CN112481328A - Preparation method of high-purity galactooligosaccharide - Google Patents
Preparation method of high-purity galactooligosaccharide Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C07H1/06—Separation; Purification
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- 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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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
Abstract
The invention discloses a preparation method of high-purity galactooligosaccharide, which comprises the following steps: preparing a lactose solution by using an acetic acid-sodium acetate buffer solution, and preparing an enzyme solution of beta-galactosidase by using the acetic acid-sodium acetate buffer solution; adding enzyme solution into the lactose solution for reaction; passing the obtained galacto-oligosaccharide mixed solution through a silica gel chromatographic column, wherein an eluent is n-butyl alcohol: ethanol: acetic acid: water 3: 1: 1: 1 by volume ratio; collecting the eluent containing the target product; drying to obtain the high-purity galactooligosaccharide. According to the invention, the silica gel is applied to the separation and purification of galactooligosaccharides for the first time, so that the process for purifying the galactooligosaccharides is greatly simplified and the production cost of the galactooligosaccharides is reduced; thereby promoting the marketization of galacto-oligosaccharide.
Description
Technical Field
The invention relates to the technical field of sugar industry. In particular to a preparation method of high-purity galacto-oligosaccharide.
Background
Common molecular structures of the galacto-oligosaccharides are 1-7 molecules of galactose at a non-reducing end and 1 molecule of glucose at a reducing end, which can selectively promote the proliferation of intestinal probiotics and can enhance the absorption of calcium and other minerals by the body. The galacto-oligosaccharide has the characteristics of low cariogenic property, low calorific value, low sweetness value and the like, naturally exists in breast milk and some fruits and vegetables, such as a small amount of onion, garlic, banana, soybean and jerusalem artichoke, is a functional oligosaccharide which is concerned in recent years, is one of the most widely accepted and safe oligosaccharides among a plurality of functional oligosaccharides, is listed as an internationally accepted and safe food additive by the U.S. food and drug administration, and is widely applied to baked foods, candies, jam, cold drinks and food preservation.
Galactooligosaccharides have particular advantages over other prebiotics (e.g. fructooligosaccharides, oligomannose, xylooligosaccharides):
(1) biological safety is widely recognized throughout the world, and in europe, galactooligosaccharides and fructooligosaccharides have been allowed to be used as food additives, meet the us standards, and are recognized as safe substances (GRAS). The galactooligosaccharide is approved as a new resource food in China and is allowed to be added into infant food, dairy products, beverages, baked food and candies;
(2) more acid and high temperature resistance, which makes it easier to use for additives;
(3) has a similar structure with partial milk-derived oligosaccharide, and has wider physiological significance for mammals;
(4) the raw material for preparing galacto-oligosaccharides can be lactose with wide sources.
The existing synthetic galactooligosaccharides comprise an enzymolysis method, a chemical synthesis method, an extraction method from natural substances, a fermentation method and a natural macromolecule hydrolysis method. The enzymolysis method has the advantages of rich substrate sources, low cost, simple and convenient operation, environmental friendliness and the like, and is the most common technology for industrially preparing galactooligosaccharides at present. Among the glycosidases, the aspergillus oryzae-derived beta-galactosidase has unique advantages: the cost is higher than that of the enterobacter coli source, and the Kluyveromyces lactis source is low; the biological safety is widely accepted, and the biological safety is allowed to be used in the food production and processing process; the pH of the biological activity of the aptamer itself is acidic, while most whey powders are also acidic in pH, which facilitates the use of an Aspergillus oryzae derived oligogalactosidase in the conversion of whey powder, a dairy by-product.
