CN108640953B - Refining method of pumpkin oligosaccharide by adopting macroporous resin - Google Patents
Refining method of pumpkin oligosaccharide by adopting macroporous resin Download PDFInfo
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Images
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- 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
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- 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
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/40—Colouring or decolouring of foods
- A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
- A23L5/43—Addition of dyes or pigments, e.g. in combination with optical brighteners using naturally occurring organic dyes or pigments, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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- A—HUMAN NECESSITIES
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- 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
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
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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
Abstract
The invention discloses a method for refining pumpkin oligosaccharide by adopting macroporous resin, which comprises the following steps: adding water into the crude extract of pumpkin oligosaccharide to obtain pumpkin oligosaccharide solution, adsorbing with chromatographic column filled with macroporous resin, and collecting eluate; standing, leaching macroporous resin with deionized water, evaporating, concentrating, and lyophilizing to obtain colorless pumpkin oligosaccharide; washing with ethanol solution, soaking with ethanol, passing NaOH solution through resin column, soaking, and washing with water to neutral; passing hydrochloric acid through a resin column, soaking, and recovering macroporous resin; rotary evaporation and freeze drying to recover color impurity and ethanol. The refining method can remove monosaccharide and protein impurities while achieving the high-efficiency decolorization of the pumpkin oligosaccharide, and the purity of the obtained pumpkin oligosaccharide is as high as 95-96%; the process has mild treatment conditions, does not need high temperature or acid-base treatment, does not cause degradation of pumpkin oligosaccharide, and can recycle the obtained colored impurity melanoidin.
Description
Technical Field
The invention belongs to the technical field of purification of pumpkin oligosaccharides, and particularly relates to a refining method of pumpkin oligosaccharides by adopting macroporous resin.
Background
Many studies have confirmed that functional sugar components such as polysaccharides and oligosaccharides in pumpkin have a blood sugar regulating function, and the functional sugar is widely recognized as a component derived from natural plants in terms of safety. The oligosaccharide (oligosaccharide) is called oligosaccharide, and is a low-degree polymeric sugar formed by connecting 2-10 monosaccharides into a straight chain or a branched chain through glycosidic bonds, and the molecular weight is 200-3000D. Because the monosaccharide molecules are different in combination position and combination type, the oligosaccharide with rich varieties is formed. The oligosaccharide has good functional characteristics, can be used as a sweet source, is not degraded by gastric acid and gastric enzyme of a human body, is not absorbed in small intestine, selectively promotes the proliferation of beneficial bacteria and inhibits pathogenic bacteria and diarrhea after entering large intestine, and also has the functions of protecting liver, reducing serum cholesterol, resisting dental caries and the like. Oligosaccharides are paid much attention to due to unique functional characteristics thereof, and have good research value and application potential in the fields of functional foods, feeds, additives and the like in the future.
At present, the pumpkin polysaccharide is prepared by a water extraction and alcohol precipitation method, and the supernatant after the precipitation of macromolecular polysaccharide in the separation process still contains a large amount of carbohydrate, which is presumed to be oligosaccharide with lower molecular weight. Generally, a membrane filtration method is adopted to effectively separate a pumpkin oligosaccharide crude product in a supernatant, however, the product is dark brown, is caused by pigments in pumpkins and colored impurities generated in the processing process, and causes obstacles to pumpkin oligosaccharide purification, structural research and industrial application.
At present, common decoloring methods in the food industry include an organic solvent decoloring method, an activated carbon adsorption decoloring method and a hydrogen peroxide decoloring method. However, when the methods are applied to pumpkin oligosaccharide decoloring, the decoloring efficiency is low, oligosaccharide structures such as oxydol are damaged, the operation cost is high, the recovery is difficult, and the like. Therefore, the research on the efficient and environment-friendly pumpkin oligosaccharide decoloring and impurity removing method has important significance.
