阅读说明：本技术 一种半乳甘露聚糖的分离纯化方法 (Separation and purification method of galactomannan ) 是由 武建 袁润 张伟 曹毅 彭满菊 马敏滢 于 2020-06-15 设计创作，主要内容包括：本发明提供一种半乳甘露聚糖的分离纯化方法,包括如下步骤：取中性甘露聚糖酶溶液、植物胶,经酶解反应后,得到酶解液；将所述酶解液进行离心,得到离心液与离心沉淀物；将所述离心液采用中空纤维膜进行过滤,按照所述中空纤维膜的截留分子量,将离心液进行分离,得到不同分子量的离心液；将所述离心沉淀物干燥,并将不同分子量的离心液分别脱色浓缩、干燥,即得到不同分子量的半乳甘露聚糖。本发明所述的半乳甘露聚糖的分离纯化方法,工艺简单,能源消耗低,能够满足膳食纤维提纯、精制的要求,能有效去除杂质,降低有效成分损失,产品纯度高,多糖得率高。(The invention provides a method for separating and purifying galactomannan, which comprises the following steps: taking a neutral mannase solution and vegetable gum, and carrying out enzymolysis reaction to obtain an enzymolysis solution; centrifuging the enzymolysis solution to obtain a centrifugate and a centrifugal precipitate; filtering the centrifugate by adopting a hollow fiber membrane, and separating the centrifugate according to the molecular weight cut-off of the hollow fiber membrane to obtain centrifugates with different molecular weights; and drying the centrifugal precipitate, and respectively decolorizing, concentrating and drying the centrifugates with different molecular weights to obtain the galactomannans with different molecular weights. The method for separating and purifying the galactomannan has the advantages of simple process and low energy consumption, can meet the requirements of purifying and refining dietary fiber, can effectively remove impurities, reduce the loss of effective components, and has high product purity and polysaccharide yield.)

1. A method for separating and purifying galactomannan is characterized by comprising the following steps:

(1) taking a neutral mannase solution and vegetable gum, and carrying out enzymolysis reaction to obtain an enzymolysis solution;

(2) centrifuging the enzymolysis liquid obtained in the step (1) to obtain a centrifugate and a centrifugal precipitate;

(3) filtering the centrifugate obtained in the step (2) by adopting a hollow fiber membrane, and separating the centrifugate according to the molecular weight cut-off of the hollow fiber membrane to obtain centrifugates with different molecular weights;

(4) and (3) drying the centrifugal precipitate in the step (2), and respectively decoloring, concentrating and drying the centrifugate with different molecular weights in the step (3) to obtain the galactomannan with different molecular weights.

2. The method for separating and purifying galactomannan of claim 1, wherein in step (1), the vegetable gum is one or more of locust bean gum, guar gum, sesbania gum, fenugreek gum, tara gum.

3. The method for separating and purifying galactomannan according to claim 2, wherein in step (1), the neutral mannase solution is prepared from neutral mannase and distilled water with pH value of 5.5-7.0; the dosage of the neutral mannase solution is 1000U per 1g of the plant gum.

4. The method for separating and purifying galactomannan of claim 3, wherein the reaction temperature of the enzymatic hydrolysis reaction in step (1) is 50 ℃ and the reaction time is 8 h.

5. The method for separating and purifying galactomannan of claim 4, further comprising the step of inactivating enzyme of the enzymatic hydrolysate obtained in step (1); the method comprises the following specific steps: and (3) placing the enzymolysis liquid in ultrahigh-temperature sterilization equipment for sterilization and inactivation.

6. The method for separating and purifying galactomannan of claim 5, wherein in step (4), the sugar dry powder of galactomannan is obtained by spray drying after decolorizing and concentrating centrifugate with different molecular weight.

7. The method for separating and purifying galactomannan of claim 6, wherein in step (3): the filtering temperature is 30-50 ℃; the feeding pressure of the filtration is 0.1-0.5Mpa, and the discharging pressure is 0.1-0.3 Mpa;

the membrane component of the hollow fiber membrane is a roll-type or tubular membrane component; the hollow fiber membrane is one or more of a polystyrene membrane, an acetate fiber membrane, a polyether sulfone membrane, a polysulfone membrane and a polyamide membrane.