Galactooligosaccharides obtained by microbial enzyme methods are generally mixed with glucose, galactose, and unreacted lactose, which may affect their physiological effects and range of applications, such as: glucose is one of the most available energy sources for most microorganisms, which will affect the selective regulation of galactooligosaccharides on the microflora of the organism. The galacto-oligosaccharide has similar components and complex structure, and is difficult to separate and purify, and the conventional separation method such as a crystallization method is difficult to apply. In addition, the common microbial fermentation method utilizes the specificity of preferentially metabolizing glucose, galactose or lactose by certain yeast cells so as to reduce the heterosugar component, but the process often introduces new impurities, such as short-chain fatty acids, so that the operation complexity of subsequent separation and purification is increased; in recent years, the membrane separation technology has wide application in the separation and concentration of substances, and the nanofiltration membranes with different apertures are selected in principle, namely monosaccharide, lactose and galacto-oligosaccharide can be separated in sequence, so that the defects that the separation membrane is easy to pollute, needs frequent sterilization and cleaning and is not suitable for large-scale industrial production are overcome; although the chromatographic column chromatography represented by sephadex or polyacrylamide gel is simple in equipment and easy to operate, the filler is expensive, the separation period is long, the product concentration is low, and the large-scale production and application of the filler are limited, but the chromatographic column chromatography of the silicagel filler reduces the production cost under the condition of ensuring the separation degree, and promotes the marketization of the galactooligosaccharide.
Chromatography was first to separate some plant pigments based on compound color, and nowadays, chromatography has evolved into a highly sensitive, efficient method of compound isolation and identification. The column packing may be classified into silica gel column chromatography, ion exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography, etc., and among them, silica gel column chromatography (positive/negative phase) is most widely used. Compared with other chromatographic methods, the column chromatography has the advantages of simple operation, economy, large sample bearing capacity and the like, and is one of the first-choice methods for separating and purifying natural products. The normal phase silica gel column chromatography packing is normal phase silica gel and is suitable for separating compounds with relatively small polarity; reverse silica gel column chromatography is suitable for separating relatively more polar compounds, such as glycosides and alkaloids. Although silica gel column chromatography has been used to separate and purify cellooligosaccharide, dextran, ricinine with single degree of polymerization, there is little research on the preparation of high-purity galactooligosaccharide.
The common filler for purifying galacto-oligosaccharide at present is polyacrylamide gel or dextran gel, and the eluent is deionized water; however, these two fillers are very expensive and cannot be used for the industrial production of purified galactooligosaccharides. It has been reported in the literature that cellooligosaccharides were separated on silica gel, the eluent being n-butanol: acetic acid: water 2: 1: 1
When the filler for purifying the galactooligosaccharide is polyacrylamide gel or glucan gel, the principle of size exclusion chromatography is used, namely the pore size of the filler is used, so that sugar components with different polymerization degrees are separated under the washing of eluent.
Disclosure of Invention
The problem to be solved by the present invention is to provide a method for preparing high purity galactooligosaccharides (about 100% galactooligosaccharides).
In order to solve the technical problems, the invention provides a preparation method of high-purity galactooligosaccharide, which comprises the following steps:
1) preparing a lactose solution by using an acetic acid-sodium acetate buffer solution (pH 5), and preparing an enzyme solution of beta-galactosidase by using the acetic acid-sodium acetate buffer solution (pH 5);
adding enzyme solution into lactose solution, and reacting at 50 + -2 deg.C (in constant temperature gas bath shaking table) for 4 + -0.5 hr to obtain galactooligosaccharide mixed solution;
the dosage ratio of the beta-galactosidase to the lactose is 40 +/-5U/g lactose;
2) and silica gel chromatography (namely, separating the mixed liquid of the galactooligosaccharide by using a silica gel chromatographic column) is adopted:
passing the galactooligosaccharide mixed solution through a silica gel chromatographic column, wherein the eluent is n-butyl alcohol: ethanol: acetic acid: water 3: 1: 1: 1 by volume ratio; the flow rate is 0.4 plus or minus 0.05 ml/min;
collecting the eluate containing the target product (i.e., the sample not containing monosaccharide or disaccharide);
3) and drying the eluent obtained in the step 2) to obtain the high-purity galactooligosaccharide.
As an improvement of the process for producing a high-purity galactooligosaccharide of the present invention:
the target products are galacto-oligosaccharide, galacto-oligosaccharide tetrasaccharide and galacto-oligosaccharide pentasaccharide.
As a further improvement of the process for producing a high-purity galactooligosaccharide of the present invention:
the silica gel in the step 2) is 200-mesh 300-mesh silica gel.
As a further improvement of the process for producing a high-purity galactooligosaccharide of the present invention:
the drying in the step 3) comprises the following steps: drying at 60 + -5 deg.C (to remove organic solvent), re-dissolving with double distilled water, and lyophilizing at (-40 deg.C) to constant weight.
As a further improvement of the process for producing a high-purity galactooligosaccharide of the present invention:
in the step 2), the collected eluate is monitored for galactooligosaccharides (change in the content of galactooligosaccharides) by thin chromatography, and the eluate containing only galactooligosaccharides is mixed.
As a further improvement of the process for producing a high-purity galactooligosaccharide of the present invention:
and step 2) adopting a wet packing column.
In the present invention,
step 1): the source of beta-galactosidase belongs to aspergillus oryzae, and the enzyme activity of the beta-galactosidase of the aspergillus oryzae source is about 10 million ALU/g.
After the reaction is finished, placing the reaction solution in 100 ℃ boiling water for 5min to inactivate the enzyme, and stopping the enzymolysis reaction to obtain galacto-oligosaccharide mixed solution;
the elution process of the step 2) is carried out at normal temperature and normal pressure; the developing agent used in the thin layer chromatography monitoring of the sample in the step 2) is prepared from n-butyl alcohol: ethanol: acetic acid: water 2: 1: 1: 1 (v/v/v/v).
The thin chromatography method for detecting the change of the sugar component (namely, the thin chromatography method is used for sequentially detecting the liquid in each centrifugal tube) comprises the following specific steps:
(4.1) first, preparing a 1mg/ml mixed standard solution (containing lactose, galactose and glucose);
(4.2) sampling 1.5 mul of eluent and the mixed standard solution obtained in the step (4.1) from top to bottom at intervals of 1cm from the bottom of the silica gel plate, ensuring that the mixed standard solution is sampled at the first position of each silica gel plate as a reference standard, drying the sampling point by using a blower, placing the HSGF254 type silica gel plate on one side of a double-tank development chamber, and adding a developing agent (n-butyl alcohol: ethanol: acetic acid: water is 2: 1: 1: 1, v/v/v/v) into the other side for pre-saturation for 20 min;
(4.3) after the pre-saturation is finished, adding a developing agent to one side of the silica gel plate, carrying out upward development, taking out the silica gel plate after the developing agent moves upward to the position 1cm away from the edge of the top, and drying;
(4.4) uniformly spraying a color developing agent, namely 95% ethanol sulfuric acid solution (ethanol: concentrated sulfuric acid is 95: 5, v/v), onto a silica gel plate, blowing for drying after 2min, placing in an oven, and developing at 110 ℃ for 10min, so as to observe the content change of the galactooligosaccharide in each centrifugal tube.
The step 3) is carried out in a thermostatic water bath at 60. + -. 5 ℃ under the condition of an external blowing device (for example, a fan) for removing the organic solvent.
In the invention process, the sugar is used as a compound with strong polarity, can be mutually adsorbed with substances with strong polarity such as silica gel, and then elutes the sample by using eluents (with different polarities) with different organic solvent ratios so as to be separated. That is, the present invention does not apply the conventional size exclusion chromatography principle, but utilizes the adsorption capacity of silica gel to saccharides with different polymerization degrees to realize separation.
The invention has the following technical advantages:
(1) the method uses the silica gel chromatographic column to separate and purify the galactooligosaccharide, and has the advantages of simple process, large sample bearing capacity and low cost.
(2) The method for preparing the high-purity galactooligosaccharide provided by the invention does not carry out any biological and chemical reaction, and has high food safety.
(3) The method provided by the invention not only can obtain a mixture of high-purity galactooligosaccharides, but also can separate and prepare a galactooligosaccharide component with single polymerization degree, and can be used for further structure exploration.
In conclusion, the method applies the silica gel to the separation and purification of the galactooligosaccharides for the first time, greatly simplifies the purification process of the galactooligosaccharides and reduces the production cost of the galactooligosaccharides; thereby promoting the marketization of galacto-oligosaccharide.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a diagram illustrating the elution of various saccharide components in the eluate of example 1;
the abscissa is the number of the centrifuge tube, the numbers on the left and right sides above each rectangle represent the tube numbers of appearance and disappearance of the sugar, respectively, the intersecting area of the rectangles indicates that the corresponding two sugars are simultaneously present in the samples, and each 2ml of eluate is collected in one centrifuge tube. Wherein the monosaccharide comprises glucose and galactose, the disaccharide comprises lactose, galactobiose and isolactose, galactooligosaccharide tri-and tetrasaccharide represents galactooligosaccharide with three and four polymerization degrees, and galactooligosaccharide pentasaccharide greater than or equal to five represents galactooligosaccharide with five polymerization degrees.
FIG. 2 shows HPLC results (■) of high purity galactooligosaccharide after freeze-drying, and the enzymatic hydrolysate, i.e., a mixed solution of low purity galactooligosaccharide (-), was used as a reference standard.
FIG. 3 is a graph showing the elution of various saccharide components in the eluate of example 2, wherein each 5ml of the eluate is collected in a centrifuge tube.
FIG. 4 is a graph showing the elution of various kinds of sugar components in the eluate of comparative example 1-1, wherein each 2ml of the eluate is collected in one centrifugal tube.
FIG. 5 is a graph showing the elution of various types of sugar components in the eluents of comparative examples 1 to 2, each 2ml of the eluents being collected in one centrifugal tube.
FIG. 6 is a graph showing the elution of various types of sugar components in the eluents of comparative examples 1 to 3, each 2ml of the eluents being collected in one centrifugal tube.
FIG. 7 is a graph showing elution of various kinds of sugar components in the eluate of comparative example 2-1, wherein each 2ml of the eluate is collected in one centrifugal tube.
FIG. 8 is a graph showing the elution of various types of sugar components in the eluate of comparative example 2-2, each 2ml of the eluate being collected in a centrifuge tube.
Detailed Description
The invention is further illustrated with reference to the following examples, without limiting the scope of the invention. The beta-galactosidase in the following examples is a source of Aspergillus oryzae, for example 10 million ALU/g galactosidase from Nanning Pompe bioengineering, Inc. can be used. The chemical reagents used were analytical grade unless otherwise specified. All manipulations were done in the laboratory.
Example 1, a method for preparing high-purity galactooligosaccharide, comprising the steps of:
1) mixed liquid for producing galacto-oligosaccharide by enzymolysis
(1.1) preparing 100ml of a 50% (i.e., 50g/100ml) lactose solution in a water bath (70 ℃) using an acetic acid-sodium acetate buffer (pH 5), and cooling to 50 ℃;
(1.2) preparing 20mg/ml of a galactosidase derived from aspergillus oryzae by using an acetic acid-sodium acetate buffer solution (pH 5) (the enzyme activity in 1ml of the enzyme solution is 2000U in total);
(1.3) adding 1ml of the galactosidase solution obtained in the step (1.2) into 100ml of the lactose solution obtained in the step (1.1), shaking uniformly, transferring to a constant-temperature air bath shaking table, keeping the temperature at 50 ℃, the rotating speed of the shaking table at 100rpm, reacting for 4 hours, putting the enzymolysis reaction solution into boiling water at 100 ℃, keeping the temperature for 5min to ensure that the enzyme loses activity, and stopping the enzymolysis reaction to obtain a solution (galactooligosaccharide mixed solution) with the content of the galactooligosaccharide of about 31.89%.
2) Silica gel chromatographic column is packed to wet process
(2.1) 30g of silica gel (200-300 mesh) was weighed out and completely dissolved in 100ml of eluent which was prepared from n-butanol: ethanol: acetic acid: water 3: 1: 1: 1(v/v/v/v) and sonicated for 10min to remove dissolved air bubbles;
(2.2) a glass chromatographic column (3cm multiplied by 30cm) is filled with eluent with the height of 5cm in advance, and then the silica gel solution (2.1) is uniformly drained into the chromatographic column by a glass rod and is gently stirred. After the silica gel is completely poured into the chromatographic column, the lower end valve is opened, the eluent is controlled to flow through the silica gel column bed at the flow rate of 0.4ml/min, and after 2 hours, the column bed is basically compacted, and the height of the column bed is about 17cm generally and is proper.
3) Galacto-oligosaccharide sample loading separation
Sucking 2ml of the galactooligosaccharide mixed solution obtained in the step (1.3) by a liquid transfer machine, and adding the galactooligosaccharide mixed solution into the silica gel column packaged in the step (2.2) in an adherent manner; opening the lower valve, adding eluent, controlling the eluent to flow through the silica gel column bed at the flow rate of 0.4ml/min, and collecting the obtained eluent; every 2ml of eluate was collected in centrifuge tubes (total 268 tubes) for subsequent detection;
the following steps (4.2) to (4.4) are respectively carried out for each tube of eluent:
4) thin layer chromatography for detecting sugar composition changes
(4.1) respectively taking 15mg of lactose, galactose and glucose standard substance, dissolving in 5ml of methanol to prepare 3mg/ml single standard substance solution, and then mixing the standard substance solutions in equal volumes to obtain 1mg/ml mixed standard substance solution;
(4.2) sampling 1.5 mul of the eluent obtained in the step 3) and the mixed standard solution obtained in the step (4.1) from top to bottom at a position 1cm away from the bottom of the silica gel plate in sequence at intervals of 1cm, ensuring that the mixed standard solution is sampled at the first position of each silica gel plate as a reference standard, drying the sampling point by using a blower, placing the HSGF254 type silica gel plate on one side of a double-groove developing chamber, and adding a developing agent (n-butyl alcohol: ethanol: acetic acid: water 2: 1: 1: 1 (v/v/v) prepared) for 20 min;
(4.3) after the pre-saturation is finished, adding a developing agent to one side of the silica gel plate, carrying out upward development, taking out the silica gel plate after the developing agent moves upward to the position 1cm away from the edge of the top, and drying;
(4.4) uniformly spraying a color developing agent, namely 95% ethanol sulfuric acid solution (ethanol: concentrated sulfuric acid: 95: 5, v/v), on a silica gel plate, blowing for drying after 2min, placing in a drying oven at 110 ℃ for color development for 10min, comparing with a thin layer chromatography result of a mixed standard solution, and qualitatively obtaining the change of sugar components in the collected centrifugal tubes, which is specifically shown in fig. 1.
5) Galactooligosaccharide solution freeze drying
Monitoring the change of the content of galactooligosaccharides in each centrifuge tube by thin chromatography, mixing samples only containing galactooligosaccharides (namely, samples of tubes 118-268), placing the mixture in a water bath kettle at a constant temperature (60 ℃), removing the organic solvent until the mixture is dried under the condition of blowing air by a fan, adding a proper amount of double distilled water for redissolving the galactooligosaccharides, and then freeze-drying the mixture at the temperature of (-40 ℃) until the weight is constant to obtain a product which is 100 percent galactooligosaccharides solid powder (242.15 mg in total) through HPLC detection, as shown in figure 2, wherein the single galactooligosaccharides, tetrasaccharides and not less than pentasaccharides are respectively 80.3mg,42.2mg and 14.7 mg.
Embodiment 2, a method for preparing high-purity galactooligosaccharide, comprising the steps of:
1) and (3) carrying out enzymolysis to produce galactooligosaccharide mixed solution: the same as in example 1.
2) Silica gel chromatographic column is packed to wet process
(2.1) 150g of silica gel (200-300 mesh) was weighed out and completely dissolved in 500ml of an eluent consisting of n-butanol: ethanol: acetic acid: water 3: 1: 1: 1(v/v/v/v) and sonicated for 10min to remove dissolved air bubbles;
(2.2) pre-loading eluent with the height of 5cm in a glass chromatographic column (6cm multiplied by 30cm), then uniformly draining the silica gel solution (2.1) into the chromatographic column by using a glass rod, and gently stirring. After the silica gel is completely poured into the chromatographic column, the lower end valve is opened, the eluent is controlled to flow through the silica gel column bed at the flow rate of 0.4ml/min, after 3 hours, the column bed is basically compacted, and the height of the column bed is about 17cm generally and is proper.