Macroporous resin is a class of organic polymer adsorbent developed in recent years and is an effective method for decoloring natural extracts. Compared with the traditional decoloring method, the macroporous resin decoloring method has the characteristics of simple operation, high separation efficiency, easy recovery, low cost, small pollution and the like.
Disclosure of Invention
The invention aims to provide a refining method of pumpkin oligosaccharide by adopting macroporous resin, thereby overcoming the defects of poor decoloring effect of colored substances, low impurity removal rate and oligosaccharide structure damage in the existing pumpkin oligosaccharide.
In order to achieve one or more of the above objects, the present invention provides, in one embodiment, a method for refining pumpkin oligosaccharides using macroporous resin, comprising the steps of:
(1) adding water into the pumpkin oligosaccharide crude extract to prepare a pumpkin oligosaccharide upper column solution; enabling the pumpkin oligosaccharide upper column solution with the column volume (BV) of 0.8-1.5 times to pass through a chromatographic column filled with macroporous resin for upper column adsorption, and then collecting effluent liquid;
(2) standing, then leaching the macroporous resin with deionized water, mixing the residual pumpkin oligosaccharide eluent in the column with the effluent liquid obtained in the step (1), and then evaporating, concentrating and freeze-drying to obtain colorless pumpkin oligosaccharide;
(3) eluting and adsorbing colored impurities on the macroporous resin by using 1.5-2.2 BV of ethanol solution; soaking in ethanol, washing with water until no alcohol smell exists, passing NaOH solution through resin column, soaking, and washing with water until neutral; then hydrochloric acid is adopted to pass through a resin column and is soaked, water is washed to be neutral, and macroporous resin is recovered; and finally, recovering the colored impurities and the ethanol by rotary evaporation and freeze drying.
In the refining method, part of monosaccharide and protein in the pumpkin oligosaccharide crude extract can be removed through macroporous resin. The specific removal principle is as follows: separating oligosaccharide and monosaccharide with different molecular weights by virtue of the molecular sieve action of the macroporous resin; according to the difference of the structure, physical property and chemical property of the protein and sugar, the adsorption and elution performances of the protein and oligosaccharide in the pumpkin oligosaccharide crude extract on the macroporous resin are different, so that part of the protein is removed; in conclusion, partial monosaccharide and protein in the pumpkin oligosaccharide are finally removed through the macroporous adsorption resin.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, wherein the mass percentage concentration of the pumpkin oligosaccharide solution on the column in step (1) is 230-280 mg/ml;
and/or enabling the pumpkin oligosaccharide upper column solution to pass through a chromatographic column filled with macroporous resin at the flow rate of 0.8-1.5 BV/h.
Under the condition of a certain amount of macroporous resin, the decolorizing effect of an experimental group with low sample loading flow rate and small sample loading amount and an experimental group with low eluting speed and large eluting liquid amount is better, but the cost is increased due to too low speed and too large amount.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, wherein the macroporous resin is pretreated before use in step (1), specifically comprising the following operations: firstly, soaking macroporous resin in deionized water, and washing with ethanol until the macroporous resin is not turbid; then soaking the mixture in ethanol, and washing the mixture by deionized water until no alcohol smell exists; and then, passing a NaOH solution with the mass percent concentration of 5% through a resin column and soaking the resin, washing with water to be neutral, passing hydrochloric acid with the mass percent concentration of 5% through the resin column and soaking the resin, and finally washing with water to be neutral to obtain the treated macroporous resin.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by adopting macroporous resin, and in the pretreatment, the soaking time by adopting deionized water and ethanol is 20-30 h.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by adopting macroporous resin, and in the pretreatment, the resin is soaked by adopting NaOH solution or hydrochloric acid for 1-4 h.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharides by using macroporous resin, in the step (2), the standing time is 45-60 min;
and/or leaching the macroporous resin by the deionized water at the speed of 1.0-3.0 BV/h.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, wherein the volume percentage concentration of the ethanol solution in the step (3) is 60-80%.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, wherein the macroporous resin is selected from one or two of ion exchange resin and nonpolar macroporous resin, preferably the nonpolar macroporous resin.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by adopting macroporous resin, wherein the nonpolar macroporous resin is selected from one or more of DM21 type macroporous adsorption resin and DM28 type macroporous adsorption resin.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by adopting macroporous resin, wherein the material of the chromatographic column is reinforced glass, and the specification is 6cm × 80 cm;
and/or, the macroporous resin is loaded into the chromatographic column at 3/4 when in use.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by adopting macroporous resin, and the aspect ratio of resin filled in the chromatographic column is 1: 10. Wherein, under the condition of a certain amount of macroporous resin, the aspect ratio of the height to the diameter of the chromatographic column is large, which is beneficial to removing monosaccharide impurities.
