阅读说明：本技术 一种从角豆水提取物中提纯d-松醇的方法 (Method for purifying D-pinitol from carob water extract ) 是由 李文德 杨浩 蒲首丞 赵虎军 楼永增 于 2019-12-07 设计创作，主要内容包括：本发明公开了一种从角豆水提取物中提纯D-松醇的方法,属于D-松醇提纯技术领域,所述提纯D-松醇的方法包括以下步骤：原料预处理：调节原料液的pH呈酸性；蔗糖水解转化：原料液中加入蔗糖酶,将蔗糖水解转化为果糖和葡萄糖；果糖沉淀：将果糖采用沉淀法去除；葡萄糖氧化：将葡萄糖氧化为葡萄糖酸；提纯D-松醇：将葡萄糖酸采用层析法分离后,减压浓缩水洗脱液得到高纯度D-松醇。本发明目的在于在使用温和的提纯方法减少杂质的同时,再分步骤尽可能多的去除升血糖因子。(The invention discloses a method for purifying D-pinitol from a water extract of carob, belonging to the technical field of purification of D-pinitol, and the method for purifying D-pinitol comprises the following steps: pretreatment of raw materials: adjusting the pH of the raw material liquid to be acidic; hydrolysis and transformation of sucrose: adding sucrase into the raw material solution to hydrolyze and convert sucrose into fructose and glucose; and (3) fructose precipitation: removing fructose by precipitation; oxidation of glucose: oxidizing glucose to gluconic acid; purifying D-pinitol: separating gluconic acid by chromatography, and concentrating water eluent under reduced pressure to obtain high-purity D-pinitol. The present invention aims to remove as much as possible of the hyperglycemic factor by a subdivided step while reducing impurities using a mild purification method.)

1. A method for purifying D-pinitol from an aqueous extract of carob beans, said method comprising the steps of:

pretreatment of raw materials: adjusting the pH of the aqueous extract of carob to acidic;

hydrolysis and transformation of sucrose: adding sucrase into the raw material solution to hydrolyze and convert sucrose into fructose and glucose;

and (3) fructose precipitation: removing fructose by precipitation;

oxidation of glucose: oxidizing glucose to gluconic acid;

purifying D-pinitol: separating gluconic acid by chromatography, concentrating water eluent under reduced pressure, and drying to obtain purified D-pinitol.

2. The method of purifying D-pinitol from an aqueous extract of carob beans as claimed in claim 1, wherein said method comprises the steps of:

pretreatment of raw materials: diluting the raw material liquid, and adding acetic acid to adjust the pH to 4-5;

hydrolysis and transformation of sucrose: heating the pretreated raw material liquid to 50-60 ℃ in a water bath, adding sucrase, performing thermal insulation hydrolysis at the stirring speed of 150-200rpm, and performing stirring reaction for 2-4h to obtain hydrolysate;

and (3) fructose precipitation: adding the hydrolysate into deionized cold water at the temperature of 2-10 ℃, wherein the volume ratio of the hydrolysate to the deionized cold water is 1 (5-7), stirring uniformly, adding supersaturated calcium hydroxide solution, standing in a refrigeration house at the temperature of 0-10 ℃ for 10h, centrifuging, and concentrating the obtained supernatant under reduced pressure;

oxidation of glucose: taking the supernatant I after decompression concentration, diluting, adding acetic acid to adjust the pH value to be 5-6, heating in a water bath, adding glucose oxidase and catalase according to the detected glucose concentration, uniformly stirring, introducing oxygen or compressed air with the flow of 1L/min, and reacting for 12-24h to obtain a reaction solution;

purifying D-pinitol: performing chromatography on the reaction solution by ion exchange resin, eluting with water to obtain an eluent containing D-pinitol, concentrating the eluent under reduced pressure, and drying to obtain purified D-pinitol;

secondary purification: dissolving the obtained D-pinitol with deionized water, and repeating the steps of sucrose hydrolysis conversion, fructose precipitation, glucose oxidation and D-pinitol purification to obtain the D-pinitol with the purity of more than 90%.

3. The method of claim 2, wherein said sucrose hydrolysis conversion step is performed in an amount of 15-25mg sucrase per 100g starting material solution.

4. The method for purifying D-pinitol from an aqueous extract of carob bean as claimed in claim 3, wherein in said fructose precipitation step, the volume ratio of the hydrolysate to the supersaturated calcium hydroxide solution is 1 (0.5-1.5), and the concentration of said supersaturated calcium hydroxide solution is 10-20%.

5. The method of claim 4, wherein the glucose oxidation step comprises diluting the first supernatant after concentration under reduced pressure to a glucose concentration of 10%.

6. The method for purifying D-pinitol from an aqueous extract of carob bean as claimed in claim 5, wherein said glucose oxidation step is performed using 3.5-4 kg/ton of glucose as glucose oxidase.