3) Galacto-oligosaccharide sample loading separation
Sucking 10ml of the galactooligosaccharide mixed solution in the step (1.3) by a liquid transfer machine, and adding the mixture into the silica gel column packaged in the step (2.2) in an adherent manner; opening the lower valve, adding eluent, controlling the eluent to flow through the silica gel column bed at the flow rate of 0.4ml/min, and collecting the obtained eluent; collecting each 5ml of eluent in a centrifugal tube (total 490 tubes) for subsequent separation detection;
the following steps (4.2) to (4.4) are respectively carried out for each tube of eluent:
4) thin chromatography detection of sugar composition changes: equivalent to example 1.
5) Galactooligosaccharide solution freeze drying
Monitoring the change of the content of galacto-oligosaccharide in each centrifuge tube by thin chromatography, mixing samples only containing galacto-oligosaccharide (namely 235 th tube 490), placing the mixture in a water bath pan at constant temperature (60 ℃), removing the organic solvent to dry under the condition of blowing by an external fan, adding a proper amount of double distilled water to re-dissolve the galacto-oligosaccharide, and then freeze-drying the mixture at the temperature of (-40 ℃) to constant weight to obtain a product which is 100 percent galacto-oligosaccharide solid powder (1.21 g in total) detected by HPLC, wherein single galacto-oligosaccharide, tetrasaccharide and more than or equal to pentasaccharide are 402.5mg,209.8mg and 74.2mg respectively.
Comparative example 1-1, eluent of example 1 was purified from "n-butanol: ethanol: acetic acid: water 3: 1: 1: 1 "changed to" n-butanol: acetic acid: water-4: 1: 1 "; the rest is equivalent to embodiment 1.
The results obtained were: 562ml of eluent (281 tubes in total) was recovered cumulatively, i.e. the volume of eluent was increased, since the polarity of eluent was decreased, making it difficult to elute the sugar from silica gel. It can also be seen that the time of occurrence of each sugar component is delayed to a different extent and the separation effect between the different sugar components is reduced, as shown in more tubes with two types of sugars present in the eluent at the same time, as shown in fig. 4. The results show that the galactooligosaccharide samples from 126 th to 281 th tubes were pure, and 233.1mg of galactooligosaccharide product was recovered and lyophilized, while 69.2mg,35.4mg and 13.6mg of single galactooligosaccharide, tetrasaccharide and pentasaccharide were obtained.
Comparative examples 1-2 the eluent of example 1 was purified from "n-butanol: ethanol: acetic acid: water 3: 1: 1: 1 "changed to" n-butanol: ethanol: acetic acid: water 3: 1: 1: 5'; the rest is equivalent to embodiment 1.
The results obtained were: the cumulative recovery of 460ml of eluent (230 tubes in total) reduced the volume of eluent due to the increased polarity of eluent, which made it easier to desorb sugars from silica gel. It can also be seen that the timing of the appearance of the individual saccharide fractions is also advanced to a different extent, but the separation between the different saccharide fractions is reduced, in particular not only by the presence of both types of saccharide in the eluate in more tubes, but also by a significantly reduced number of tubes in which only a single saccharide fraction is present in the eluate, as compared to example 1, with the result detailed in FIG. 5. The results show that the galactooligosaccharide sample from tube 106 to 230 is pure, 220.5mg of galactooligosaccharide product is obtained by recovery and freeze-drying, and the single galactooligosaccharide, tetrasaccharide and not less than pentasaccharide are 49.2mg,36.6mg and 12.2mg respectively.
Comparative examples 1-3 the eluent of example 1 was purified from "n-butanol: ethanol: acetic acid: water 3: 1: 1: 1 "changed to" n-butanol: acetic acid: water 2: 1: 1 "; the rest is equivalent to embodiment 1.
The results obtained were: the cumulative recovery of 516ml of eluent (258 tubes total) reduced the volume of eluent due to the increased polarity of eluent, which made it easier to elute the sugar from the silica gel. It can also be seen that each sugar component elutes in advance and that although the number of tubes in which both types of sugars are present in the eluate is reduced, the amount of dissolved sugar component is high, which ultimately also results in a reduction in the total GOS and the recovery of its individual components, as detailed in fig. 6. The results show that tubes 108 to 258 are pure galacto-oligosaccharide samples, and that recovery and lyophilization yielded 228.3mg of galacto-oligosaccharide product, while single galacto-oligosaccharides, tetrasaccharides, and not less than pentasaccharides, 75.4mg,36.2mg, and 14.1mg, respectively, were obtained.