In a preferred embodiment of the present invention, the present invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, wherein the preparation method of the crude extract of pumpkin oligosaccharide in step (1) comprises:
(1) removing seeds of pumpkins, cutting the pumpkins into pieces, adding water, pulping, extracting with hot water, centrifuging, taking supernate, and concentrating the supernate to 1/2-1/5 of the volume of the original supernate to obtain concentrated solution;
(2) adding ethanol for precipitation, centrifuging and taking supernatant;
(3) diluting the supernatant, filtering with 20 μm filter bag, passing the filtrate through ultrafiltration section, wherein the cut-off molecular weight of the ultrafiltration membrane is 3000Da, passing the permeate through nanofiltration section, and the cut-off molecular weight of the nanofiltration membrane is 300Da, and the cut-off liquid is crude extract of pumpkin oligosaccharide.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, in the preparation method of the pumpkin oligosaccharide crude extract, the temperature of hot water in the step (1) is 80-90 ℃, and the time of hot water extraction is 1-3 h.
In a preferred embodiment of the present invention, the present invention provides a method for refining pumpkin oligosaccharide by using macroporous resin, in the preparation method of the crude extract of pumpkin oligosaccharide, the volume percentage concentration of the ethanol solution in step (2) is 70-90%, preferably 80%.
In a preferred embodiment of the invention, the invention provides a refining method of pumpkin oligosaccharide by using macroporous resin, and in the preparation method of the pumpkin oligosaccharide crude extract, the volume ratio of the ethanol solution in the step (2) to the concentrated solution obtained in the step (1) is 2-5: 1.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention applies the macroporous resin adsorption technology to the production process of the pumpkin oligosaccharide, the macroporous resin adsorbs colored impurities in the solution, and the molecular sieve has certain removing effect on monosaccharide impurities in the pumpkin oligosaccharide crude product, so that the high-efficiency decoloration of the pumpkin oligosaccharide is realized, the monosaccharide and protein impurities can be removed at the same time, and the colored impurities can be recycled; the purity of the obtained pumpkin oligosaccharide is high and reaches 94-96%, no obvious impurity is found through high performance liquid chromatography detection, and the quality of the pumpkin oligosaccharide product is further improved.
(2) In the refining method adopted by the invention, the impurities and the color of the pumpkin oligosaccharide can be efficiently removed without adjusting the pH value when the pumpkin oligosaccharide upper column solution is prepared; the refining method has mild treatment conditions, low energy consumption and less generated waste, does not need high temperature or acid-base treatment in the macroporous resin decoloring process, does not cause degradation of the pumpkin oligosaccharide, and can prove that the structure of the main component oligosaccharide is not changed through infrared spectrum comparison of the pumpkin oligosaccharide before and after refining.
(3) The colored impurities removed by the refining method are colored matter melanoidin generated by Maillard reaction in the extraction process of the pumpkin oligosaccharide, compared with the traditional activated carbon decoloring and hydrogen peroxide decoloring methods, the method can collect oligosaccharide decoloring liquid (water washing) and colored impurity eluent (70% ethanol washing) respectively, and can recover the colored components (the recovery rate is 79%) while keeping the structural characteristics and properties of the oligosaccharide; in addition, as the melanoidin which is a color product of the Maillard reaction has antioxidant, anti-caries and blood sugar regulating effects in some researches, the recovered colored component has better application prospect in the aspects of health food raw materials and functional additives.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application. In the drawings:
FIG. 1 is a comparison of color of pumpkin oligosaccharide solution according to the present invention before and after refining.