7. The method of claim 6, wherein the amount of catalase used in said step of oxidizing glucose is 2.5-3 kg/ton of glucose.

8. The method for purifying D-pinitol from an aqueous extract of carob bean as claimed in claim 7, wherein said step of oxidizing glucose is carried out by heating in a water bath to a temperature of 34-35 ℃, incubating, and oxidizing glucose.

9. The method for purifying D-pinitol from a water extract of carob bean as claimed in any one of claims 1 to 8, wherein said fructose precipitation step further comprises concentrating the supernatant first under reduced pressure to 2 times the volume of the feed solution, adding a supersaturated calcium hydroxide solution of 20% concentration 1.5 times the volume of the feed solution, stirring well, standing in a freezer at 4 ℃ for 10h, centrifuging, and concentrating the supernatant second under reduced pressure after centrifuging.

Technical Field

The invention relates to the technical field of D-pinitol purification, in particular to a method for purifying D-pinitol from carob water extract.

Background

D-pinitol (D-pinitol), IUPC (1S, 2S, 4S, 5R) -6-methoxycyclo hexane.1, 2, 3, 4, 5.pentol, CAS registry number 10284-63-6, named as D-Inositol methyl ether, 3-methyl Inositol (mateze), Ketinil, methyl Inositol, pinitol, physostigol, Sennite, L-chiro-Inositol, pinite, and pinecophor, and the molecular formula is C₇H₁₄₀O₆The D-pinitol has a molecular weight of 194.18, is white granular crystal in methanol, is odorless, sweet, soluble in water, insoluble in methanol and ethanol, and insoluble in organic solvents such as chloroform, acetone, diethyl ether, etc. The melting point of the D-pinitol is 186-188 ℃; the specific optical rotation was 65.5. The structure is as follows:

d-pinitol is a substance having a variety of important physiological activities, and has been reported to treat hyperglycemia, metabolic disorders, cough, enhance the retention of sarcosine, protect the liver, and have an antitumor effect.

D-pinitol can be prepared by chemical synthesis, but chemical synthesis has the problems of residues of various chemical reaction raw materials and safety risks, and the food grade is mainly prepared by separating and purifying plants naturally at present. D-pinitol is naturally present in the pollen of Pinaceae plants such as sugar pine, radix Puerariae, Central east carob bean, semen Ginkgo, and Leguminosae plant Lespedeza cuneata, and is also found in senna leaf, herba Dianthi, white bark pine cone, Pindorw leaf, and Madagasca latex. Moreover, researches show that the water extract of the green carob bean in the middle east has about 5-6 percent of D-pinitol content, but has about 39 percent of water content, about 13 percent of fructose content, about 13 percent of glucose content and about 22.3 percent of sucrose content, has more impurities, and has excessively high sugar content and high contents of blood sugar increasing factors of fructose, sucrose and glucose for people who are not suitable for taking sugar, such as diabetics, so that the problem of reducing the content of the impurities in the extract and the amount of the blood sugar increasing factors while purifying the D-pinitol is urgently needed to be solved.

At present, the method for extracting D-pinitol commonly uses a solvent method and an ultrasonic method, and purification is carried out by using ion exchange resin, a precipitator and the like after extraction, but the methods have poor purification effect, high impurity content and almost no removal of blood glucose increasing factors. Therefore, there is an urgent need for a safe and mild purification method of D-pinitol, which can convert the blood sugar increasing factor into a factor having no influence on blood sugar of human body and beneficial to health.

Disclosure of Invention

In view of the above, the present invention provides a method for purifying D-pinitol from a water extract of carob beans, which comprises the steps of reducing impurities by using a mild enzymatic purification method, and sequentially performing sucrose hydrolysis conversion, fructose precipitation and glucose oxidation to remove as many as possible of blood glucose increasing factors.

A method for purifying D-pinitol from an aqueous extract of carob beans, said method comprising the steps of:

pretreatment of raw materials: adjusting the pH of the aqueous extract of carob to acidic;

hydrolysis and transformation of sucrose: adding sucrase into the raw material solution to hydrolyze and convert sucrose into fructose and glucose;

and (3) fructose precipitation: removing fructose by precipitation;

oxidation of glucose: oxidizing glucose to gluconic acid;

purifying D-pinitol: separating gluconic acid by chromatography, concentrating water eluent under reduced pressure, and drying to obtain high-purity D-pinitol.

Further, the method for purifying D-pinitol includes the following steps:

pretreatment of raw materials: diluting the raw material liquid, and adding acetic acid to adjust the pH to 4-5; adjusting the pH value to reach the optimal pH value of the catalytic reaction of the sucrase, and ensuring that the subsequent sucrose can be hydrolyzed and converted into fructose and glucose as much as possible;

hydrolysis and transformation of sucrose: heating the pretreated raw material liquid to 50-60 ℃ in a water bath, adding sucrase, performing thermal insulation hydrolysis at the stirring speed of 150-200rpm, and performing stirring reaction for 2-4h to obtain hydrolysate; the detection shows that the hydrolysis rate of the sucrose is 95-97.6%.

