB and PC is highest and can reach 63%, which indicates that the increase of the total sugar content and the purity improvement of the polysaccharide are beneficial to enhancing the adsorption effect of the polysaccharide on cholate.
Compared with the moringa oleifera leaf polysaccharide P5 prepared by the TCA method, the other four polysaccharides can better inhibit the solubility of cholesterol in cholate micelles generated in gastrointestinal digestion, and can effectively reduce the absorption of the cholesterol by more than 58%. In conclusion, the double-enzymolysis double-resin combined method can improve the activity of the moringa oleifera leaf polysaccharide in inhibiting excessive digestion and lipid absorption of the gastrointestinal tract, and plays a role in regulating and controlling lipid absorption.
In general, the total sugar content of the moringa oleifera leaf polysaccharides P5, P6 and P7 in the comparative examples 6, 7 and 8 can reach more than 70%, but the former two preparation methods use strong acid with strong corrosiveness and organic reagent, so that the application of the moringa oleifera leaf polysaccharides in foods is limited, and the latter are obtained by anion exchange chromatography in the traditional polysaccharide purification method, and the preparation time is long, the yield is extremely low, and the application feasibility is low. The double enzyme-double resin combination method of the obtained examples is optimized on the basis of comparative examples 2, 3, 4 and 5, so that the total sugar content and the purity of the polysaccharide can be greatly improved to 80%, the yield can be effectively ensured to 2.5% by removing impurities, the polysaccharide retention rate is ensured to 90%, and the application of the high-purity high-activity polysaccharide in various food systems can be realized. The glucose dialysis delay index of the moringa oleifera leaf polysaccharides PA, PB and PC is highest and is more than 45% when the concentration in the final digestion volume is 10 mg/mL; when the concentration in the final digestion volume is 5mg/mL, the cholate adsorption rate and the cholesterol micelle dissolution inhibition rate of the moringa oleifera leaf polysaccharides PA, PB and PC are also highest and are more than 58%. The polysaccharide is the main active ingredient in the moringa leaves, the purity of the polysaccharide is improved, the activity can be correspondingly improved, compared with the moringa leaf polysaccharide extract MEB (CN 202210833825.6) before purification, the color is lighter, the total sugar content is improved by 52.50%, the purity is improved by 49.90%, the glucose dialysis delay index is improved by 20.88%, the cholate adsorption rate is improved by 17.39%, the cholesterol micelle dissolution inhibition rate is improved by 3.07%, and the dual-enzyme dual-resin combined method can effectively improve the purity of the moringa leaf polysaccharide and regulate the glycolipid absorption activity. The double enzymolysis in the embodiment ensures that protein substances are fully degraded into small molecular peptides through long-time enzymolysis, and the small molecular peptides are removed through two steps of ultrafiltration and dialysis; the double resin method can make the polysaccharide more colorless and transparent, and can absorb salt besides small molecular pigment substances to further increase the occupancy rate of the polysaccharide. The moringa oleifera leaf polysaccharide finally obtained in the embodiment has higher purity and can regulate the absorption activity of glycolipid, and has the potential of being applied to various foods and health care products as food ingredients.
The invention provides a method for improving purity and activity of moringa oleifera leaf polysaccharide. The preparation method adopts double protease enzymolysis to fully degrade protein into small molecular peptide, uses nonionic macroporous resin to adsorb phenolic substances before enzymolysis, uses ionic macroporous resin to adsorb enzymolysis products and salt, and then separates the small molecular peptide through ultrafiltration, dialysis and other processes to obtain the moringa oleifera leaf polysaccharide with high purity, strong absorption activity of regulating glycolipid, the preparation process is simple and mild, the polysaccharide loss rate is low, the technical bottleneck of difficult purification of plant polysaccharide is broken through, the yield and application prospect of the high-purity high-activity polysaccharide are greatly improved, the required processing equipment is simple and easy to operate, the food processing specification is met, the potential of industrial popularization and application is provided, and the technical advantage and the advancement are obvious.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and one skilled in the art will realize that the present invention discloses a method for improving purity and activity of moringa oleifera leaf polysaccharide without departing from the spirit of the present invention.
Claims (10)
1. The preparation method of the moringa oleifera leaf polysaccharide with high purity and high activity is characterized by comprising the following steps of:
(1) Preparing a moringa oleifera leaf polysaccharide extract: preparing an extracting solution 2 in the Chinese patent application claim 1 with the application number of CN202210833825.6, namely, the polysaccharide extracting solution 1;
(2) Resin adsorption and filtration: adding nonionic macroporous resin into the polysaccharide extract 1 obtained in the step (1), stirring at a constant speed, and filtering to obtain a polysaccharide extract 2;
(3) Protease enzymolysis: regulating the pH value of the polysaccharide extract 2 obtained in the step (2), adding Alcalase enzyme and pancreatin, stirring at constant temperature for enzymolysis, and obtaining a polysaccharide extract 3;
(4) Resin adsorption, filtration and centrifugation: adding ion macroporous resin into the polysaccharide extract 3 obtained in the step (3), uniformly stirring, filtering, and centrifuging to obtain polysaccharide extract 4;
(5) Ultrafiltration and concentration: carrying out ultrafiltration separation on the polysaccharide extract 4 obtained in the step (4), and concentrating to obtain a polysaccharide extract 5;
(6) And (3) dialysis: dialyzing polysaccharide extract 5 obtained in step (5) against polysaccharide extract 6;
(7) And (3) freeze drying: and (3) freeze-drying the polysaccharide extract 6 obtained in the step (6) to obtain moringa oleifera leaf polysaccharide.
2. The method for preparing the high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (1), the preparation method of the polysaccharide extract 1 is specifically as follows:
1) Crushing by liquid nitrogen: crushing the dried moringa leaves into dry powder by a liquid nitrogen crushing device to obtain moringa leaf dry powder;
2) Enzymatic hydrolysis of cellulase: uniformly mixing the moringa leaf dry powder obtained in the step 1) with water, regulating the pH, adding cellulase after fully and uniformly mixing, and stirring at constant temperature for enzymolysis to obtain a suspension 1;
3) Heating and extracting: heating and extracting the suspension 1 to obtain a suspension 2;
4) And (3) yeast fermentation: adding active dry yeast into the suspension 2, and fermenting at constant temperature to obtain a suspension 3;
5) Centrifuging and concentrating: centrifuging the suspension 3, and concentrating the supernatant under reduced pressure to obtain an extract A;
6) Ethanol precipitation: adding ethanol solution into the extract A, mixing to obtain mixed solution, standing, centrifuging, collecting precipitate, adding water, heating for dissolving, and concentrating under reduced pressure to obtain polysaccharide extract 1.
3. The method for preparing the high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (2), the nonionic macroporous resin is XAD-16 macroporous resin, the ratio of the mass of the nonionic macroporous resin to the volume of the polysaccharide extract 1 in the step (1) is 40-60 g/L, the stirring speed is 100-120 r/min, the stirring temperature is 50-60 ℃, the stirring time is 1-2 h, and the mesh number of the filtering cloth is 80-120 meshes.
4. The preparation method of the high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (3), a sodium hydroxide solution is used for adjusting the pH value of a polysaccharide extract 2 to 7.0-8.0, the mass of Alcalase enzyme added is 1-3% of the mass of protein in the polysaccharide extract 2 obtained in the step (2), the mass of pancreatin added is 1-3% of the mass of protein in the polysaccharide extract 2 obtained in the step (2), the enzymolysis temperature is 50-60 ℃, the stirring speed is 120-180 r/min, and the enzymolysis time is 24-36 h.
5. The method for preparing the high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (4), the ionic macroporous resin is D-280 macroporous resin, and the ratio of the mass of the added ionic macroporous resin to the volume of the polysaccharide extract 3 in the step (3) is 40-60 g/L.
6. The method for preparing high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (4), the stirring speed is 100-120 r/min, the stirring temperature is 50-60 ℃, the stirring time is 1-2 h, the mesh number of the filtering cloth is 80-120 mesh, the centrifugation temperature is 4-8 ℃, the centrifugal force is 6000-8000 g, and the centrifugation time is 15-25 min.
7. The method for preparing high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (5), ultrafiltration is performed by adopting an ultrafiltration membrane with a molecular weight of 8000-12000 Da, the number of times of ultrafiltration is 5-7 times, the polysaccharide extract 5 is an ultrafiltration retentate with a molecular weight of >10000Da, the concentration temperature is 50-60 ℃, and the solid content in the polysaccharide extract 5 is 2-4 wt%.
8. The method for preparing the high-purity and high-activity moringa oleifera leaf polysaccharide according to claim 1, wherein in the step (6), the polysaccharide extract 5 is put into a dialysis bag with a cut-off molecular weight of 8000-14000 Da, is dialyzed for 24-48 hours in deionized water with a temperature of 4-10 ℃, water is changed every 8-12 hours, and the polysaccharide extract 6 is liquid in the dialysis bag.
9. The moringa oleifera leaf polysaccharide with high purity and high activity prepared by the preparation method of any one of claims 1-8.
10. The use of a high purity and high activity Moringa oleifera leaf polysaccharide of claim 9 in foods, health products or pharmaceuticals for reducing blood glucose and controlling blood lipid.
Priority Applications (1)
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