FIG. 2 is a High Performance Liquid Chromatogram (HPLC) for detecting the molecular weight of pumpkin oligosaccharide solution obtained in example 1 of the present invention.
Fig. 3 is a uv-vis full-wavelength scan of the pumpkin oligosaccharide solution obtained in example 2 according to the present invention.
FIG. 4 is an infrared spectrum of pumpkin oligosaccharide solution obtained in example 1 of the present invention.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The materials, reagents and the like used in the above examples are commercially available unless otherwise specified.
Experimental equipment and conditions adopted for ultraviolet visible light full-wavelength scanning are as follows: UV-visible spectrophotometer UV-visspectrophotometer (T6-1650E).
Experimental equipment and conditions adopted by the molecular weight high performance liquid chromatography are as follows: the German Noel Knauer high performance liquid chromatograph has the sample introduction amount of 20ul, the detection time of 20min, the sample introduction concentration of 1 percent and the column temperature of 38 ℃.
Experimental equipment and conditions adopted by infrared spectroscopy: a fourier infrared spectrometer Nicolet 6700FTIR spectrometer (Madison, WI, u.s.a).
Example 1
A refining method of pumpkin oligosaccharide by adopting macroporous resin comprises the following steps:
(1) macroporous resin pretreatment: soaking DM28 type macroporous adsorbent resin in deionized water for 24h, washing with ethanol until the resin is not turbid, soaking in ethanol for 24h, washing with water until no alcohol smell exists, passing 5% NaOH solution through a resin column and soaking for 2h, washing with water until the resin is neutral, passing 5% HCl through the resin column and soaking for 4h, washing with water until the resin is neutral, and keeping at 4 ℃ for later use; when in use, the filler is arranged at 3/4 of a chromatographic column (6cm multiplied by 80cm) to ensure that the filler is even and flat and has no bubbles;
(2) adding water into the crude extract of pumpkin oligosaccharide to prepare a solution with the concentration of 250mg/ml, passing the solution with the column volume (BV) of 1.0 time through a chromatographic column filled with macroporous resin at the flow rate of 1.0BV/h, carrying out column adsorption, and collecting the effluent;
(3) standing for 50min after the pumpkin oligosaccharide solution completely passes through a separation column filled with macroporous resin, eluting the macroporous resin adsorbed with colored impurities by using 2.0BV deionized water at the speed of 1.5BV/h, combining the residual pumpkin oligosaccharide elution in the column with the effluent liquid obtained in the step (2), evaporating and concentrating, and freeze-drying to obtain colorless pumpkin oligosaccharide;
(4) then 2.0BV of ethanol solution with the volume percentage concentration of 70 percent is used for eluting the colored impurities adsorbed on the macroporous resin at the speed of 1.5 BV/h;
(5) after each operation is finished, carrying out macroporous resin cleaning and recovery, soaking for 24h by adopting ethanol, washing by using water until no alcohol smell exists, passing a 5% NaOH solution through a resin column and soaking for 2h, washing to be neutral, passing 5% HCl through the resin column and soaking for 2h, washing to be neutral, and recovering macroporous resin; and finally, recovering the colored impurities and the ethanol by adopting rotary evaporation and freeze drying.
The preparation method of the pumpkin oligosaccharide crude extract comprises the following steps: removing seeds of pumpkins, cutting the pumpkins into pieces, adding 2-5 times of water by weight, pulping, extracting for 2 hours by adopting hot water at 85 ℃, centrifuging to obtain supernate, and concentrating to 1/2 of the volume of the original supernate;
then, adding 80% ethanol with 2 times volume of the mixture for precipitation, and centrifuging to obtain supernatant; diluting the supernatant after extracting the pumpkin polysaccharide and filtering the supernatant through a filter bag with the diameter of 20 mu m;
the filtrate firstly passes through an ultrafiltration section, the molecular weight cutoff of the ultrafiltration membrane is 3000Da, and then passes through a nanofiltration section, the molecular weight cutoff of the nanofiltration membrane is 300Da, and the liquid to be cut off is the pumpkin oligosaccharide crude extract.