8. The method for separating and purifying galactomannan of claim 7, wherein in step (3), the hollow fiber membranes comprise hollow fiber membranes with molecular weight cut-off of 200kDa, 50kDa and 2kDa, respectively, and centrifugate with molecular weight of more than 200kDa, centrifugate with molecular weight of less than 50kDa and centrifugate with molecular weight of less than 2kDa are obtained after the cut-off.

9. The method for separating and purifying galactomannan of claim 8, wherein in step (4), the centrifugation sediment in step (2) is dried together with the centrifugate with molecular weight of more than 200kDa, and the centrifugate with molecular weight of less than 200kDa, the centrifugate with molecular weight of less than 50kDa and the centrifugate with molecular weight of less than 2kDa in step (3) are decolorized, concentrated and dried respectively.

10. The method for separating and purifying galactomannan of claim 9, wherein the hollow fiber membrane further comprises a hollow fiber membrane with a molecular weight cut-off of 300kDa in step (3) when the substrate concentration of the vegetable gum is greater than or equal to 0.15 kg/L.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for separating and purifying galactomannan.

Background

The dietary fiber is the total of edible plant components, carbohydrates and similar substances which can be digested and absorbed by human small intestine and partially or completely fermented in human large intestine. Generally refers to polysaccharide food components which are not easy to digest by digestive enzymes, carbohydrate with the polymerization degree of more than or equal to 3 and lignin, and mainly comes from the cell wall of plants (China society for Nutrition). Based on the above definition, dietary fiber includes many substances that are not digested by the human small intestine, such as cellulose, hemicellulose, gums, β -glucan, pectin, lignin, glucan, levan, resistant starch, dextrin, and the like.

Dietary fiber is classified into 2 basic types, based on its solubility in water, namely: water Soluble Dietary Fiber (SDF) and insoluble dietary fiber (NDF). Wherein the water soluble dietary fiber is soluble in warm water or hot water, and the part of the fiber which can be reprecipitated by 4 times of 95% ethanol in water solution is mainly stored substances and secretion in cell walls, and also comprises microbial polysaccharide and synthetic polysaccharide, which mainly comprise some gum substances, such as pectin, gum, mucilage, galactomannose, glucan, sodium alginate, carboxymethyl cellulose, fungal polysaccharide and the like, and part of hemicellulose.

The chemical composition and structure of dietary fiber determine its physicochemical properties, and dietary fiber is not digested and absorbed in human body, but has important physiological functions. Specifically, the following can be divided into: (1) promoting gastrointestinal motility and relieving constipation. The dietary fiber promotes gastrointestinal peristalsis, shortens the retention time of chyme in intestinal tracts and accelerates the excretion of excrement. Dietary fiber is fermented in the intestinal lumen by enzymes produced by bacteria, which break down into monosaccharides and then produce short chain fatty acids. The short chain fatty acid is utilized to generate CO2 in intestinal lumen, increase acidity and fecal amount, and accelerate intestinal content transfer in colon to facilitate excretion of feces, thereby preventing constipation. (2) Clearing away the doffer. The dietary fiber contains pectin, gum, algal polysaccharide and other structures, so that the viscosity of contents in the small intestine can be obviously increased, and the dietary fiber contains a large number of groups such as hydroxyl, carboxyl and the like, so that the absorption of heavy metals such as mercury, cadmium, lead and the like and toxic substances such as nitrosamine and the like in the food in the intestinal tract can be effectively reduced; under the condition of pH close to the stomach, the fruit fiber containing more water-soluble fiber has stronger adsorption capacity. In addition, the dietary fiber can inhibit the growth and reproduction of anaerobic bacteria, and is beneficial to the synthesis of vitamins for human body by intestinal beneficial bacteria. In addition, the dietary fiber can reduce the transfer of pathogenic bacteria from the rear intestine to the front intestine, the water-insoluble dietary fiber has strong capability of adsorbing toxin and pathogenic bacteria to harmful substances, and other bioactive substances such as phytic acid and the like are carried, so that the formation of cancer can be inhibited, and the incidence rate of colon cancer can be reduced. (3) Stabilize blood glucose concentration and lower blood cholesterol level. The mucus formed by dietary fiber forms a collagen layer in stomach, and reduces the emptying rate of stomach. The composition can block digestive enzyme from contacting with actual work in intestines, slow down small intestine contraction, influence glucose absorption, make glucose comfortable, reduce effective concentration of intestinal juice glucose, influence alpha-amylase to starch degradation, reduce glucose release rate in intestinal juice, improve peripheral tissue sensitivity to insulin, reduce body action to insulin, and reduce glucose absorption rate. (4) Stabilizing the balance of intestinal flora. The intestinal microecological system of human body includes microbial community and its microenvironment, and the intestinal wall has distributed powerful immune system with intestinal mucosa as boundary, and these two systems have the same action and mutual restriction and are in dynamic balance state. The human intestinal micro-ecology plays an important role in promoting the development of an immune system, maintaining a normal immune function, synergistically antagonizing pathogen invasion and the like. The health dietary habit is kept, and the intake of foods rich in dietary fiber, such as coarse cereals, coarse grains, mushroom foods, buckwheat, oat flour and the like, is properly increased, so that the growth of intestinal probiotics can be promoted, and the reproduction of pathogenic bacteria can be inhibited. (5) Fermentation performance. The anaerobic bacteria and yeast can decompose dietary fiber, the fermentation condition of which is related to the degree of the bacteria contacting substrates and the individual chemical structure of polysaccharide, wherein the structural characteristics of the monosaccharide and uronic acid in polysaccharide molecules, such as the type, the order of magnitude and the bonding mode, largely determine the fermentation condition of the fiber in intestinal tract. Dietary fiber from the same source is more fermentable in smaller particles than in larger particles and more fermentable in the matrix than in the matrix when taken alone. Soluble, easily fermentable dietary fibers are fermented in the colon and provide nutrients for microorganisms in the colon, and short chain fatty acids produced by fiber fermentation in turn inhibit the growth of pathogenic bacteria and contribute to stabilizing the intestinal flora balance. In addition, butyric acid can provide energy for colon mucosal cells, promote the proliferation of epithelial cells, maintain the integrity of intestinal mucosa, thereby being beneficial to the absorption of nutrients and preventing diarrhea. Intestinal saprophytic bacteria easily grow in a high pH environment, and some beneficial bacteria in colon degrade dietary fiber to generate short chain fatty acid, so that the pH value in the intestinal tract is reduced, and the growth of saprophytic bacteria is inhibited. (6) The dietary fiber has water-swelling property. The dietary fiber has hydrophilic polar groups, and thus has water retention and swelling properties. The dietary fiber has small specific weight, increases volume after meeting water, has volume effect on intestinal tracts, fills the gastric cavity after eating, needs longer time for digestion, prolongs the gastric emptying time, and reduces abnormal behaviors caused by animal hunger.

In summary, dietary fiber is a polysaccharide that is neither digested nor absorbed by the gastrointestinal tract nor produces energy. The physiological function of the dietary fiber is that the substances can stimulate the intestinal tract to wriggle, is beneficial to the discharge of excrement and can prevent constipation, rectal cancer, hemorrhoids and varicose veins of lower limbs; can be used for preventing cardiovascular diseases such as atherosclerosis and coronary heart disease; the product has satiety effect, is beneficial to obesity patients to eat, and can be used as a weight-reducing product; improving sugar tolerance, regulating blood sugar level of diabetic patients, and can be used as food for diabetic patients; improving intestinal flora and maintaining intestinal flora balance.

The mannooligosaccharide is oligosaccharide formed by connecting 2-10 mannose through beta-1, 4-glycosidic bond, and the mannooligosaccharide derived from guar gum also contains galactose side chains connected by alpha-1, 6-glycosidic bond. The mannan oligosaccharide has the characteristics of stable property, low calorie, blood sugar reduction and the like, and is a new-generation functional food preparation. Therefore, the guar gum-derived galactomannan and the soluble dietary fiber have very wide market prospects. In the guar gum-derived galactomannan preparation process, flocculation precipitation and centrifugal separation methods are mostly adopted for impurity removal and decoloration of the obtained polysaccharide extract, but the flocculation method is high in cost and low in efficiency, impurities introduced by adding a flocculating agent need to be removed in the subsequent process, and meanwhile, a part of galactomannan is adsorbed by precipitates, so that the yield is reduced. The deproteinization method of polysaccharide is commonly used in a Savage method, a trichloroacetic acid (TCA) method, a TCA-n-butanol method, an enzymatic method, a hydrochloric acid method and the like, and the polysaccharide source is different, but the polysaccharide source has the problems of large solvent dosage, residual organic solvent, introduction of impurities and the like.

In order to solve the above problems, chinese patent document CN105861595A discloses a method for rapidly preparing galactomannan from guar gum, which comprises the steps of guar gum sheet cleaning, crushing, enzymolysis, ultrafiltration, decolorization membrane filtration, filtrate concentration, drying, and micronization. Wherein, the mode of ultrafiltration and decoloration membrane filtration is adopted, so that higher yield and product purity can be obtained. However, the yield and purity of the galactomannan obtained by the above method are still not satisfactory, and it is not possible to separate products having different molecular weights.

Disclosure of Invention

The technical problem to be solved by the invention is that the method for preparing the galactomannan in the prior art has the problems of poor product yield and product purity and incapability of separating products with different molecular weights, and further provides a method for separating and purifying the galactomannan, which can effectively remove impurities, reduce the loss of effective components and can separate the products with different molecular weights.

In order to solve the above problems, the present invention provides a method for isolating and purifying galactomannan, comprising the steps of:

(1) taking a neutral mannase solution and vegetable gum, and carrying out enzymolysis reaction to obtain an enzymolysis solution;

(2) centrifuging the enzymolysis liquid obtained in the step (1) to obtain a centrifugate and a centrifugal precipitate;

(3) filtering the centrifugate obtained in the step (2) by adopting a hollow fiber membrane, and separating the centrifugate according to the molecular weight cut-off of the hollow fiber membrane to obtain centrifugates with different molecular weights;

(4) and (3) drying the centrifugal precipitate in the step (2), and respectively decoloring, concentrating and drying the centrifugate with different molecular weights in the step (3) to obtain the galactomannan with different molecular weights.

Preferably, in the step (1), the vegetable gum is one or more of locust bean gum, guar gum, sesbania gum, fenugreek gum and tara gum.

Preferably, in the step (1), the neutral mannase solution is prepared by using distilled water with the pH value of 5.5-7.0 and neutral mannase; the dosage of the neutral mannase solution is 1000U per 1g of the plant gum.

Preferably, in the step (1), the reaction temperature of the enzymolysis reaction is 50 ℃, and the reaction time is 8 h.

Preferably, the method further comprises the step of carrying out enzyme inactivation on the enzymolysis liquid obtained in the step (1); the method comprises the following specific steps: and (3) placing the enzymolysis liquid in ultrahigh-temperature sterilization equipment for sterilization and inactivation.

Preferably, in the step (4), after the centrifugate with different molecular weights is decolorized and concentrated, spray drying is adopted to obtain the galactomannan sugar dry powder.

Preferably, in step (3): the filtering temperature is 30-50 ℃; the feeding pressure of the filtration is 0.1-0.5Mpa, and the discharging pressure is 0.1-0.3 Mpa;

the membrane component of the hollow fiber membrane is a roll-type or tubular membrane component; the hollow fiber membrane is one or more of a polystyrene membrane, an acetate fiber membrane, a polyether sulfone membrane, a polysulfone membrane and a polyamide membrane.

Preferably, in the step (3), the hollow fiber membranes comprise hollow fiber membranes with molecular weight cut-off of 200kDa, 50kDa and 2kDa, and the centrifugate with molecular weight greater than 200kDa, the centrifugate with molecular weight less than 50kDa and the centrifugate with molecular weight less than 2kDa are obtained after the cut-off.

Preferably, in the step (4), the centrifugal precipitate in the step (2) and the centrifugate with the molecular weight of more than 200kDa are dried together, and the centrifugate with the molecular weight of less than 200kDa, the centrifugate with the molecular weight of less than 50kDa and the centrifugate with the molecular weight of less than 2kDa in the step (3) are decolorized, concentrated and dried respectively.

Preferably, when the substrate concentration of the plant gum is greater than or equal to 0.15kg/L, the hollow fiber membranes further comprise hollow fiber membranes with the molecular weight cutoff of 300kDa in the step (3).

Compared with the prior art, the invention has the following beneficial effects:

(1) the method for separating and purifying the galactomannan has the advantages of simple process, low energy consumption, cost reduction, no phase change in the process, original material characteristics maintenance, avoidance of organic reagent and no solvent residue, and can meet the requirements of purification and refining of dietary fiber;

(2) the method for separating and purifying the galactomannan adopts a membrane separation technology, has high efficiency, can effectively remove impurities and reduce the loss of effective components, and the loss rate of the galactomannan is less than or equal to 10 percent; the product purity can reach more than 99 percent, the polysaccharide yield is high, and the product yield can be improved by 20 to 50 percent;

(3) the galactomannan separation and purification method provided by the invention has the advantages that centrifugal liquids with different molecular weights are intercepted through the arrangement of the plurality of hollow fiber membranes, the fractional separation can be realized, the continuous and automatic operation is easy, and the production period is shortened;

(4) the method for separating and purifying the galactomannan can establish a method easy for quick detection, and analyze and judge the finally prepared product through intermediate detection so as to conveniently realize the control of the product quality.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples of the present invention, those who do not specify specific conditions are performed according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the embodiments, manufacturers and models of specific reagents are indicated, which are also only examples, and the implementation of the technical scheme and the implementation of the technical effect of the invention are not affected by the raw materials of different manufacturers and models.

The method for separating and purifying the galactomannan comprises the following steps:

(1) taking a neutral mannase solution and vegetable gum, and carrying out enzymolysis reaction to obtain an enzymolysis solution; and (3) placing the enzymolysis liquid in ultra-high temperature sterilization equipment UHT for sterilization and inactivation.

Wherein the vegetable gum is one or more of locust bean gum, guar gum, sesbania gum, fenugreek gum and tara gum; the neutral mannase is obtained commercially, and the neutral mannase solution is prepared from distilled water with the pH value of 5.5-7.0 and neutral mannase; the dosage of the neutral mannase solution is 1000U per 1g of the plant gum.

(2) Centrifuging the enzymolysis liquid obtained in the step (1) to obtain a centrifugate and a centrifugal precipitate; detection shows that the centrifugate after centrifugation has sugar concentration of 5-25% and viscosity of 10-200 cps.

(3) Filtering the centrifugate obtained in the step (2) by adopting a hollow fiber membrane, and separating the centrifugate according to the molecular weight cut-off of the hollow fiber membrane to obtain centrifugates with different molecular weights;

wherein the filtering temperature is 30-50 ℃; the feeding pressure of the filtration is 0.1-0.5Mpa, and the discharging pressure is 0.1-0.3 Mpa; the membrane component of the hollow fiber membrane is a roll-type or tubular membrane component; the hollow fiber membrane is one or more of a polystyrene membrane, an acetate fiber membrane, a polyether sulfone membrane, a polysulfone membrane and a polyamide membrane.

As a preferred implementation mode of the invention, the hollow fiber membranes comprise hollow fiber membranes with molecular weight cut-off of 200kDa, 50kDa and 2kDa respectively, and centrifugate with molecular weight of more than 200kDa, centrifugate with molecular weight of less than 50kDa and centrifugate with molecular weight of less than 2kDa are obtained after the cut-off.

When the substrate concentration of the vegetable gum is greater than or equal to 0.15kg/L, in the step (3), the hollow fiber membranes further comprise hollow fiber membranes with the molecular weight cutoff of 300 kDa.

(4) And (3) drying the centrifugal precipitate in the step (2), and respectively decoloring, concentrating and drying the centrifugate with different molecular weights in the step (3) to obtain the galactomannan with different molecular weights.

And (3) drying the centrifugal precipitate and centrifugate with the molecular weight of more than 200kDa in the step (2), decoloring and concentrating the centrifugate with the molecular weight of less than 200kDa, the centrifugate with the molecular weight of less than 50kDa and the centrifugate with the molecular weight of less than 2kDa in the step (3) respectively, and performing spray drying to obtain the galactomannan sugar dry powder.