Comparative example 2-1, the silica gel of example 1 is changed from 200-.
The results obtained were: the cumulative recovery of 556ml of eluent (278 tubes in total), i.e., the increase in the volume of eluent, was due to the difficulty in desorbing the sugar from the silica gel, since the specific surface area of silica gel was relatively large and the adsorption force for the sugar was increased after the diameter of silica gel was decreased. It can also be seen that the time of appearance of the individual sugar components is delayed to a different extent and the separation effect between the different sugar components is reduced, the results are shown in detail in FIG. 7. The results show that the galactooligosaccharide sample from the 124 th to 278 th tubes is pure, 235.8mg of galactooligosaccharide product is obtained by recovery and freeze-drying, and single galactooligosaccharide, tetrasaccharide and more than or equal to pentasaccharide are 71.2mg,28.4mg and 13.5mg respectively.
Comparative example 2-2, the silica gel of example 1 is changed from 200-.
The results obtained were: the eluent is collected up to 478ml (239 tubes in total), i.e. the volume of the eluent is reduced, because the specific surface area is relatively reduced after the diameter of the silica gel is increased, the adsorption capacity of the eluent on the sugar is reduced, and therefore the eluent can more easily desorb the sugar from the silica gel. At the same time, we can see that the appearance time of each sugar component is also advanced to different degrees, but the separation effect between different sugar components is reduced, and the result is shown in detail in FIG. 8. The results show that the galactooligosaccharide sample is pure from the 113 th tube to the 239 th tube, 218.5mg of galactooligosaccharide product is obtained by recovery and freeze-drying, and single galactooligosaccharide, tetrasaccharide and more than or equal to pentasaccharide are 45.2mg,36.6mg and 13.2mg respectively.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (7)
1. The preparation method of the high-purity galactooligosaccharide is characterized by comprising the following steps:
1) preparing a lactose solution by using an acetic acid-sodium acetate buffer solution, and preparing an enzyme solution of beta-galactosidase by using the acetic acid-sodium acetate buffer solution;
adding enzyme solution into lactose solution, and reacting at 50 + -2 deg.C for 4 + -0.5 hr to obtain galactooligosaccharide mixed solution;
the dosage ratio of the beta-galactosidase to the lactose is 40 +/-5U/g lactose;
2) and performing silica gel chromatography:
passing the galactooligosaccharide mixed solution through a silica gel chromatographic column, wherein the eluent is n-butyl alcohol: ethanol: acetic acid: water 3: 1: 1: 1 by volume ratio; the flow rate is 0.4 plus or minus 0.05 ml/min;
collecting the eluent containing the target product;
3) and drying the eluent obtained in the step 2) to obtain the high-purity galactooligosaccharide.
2. The method for producing high-purity galactooligosaccharide according to claim 1, wherein:
the target products are galacto-oligosaccharide, galacto-oligosaccharide tetrasaccharide and galacto-oligosaccharide pentasaccharide.
3. The method for producing a high-purity galactooligosaccharide according to claim 1 or 2, wherein:
the silica gel in the step 2) is 200-mesh 300-mesh silica gel.
4. The method for producing high-purity galactooligosaccharide according to claim 3, wherein:
the drying in the step 3) comprises the following steps: drying at 60 + -5 deg.C, re-dissolving with double distilled water, and lyophilizing.
5. The method for producing high-purity galactooligosaccharide according to claim 4, wherein:
in the step 2), the galactooligosaccharide in the collected eluent is monitored by thin chromatography, and the eluent only containing the galactooligosaccharide is mixed.
6. The method for producing high-purity galactooligosaccharide according to any one of claims 1 to 5, wherein:
acetic acid-sodium acetate buffer with pH 5 was used.
7. The method for producing high-purity galactooligosaccharide according to any one of claims 1 to 5, wherein:
and step 2) adopting a wet packing column.
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