The model of macroporous resin used in the implementation is DM28 type macroporous adsorption resin; the chromatography column material is reinforced glass with specification of (6cm × 80cm), and the diameter-height ratio of the filled resin is 1: 10; after passing through a chromatographic column filled with macroporous resin, the standing time is 55 min.
For example, as shown in fig. 1, the color of the pumpkin oligosaccharide solution prepared in this embodiment before and after refining is 92.6% decolorized by macroporous resin refining, and as can be seen from the color comparison in fig. 1, the dark brown solution becomes a nearly transparent solution after macroporous resin refining, so that the refining method of this embodiment achieves a better decolorization effect.
As shown in a high performance liquid chromatogram for detecting the molecular weight of the pumpkin oligosaccharide liquid obtained in example 1 in the figure 2, the molecular weight distribution of the refined pumpkin oligosaccharide liquid is concentrated in 1000-1300 Da, the molecular weight range belongs to the molecular weight distribution range of oligosaccharide, the purity is 96%, and the decolorization rate is 92.4%.
The infrared spectrogram of the pumpkin oligosaccharide solution obtained in the example 1 in fig. 4 shows that the chemical structures of the main components of the pumpkin oligosaccharide before and after decolorization are not significantly different, which indicates that the macroporous adsorption resin method does not damage the structure of the pumpkin oligosaccharide during the refining process, and can well protect the functional groups in the pumpkin oligosaccharide.
Example 2
A refining method of pumpkin oligosaccharide by adopting macroporous resin comprises the following steps:
(1) macroporous resin pretreatment: soaking DM21 type macroporous adsorbent resin in deionized water for 30h, washing with ethanol until the resin is not turbid, soaking in ethanol for 30h, washing with water until no alcohol smell exists, passing 5% NaOH solution through a resin column and soaking for 4h, washing with water until the resin is neutral, passing 5% HCl through the resin column and soaking for 4h, washing with water until the resin is neutral, and keeping at 4 ℃ for later use; when in use, the filler is filled into 3/4 parts of the chromatographic column to ensure that the filler is uniform and flat and has no bubbles;
(2) adding water into the crude extract of pumpkin oligosaccharide to prepare a solution with the concentration of 280mg/ml, passing the solution with the column volume (BV) of 1.5 times of that of the crude extract through a chromatographic column filled with macroporous resin at the flow rate of 1.5BV/h, carrying out column adsorption, and collecting the effluent;
(3) standing for 60min after the pumpkin oligosaccharide solution completely passes through a separation column filled with macroporous resin, eluting the macroporous resin adsorbed with colored impurities by using 2.5BV deionized water at the speed of 1.0BV/h, combining the residual pumpkin oligosaccharide elution in the column with the effluent liquid obtained in the step (2), evaporating and concentrating, and freeze-drying to obtain colorless pumpkin oligosaccharide;
(4) then 2.2BV of ethanol solution with the volume percentage concentration of 80 percent is used for eluting the colored impurities adsorbed on the macroporous resin at the speed of 1.0 BV/h;
(5) after each operation is finished, carrying out macroporous resin cleaning and recovery, soaking for 24h by adopting ethanol, washing by using water until no alcohol smell exists, passing a 5% NaOH solution through a resin column and soaking for 4h, washing by using water until the solution is neutral, passing 5% HCl through the resin column and soaking for 4h, and washing by using water until the solution is neutral; finally, recovering the macroporous resin; and finally, recovering the colored impurities and the ethanol by adopting rotary evaporation and freeze drying.
The preparation method of the pumpkin oligosaccharide crude extract comprises the following steps: removing seeds of fructus Cucurbitae Moschatae, cutting into pieces, pulping with 5 times of water, extracting with 90 deg.C hot water for 2 hr, centrifuging to obtain supernatant, and concentrating to 1/5 of the original supernatant;
then, adding 80% ethanol with 5 times volume of the mixture for precipitation, and centrifuging to obtain supernatant; diluting the supernatant after extracting the pumpkin polysaccharide and filtering the supernatant through a filter bag with the diameter of 20 mu m;
the filtrate firstly passes through an ultrafiltration section, the molecular weight cutoff of the ultrafiltration membrane is 3000Da, and then passes through a nanofiltration section, the molecular weight cutoff of the nanofiltration membrane is 300Da, and the liquid to be cut off is the pumpkin oligosaccharide crude extract.
The model of the macroporous resin used in the implementation is DM21 type macroporous adsorption resin; the chromatography column material is reinforced glass with the specification of 6cm multiplied by 80cm, and the diameter-height ratio of the filled resin is 1: 10; standing for 50min after passing through chromatography column filled with macroporous resin.
The decolorization rate of the pumpkin oligosaccharide prepared in this example is 89.1%, the purity is 95%, and according to the ultraviolet-visible spectrum comparison before and after the decolorization of the pumpkin oligosaccharide liquid shown in fig. 3, the absorption peak of protein at 280nm is reduced, so that the protein in the crude extract of the pumpkin oligosaccharide can be removed in this example 2.
Example 3
The crude extract of squash oligosaccharide used in this example was the crude extract of squash oligosaccharide prepared in example 1.
A refining method of pumpkin oligosaccharide by adopting macroporous resin comprises the following steps:
(1) macroporous resin pretreatment: soaking DM28 type macroporous adsorbent resin in deionized water for 20h, washing with ethanol until the resin is not turbid, soaking in ethanol for 20h, washing with water until no alcohol smell exists, passing 5% NaOH solution through a resin column and soaking for 1h, washing with water until the resin is neutral, passing 5% HCl through the resin column and soaking for 1h, washing with water until the resin is neutral, and keeping at 4 ℃; when in use, the filler is arranged at 3/4 of a chromatographic column (6cm multiplied by 80cm) to ensure that the filler is even and flat and has no bubbles;
(2) adding water into the crude extract of pumpkin oligosaccharide to prepare a solution with the concentration of 230mg/ml, passing the solution with the column volume (BV) of 0.8 times of that of the crude extract through a chromatographic column filled with macroporous resin at the flow rate of 0.8BV/h, carrying out column adsorption, and collecting the effluent;
(3) standing for 45min after the pumpkin oligosaccharide solution completely passes through a separation column filled with macroporous resin, then eluting the macroporous resin adsorbed with colored impurities by using 1.5BV deionized water at the speed of 1.0BV/h, combining the residual pumpkin oligosaccharide elution in the column with the effluent liquid obtained in the step (2), evaporating and concentrating, and freeze-drying to obtain colorless pumpkin oligosaccharide;
(4) then 2.2BV of 60 percent ethanol solution with volume percentage concentration is used to elute the colored impurities adsorbed on the macroporous resin at the speed of 1.5 BV/h;
(5) after each operation is finished, carrying out macroporous resin cleaning and recovery, soaking for 24h by adopting ethanol, washing by using water until no alcohol smell exists, passing a 5% NaOH solution through a resin column and soaking for 2h, washing to be neutral, passing 5% HCl through the resin column and soaking for 2h, washing to be neutral, and recovering macroporous resin; and finally, recovering the colored impurities and the ethanol by adopting rotary evaporation and freeze drying.
The pumpkin oligosaccharide decoloring rate and the purity of the pumpkin oligosaccharide prepared by the embodiment are 88.6% and 94%.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (7)
1. A refining method of pumpkin oligosaccharide by adopting macroporous resin is characterized by comprising the following steps:
(1) adding water into the pumpkin oligosaccharide crude extract to prepare a pumpkin oligosaccharide upper column solution; enabling the pumpkin oligosaccharide upper column solution with the column volume (BV) of 0.8-1.5 times to pass through a chromatographic column filled with macroporous resin for upper column adsorption, and then collecting effluent liquid;
(2) standing, then leaching the macroporous resin with deionized water, mixing the residual pumpkin oligosaccharide eluent in the column with the effluent liquid obtained in the step (1), and then evaporating, concentrating and freeze-drying to obtain colorless pumpkin oligosaccharide;
(3) eluting and adsorbing colored impurities on the macroporous resin by using 1.5-2.2 BV of ethanol solution; soaking in ethanol, washing with water until no alcohol smell exists, passing NaOH solution through resin column, soaking, and washing with water until neutral; then hydrochloric acid is adopted to pass through a resin column and is soaked, water is washed to be neutral, and macroporous resin is recovered; finally, carrying out rotary evaporation and freeze drying to recover colored impurities and ethanol;
the preparation method of the pumpkin oligosaccharide crude extract in the step (1) comprises the following steps:
(1) removing seeds of pumpkins, cutting the pumpkins into pieces, adding water, pulping, extracting with hot water, centrifuging, taking supernate, and concentrating the supernate to 1/2-1/5 of the volume of the original supernate to obtain concentrated solution; (2) adding ethanol for precipitation, centrifuging and taking supernatant; (3) diluting the supernatant, filtering with a 20 μm filter bag, passing the filtrate through an ultrafiltration section, wherein the cut-off molecular weight of the ultrafiltration membrane is 3000Da, passing the permeate through a nanofiltration section, and the cut-off molecular weight of the nanofiltration membrane is 300Da, wherein the cut-off liquid is crude extract of pumpkin oligosaccharide;
the macroporous resin in the step (1) is pretreated before use, and the specific operation is as follows: firstly, soaking macroporous resin in deionized water, and washing with ethanol until the macroporous resin is not turbid; then soaking the mixture in ethanol, and washing the mixture by deionized water until no alcohol smell exists; then NaOH solution with the mass percentage concentration of 5% passes through the resin column and soaks the resin, water washing is carried out until the resin is neutral, hydrochloric acid with the mass percentage concentration of 5% passes through the resin column and soaks the resin, and finally water washing is carried out until the resin is neutral, thus obtaining the treated macroporous resin;
the macroporous resin is nonpolar macroporous resin, and the nonpolar macroporous resin is selected from one or more of DM21 type macroporous adsorption resin and DM28 type macroporous adsorption resin.
2. The refining method of claim 1, wherein the mass percent concentration of the pumpkin oligosaccharide solution on the column in step (1) is 230-280 mg/ml.
3. The refining method according to claim 1, wherein the ethanol solution in the step (3) has a concentration of 60 to 80% by volume.
4. The purification method according to claim 1, wherein the material of the chromatography column is a tempered glass having a specification of 6cm x 80 cm.
5. The refining method of claim 1, wherein the ethanol solution in step (2) of the preparation method of the crude pumpkin oligosaccharide extract has a volume percentage concentration of 70-90%.
6. The refining method of claim 5, wherein the ethanol solution has a concentration of 80% by volume.
7. The refining method according to claim 1, wherein the volume ratio of the ethanol solution in the step (2) to the concentrated solution obtained in the step (1) is 2-5: 1.
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| CN112899326A (en) * | 2021-03-31 | 2021-06-04 | 新疆大学 | Method for preparing melon peel pectin oligosaccharide with antibacterial activity by using immobilized enzyme |
| CN114210312B (en) * | 2021-12-17 | 2024-03-22 | 金七药业股份有限公司 | Regeneration treatment method for macroporous resin low-temperature freezing |
| CN114163486A (en) * | 2021-12-22 | 2022-03-11 | 云南三七科技有限公司 | Method for preparing oligosaccharide from pseudo-ginseng |
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