After the sucrose is hydrolyzed and converted, the sucrose is converted into fructose and glucose, and the content of blood glucose increasing factors is reduced;

and (3) fructose precipitation: adding the hydrolysate into deionized cold water at 2-10 deg.C, wherein the volume ratio of the hydrolysate to the deionized cold water is 1 (5-7), stirring, adding supersaturated calcium hydroxide solution, standing in a cold storage at 0-10 deg.C for 10 hr, centrifuging, and concentrating the obtained supernatant under reduced pressure.

Calcium hydroxide is used for precipitating fructose, a large amount of glucose remains in the solution at the moment, and the content of blood glucose increasing factors is further reduced;

oxidation of glucose: taking the supernatant I after decompression concentration, diluting, adjusting the pH value to be 5-6, heating in a water bath, preserving heat, adding glucose oxidase and catalase according to the detected glucose concentration after the supernatant I reaches the optimal reaction temperature of enzyme catalysis, uniformly stirring, and simultaneously introducing oxygen or compressed air with the flow of 1L/min for 12-24h to obtain a reaction solution;

purifying D-pinitol: performing chromatography on the reaction solution by ion exchange resin, eluting with water to obtain an eluent containing D-pinitol, concentrating the eluent under reduced pressure, and drying to obtain purified D-pinitol; d293 anion exchange resin is selected as the ion exchange resin;

secondary purification: dissolving the obtained D-pinitol with deionized water, and repeating the steps of sucrose hydrolysis conversion, fructose precipitation, glucose oxidation and D-pinitol purification to obtain the D-pinitol with the purity of more than 90%.

Further, in the sucrose hydrolysis conversion step, the addition amount of sucrase is 15-25mg/100g of raw material solution.

Further, in the fructose precipitation step, the volume ratio of the hydrolysate to the supersaturated calcium hydroxide solution is 1 (0.5-1.5), and the concentration of the supersaturated calcium hydroxide solution is 10-20%.

Further, in the glucose oxidation step, the supernatant I after the concentration under reduced pressure is taken and diluted until the concentration of the glucose is 10%.

Further, the glucose is oxidized, the dosage of the glucose oxidase is 3.5-4 kg/ton glucose, and the dosage of the catalase is 2.5-3 kg/ton glucose.

Further, in the fructose precipitation step, after the supernatant I is subjected to reduced pressure concentration, adding a fructose adsorbent into the supernatant I, stirring at the speed of 200rpm for 8-10h, and then filtering the fructose adsorbent, wherein the fructose adsorbent is obtained by carrying out impurity removal after volcanic rock activation pretreatment and then loading calcium ions.

Further, the fructose precipitation step also comprises the steps of carrying out reduced pressure concentration on the supernatant I to 2 times of the volume of the raw material liquid, then adding a saturated calcium hydroxide solution with the volume of 1.5 times of the volume of the raw material liquid and the concentration of 20%, uniformly stirring, standing in a refrigeration house at 4 ℃ for 10 hours, then centrifuging, carrying out reduced pressure concentration on the supernatant II obtained after centrifuging, and treating the reduced pressure concentrated supernatant II in the glucose oxidation step. When the fructose precipitation rate in the detected solution is less than or equal to 90 percent, secondary precipitation is needed, redundant fructose in the solution is removed, and the content of the fructose in the solution is further reduced.

Further, in the step of glucose oxidation, the temperature is heated to 34-35 ℃ in a water bath, and the temperature is preserved to carry out glucose oxidation.

Further, the specific operations of reduced pressure concentration in the steps of fructose precipitation, glucose oxidation and D-pinitol purification are that the supernatant is vacuumized, reduced pressure is reduced to (-0.06) - (-0.1) Mpa, and the supernatant is concentrated in the environment of lower than 60 ℃. The preferred temperature is 55 ℃.

Has the advantages that:

1. the method has the advantages of converting sucrose into fructose and glucose, removing fructose precipitate, converting glucose into gluconic acid, simplifying steps, reducing impurities, reducing the content of blood glucose-raising factors, and purifying D-pinitol.

2. The method adopts a safe and mild enzyme method to remove sucrose and glucose in the blood glucose increasing factor step by step, has high conversion degree, avoids introducing other impurities and solvents, and improves the edible safety.

3. The glucogenic factor is safely converted into gluconic acid which is used as a byproduct together with fructose, so that the practical degree and the development value of the method for purifying the D-pinitol are improved, and the method is convenient to popularize.

Drawings

FIG. 1 is a spectrum of a raw material liquid of example 1;

FIG. 2 is a spectrum of example 1 after removal of sucrose (end of step (2));

FIG. 3 is a spectrum of D-pinitol after purification in example 1;

FIG. 4 is a control spectrum of example 1.

Detailed Description

The present invention will be described in detail below with reference to specific examples: