CN104017104A - Preparation method for soluble and highly substituted phosphorylated yeast glucan - Google Patents
Preparation method for soluble and highly substituted phosphorylated yeast glucan Download PDFInfo
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- CN104017104A CN104017104A CN201410213080.9A CN201410213080A CN104017104A CN 104017104 A CN104017104 A CN 104017104A CN 201410213080 A CN201410213080 A CN 201410213080A CN 104017104 A CN104017104 A CN 104017104A
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Abstract
The invention relates to a preparation method for soluble and highly substituted phosphorylated yeast glucan. The preparation method comprises the following steps of (1) mixing powder yeast glucan raw materials and sodium hexametaphosphate or potassium hexametaphosphate according to a mass ratio of 1:1.5-15 and processing for 8-40 min with a mechanical chemistry method by using a planetary ball mill; (2) dissolving a glucan mixture obtained by the step (1) in water to obtain a solution according to a mass ratio of the glucan mixture to water being 1:6-18 and removing insoluble substances by centrifugation to obtain the solution if precipitate is produced; and (3) subjecting the solution obtained in the step (2) to dialysis or ultrafiltration to remove redundant sodium hexametaphosphate or potassium hexametaphosphate, and thus a phosphorylated yeast glucan solution is obtained. The preparation method is simple and convenient, fast and efficient, is in no use of an organic solvent and strong acid and strong alkali reagents, and is beneficial to large-scale production.
Description
Technical field
The present invention relates to dextran technical field, especially relate to a kind of method solubility, high substitution value phosphorylation yeast glucan of preparing.
Background technology
Yeast callose (being called for short yeast beta-dextran or yeast glucan) is that a kind of good immunological effect is replied agent, have regulate immunizing power, antitumor, anti-oxidant, promote the physiological functions such as wound healing, radioprotective, reduction blood fat, but it is water insoluble, this has seriously limited the application of yeast glucan in the fields such as food, medicine, healthcare products.
In order to improve the solubleness of yeast glucan, Chinese scholars has been carried out modification and study on the modification to it.These methods are mainly by connect some polar groups in dextran, as phosphate group, carboxymethyl group, sulfate group increase its solubleness.But this class reaction all will be carried out conventionally in strong acid, highly basic or organic phase.Especially common phosphorylation modification and sulphation modification, need to consume a large amount of strong acid, highly basic and organic solvent, and not only cost is high, seriously polluted, also may cause potential safety hazard, is unfavorable for industrialized scale operation.
Traditional yeast glucan phosphorylation modification mainly, by add Phosphation reagent in acidic solution, is induced the carrying out of phosphating reaction under hot conditions.Because this class reaction is all carried out in the liquid environment of acidity or high temperature, yeast glucan is easily hydrolyzed, so the phosphorylation modification inefficiency of yeast glucan, and the substitution value of the phosphorylation dextran obtaining is conventionally all lower, generally lower than 0.2.The method of therefore a kind of efficient, environmental protection of research invention, simple phosphorylation modification yeast glucan is very necessary.
Summary of the invention
For the problem such as phosphorylation modification yeast glucan inefficiency, substitution value be low, seriously polluted, the applicant provides the preparation method of a kind of solubility, high substitution value phosphorylation yeast glucan.That present method has is easy, quick, efficiently, not with an organic solvent with the feature of strong acid, highly basic reagent, be conducive to industrialized large-scale production.
Technical scheme of the present invention is as follows:
The preparation method of a kind of solubility, high substitution value phosphorylation yeast glucan, adopt mechanochemistry method, in solid system, take Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium is derivatization reagent, by solid-state chemical reaction, yeast glucan is carried out to phosphorylation modification and modification, and detailed process is as follows:
(1) after 1:1.5 ~ 15 mix in mass ratio by Powdered yeast glucan raw material and Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium, adopt planetary ball mill to process 8 ~ 40 min by mechanochemistry method;
(2) by the dextran mixture lysate that obtains soluble in water obtaining after step (1) processing, the mass ratio of dextran mixture and water is 1:6 ~ 18; If the precipitation of generation, by the centrifugal insolubles of removing, thereby obtains lysate;
(3) lysate step (2) being obtained is dialysed or ultrafiltration, removes unnecessary Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium, obtains phosphorylation yeast glucan solution.
After step (3) gained phosphorylation yeast glucan solution is concentrated, can obtain phosphorylation yeast glucan concentrated solution; Step (3) gained phosphorylation yeast glucan solution is sprayed after dry or lyophilize, can obtain phosphorylation yeast glucan dry powder.The phosphate group substitution value of the yeast glucan making is 0.6 ~ 2.4.
The technique effect that the present invention is useful is:
The present invention is from phosphorylation, sulfation and carboxymethylation yeast glucan raw material are prepared in the method for solubility yeast glucan, pick out and adopt Sodium hexametaphosphate 99 and hexa metaphosphoric acid potassium to carry out phosphorylation modification yeast glucan raw material, not with an organic solvent and strong acid, highly basic reagent, almost do not pollute, the phosphorylation yeast glucan making can be completely water-soluble, there is higher phosphate group substitution value, reach as high as 240%, and there is good immunocompetence, therefore be expected to be effectively applied to food, healthcare products, medicine, in the industries such as feed, promote yeast glucan as the using value of biological immunomodulator.
Embodiment
Carry out by the following examples further to illustrate the present invention, the following example is for illustration purpose but not for limiting the scope of the invention.In following examples, used yeast dextran raw material is the yeast beta-dextran GNP-80 that Shanghai Jiekangnuo Biotechnology Co., Ltd. produces.
Embodiment 1: mechanochemical reaction is prepared phosphorylation yeast glucan scheme 1
After yeast glucan mixes with mass ratio 1:2 with Sodium hexametaphosphate 99, adopt planetary mills to carry out mechanical activation processing, power is 30 Hz, and the treatment time is 10 min; Yeast glucan mixture after processing is dissolved in after water with mass ratio 1:10, uses whizzer centrifugal 20 min under 4000 rpm rotating speeds, and wherein 82% yeast glucan is water-soluble, and 18% yeast glucan is water insoluble; Water-soluble lysate water is repeatedly dialysed, to remove unnecessary Sodium hexametaphosphate 99.Collect dialyzate, it is carried out to lyophilize processing, obtain phosphorylation yeast glucan.(phosphate group) substitution value of the phosphorylation dextran of preparation is 0.6 thus.
By phosphorylation dextran be dissolved in deionized water to concentration be 100 g/ml, through 0.22 μ m membrane filtration degerming.Utilize phosphorylation dextran solution Stimulated Macrophages RAW264.7 TNF secretion-α, secretory volume reaches 31 ng/mL, is 3 times of blank group, and phosphorylation dextran stimulating expression of macrophage secrete cytokines TNF-α is significantly described.
Embodiment 2: mechanochemical reaction is prepared phosphorylation yeast glucan scheme 2
After yeast glucan mixes with mass ratio 1:1.5 with hexa metaphosphoric acid potassium, adopt planetary mills to carry out mechanical activation processing, power is 40 Hz, and the treatment time is 8 min; Yeast glucan mixture after processing is dissolved in after water with mass ratio 1:10, uses whizzer centrifugal 20 min under 4000 rpm rotating speeds, and wherein 76% yeast glucan is water-soluble, and 24% yeast glucan is water insoluble; Water-soluble lysate water is repeatedly dialysed, to remove unnecessary hexa metaphosphoric acid potassium.Collect dialyzate, it is carried out to lyophilize processing, obtain phosphorylation yeast glucan.(phosphate group) substitution value of the phosphorylation dextran of preparation is 0.65 thus.
By phosphorylation dextran be dissolved in deionized water to concentration be 500 g/ml, through 0.22 μ m membrane filtration degerming.Utilize phosphorylation dextran solution Stimulated Macrophages RAW264.7 secretion IL-6, secretory volume reaches 0.3 ng/mL, is 5 times of blank group, and phosphorylation dextran stimulating expression of macrophage secrete cytokines IL-6 is significantly described.
Embodiment 3: mechanochemical reaction is prepared phosphorylation yeast glucan scheme 3
After yeast glucan mixes with mass ratio 1:5 with Sodium hexametaphosphate 99, adopt planetary mills to carry out mechanical activation processing, power is 30 Hz, and the treatment time is 40 min; Yeast glucan mixture after processing is dissolved in after water with mass ratio 1:8, uses whizzer centrifugal 20 min under 4000 rpm rotating speeds, and wherein 87% yeast glucan is water-soluble, and 13% yeast glucan is water insoluble; Water-soluble lysate water is repeatedly dialysed, to remove unnecessary Sodium hexametaphosphate 99.Collect dialyzate, it is carried out to lyophilize processing, obtain phosphorylation yeast glucan.(phosphate group) substitution value of the phosphorylation dextran of preparation is 2.4 thus.
By phosphorylation dextran be dissolved in deionized water to concentration be 500 g/ml, through 0.22 μ m membrane filtration degerming.Utilize phosphorylation dextran solution Stimulated Macrophages RAW264.7 secretion nitrogen protoxide (NO), secretory volume reaches 4.8 mM, is 4 times of blank group, and phosphorylation dextran stimulating expression of macrophage secretory cell messenger molecule NO is significantly described.
Embodiment 4: mechanochemical reaction is prepared phosphorylation yeast glucan scheme 4
After yeast glucan mixes with mass ratio 1:15 with Sodium hexametaphosphate 99, adopt planetary mills to carry out mechanochemistry processing, power is 40 Hz, and the treatment time is 12 min; Yeast glucan mixture after processing is dissolved in after water with mass ratio 1:15, uses whizzer centrifugal 20 min under 4000 rpm rotating speeds, and wherein 70% yeast glucan is water-soluble, and 30% yeast glucan is water insoluble; Water-soluble lysate water is carried out to repeatedly ultrafiltration, to remove unnecessary Sodium hexametaphosphate 99.Collect ultrafiltrated, it is carried out to spray drying treatment, obtain phosphorylation yeast glucan.(phosphate group) substitution value of the phosphorylation dextran of preparation is 1.5 thus.
By phosphorylation dextran be dissolved in deionized water to concentration be 100 g/ml, through 0.22 μ m membrane filtration degerming.Utilize phosphorylation dextran solution Stimulated Macrophages RAW264.7 TNF secretion-α, secretory volume reaches 70 ng/mL, is 7 times of blank group, and phosphorylation dextran stimulating expression of macrophage secrete cytokines TNF-α is significantly described.
The mensuration of yeast glucan concentration: adopt the anthrone method of measuring plant sugar concentration.
The mensuration of phosphate group substitution value: adopt fixing phosphorus method to measure the volumetric molar concentration of phosphate group in phosphorylation yeast glucan, the phosphate group volumetric molar concentration of phosphorylation yeast glucan is phosphate group substitution value with the ratio of its glucose group volumetric molar concentration.
The detection of mouse macrophage RAW 264.7 emiocytosis cytokine IL-6: (1) cell cultures: by 2 * 10
6individual/mL lymphocyte suspension adds in 96 orifice plates, and every hole adds 180 μ L, adds 20 μ L sample liquid simultaneously, with the LPS solution of 20 μ L perfect mediums and 20 μ L 1 μ g/mL, makes respectively blank and positive control.In 37 ℃, containing 5% CO
2under condition, cultivate 24 h.(2) cytokine IL-6 measures: collect the supernatant liquor in 96 orifice plates of above-mentioned cell cultures, utilize ELSIA cytokine test test kit to detect, specific experiment step reference reagent box specification sheets.
The detection of mouse macrophage RAW 264.7 emiocytosis cytokine NO: (1) cell cultures: with the cell culture processes in the detection of IL-6, cell cultures 48 h.(2) messenger molecule NO measures---Griess method: collect supernatant liquor 50 L in 96 orifice plates of above-mentioned cell cultures, react 10 min with 50 L Griess reagent (1% sulfanilamide (SN): be dissolved into the 0.1%N-1-naphthyl ethylenediamine hydrochloride=1:1 in 2.5% phosphoric acid), utilize microplate reader to measure absorbance under 540 nm.Take 100 M Sodium Nitrites as standard substance preparation standard curve, utilize typical curve to calculate NO content.
The detection of mouse macrophage RAW 264.7 emiocytosis cytokine TNF-α: (1) cell cultures: with the cell culture processes in the detection of IL-6, cell cultures 24 h.(2) cytokine TNF-α is measured: collect the supernatant liquor in 96 orifice plates of above-mentioned cell cultures, utilize ELSIA cytokine test test kit to detect, specific experiment step reference reagent box specification sheets.
Above-described is only the preferred embodiment of the present invention, the invention is not restricted to above embodiment.Protection scope of the present invention is appreciated that the oher improvements and changes that those skilled in the art directly derive or associate without departing from the inventive concept of the premise, within should be included in.
Claims (3)
1. the preparation method of a solubility, high substitution value phosphorylation yeast glucan, it is characterized in that adopting mechanochemistry method, in solid system, take Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium is derivatization reagent, by solid-state chemical reaction, yeast glucan is carried out to phosphorylation modification and modification, detailed process is as follows:
(1) after 1:1.5 ~ 15 mix in mass ratio by Powdered yeast glucan raw material and Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium, adopt planetary ball mill to process 8 ~ 40 min by mechanochemistry method;
(2) by the dextran mixture lysate that obtains soluble in water obtaining after step (1) processing, the mass ratio of dextran mixture and water is 1:6 ~ 18; If the precipitation of generation, by the centrifugal insolubles of removing, thereby obtains lysate;
(3) lysate step (2) being obtained is dialysed or ultrafiltration, removes unnecessary Sodium hexametaphosphate 99 or hexa metaphosphoric acid potassium, obtains phosphorylation yeast glucan solution.
2. solubility according to claim 1, high substitution value phosphorylation yeast glucan preparation method, after it is characterized in that step (3) gained phosphorylation yeast glucan solution to concentrate, can obtain phosphorylation yeast glucan concentrated solution; Step (3) gained phosphorylation yeast glucan solution is sprayed after dry or lyophilize, can obtain phosphorylation yeast glucan dry powder.
3. solubility according to claim 1, high substitution value phosphorylation yeast glucan preparation method, is characterized in that the phosphate group substitution value of the yeast glucan that makes is 0.6 ~ 2.4.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107674129A (en) * | 2017-09-04 | 2018-02-09 | 珠海伊斯佳科技股份有限公司 | Schizophan phosphorylated derivative and preparation method thereof, application |
| CN114014949A (en) * | 2021-11-15 | 2022-02-08 | 戚春建 | Preparation method of water-soluble yeast glucan |
| CN116589609A (en) * | 2023-07-13 | 2023-08-15 | 天津永续新材料有限公司 | Preparation method of phosphorylated nano-chitin based on mechanochemical method and phosphorylated nano-chitin |
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| CN102838688A (en) * | 2012-09-12 | 2012-12-26 | 江南大学 | Preparation method of soluble yeast glucan |
| WO2013146669A1 (en) * | 2012-03-28 | 2013-10-03 | 国立大学法人岡山大学 | Method for producing phosphorylated polysaccharide |
| CN103524638A (en) * | 2013-10-16 | 2014-01-22 | 江南大学 | Method for preparing soluble yeast glucan |
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| WO2013146669A1 (en) * | 2012-03-28 | 2013-10-03 | 国立大学法人岡山大学 | Method for producing phosphorylated polysaccharide |
| CN102838688A (en) * | 2012-09-12 | 2012-12-26 | 江南大学 | Preparation method of soluble yeast glucan |
| CN103524638A (en) * | 2013-10-16 | 2014-01-22 | 江南大学 | Method for preparing soluble yeast glucan |
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| 潘志东: "矿渣水泥和魔芋葡甘聚糖的振动机械力化学效应", 《中国博士学位论文全文数据库 工程科技Ⅰ辑》, no. 12, 15 December 2011 (2011-12-15), pages 131 - 142 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674129A (en) * | 2017-09-04 | 2018-02-09 | 珠海伊斯佳科技股份有限公司 | Schizophan phosphorylated derivative and preparation method thereof, application |
| CN107674129B (en) * | 2017-09-04 | 2020-12-15 | 珠海伊斯佳科技股份有限公司 | Schizophyllan phosphorylated derivative and preparation method and application thereof |
| CN114014949A (en) * | 2021-11-15 | 2022-02-08 | 戚春建 | Preparation method of water-soluble yeast glucan |
| CN114014949B (en) * | 2021-11-15 | 2022-08-30 | 戚春建 | Preparation method of water-soluble yeast glucan |
| CN116589609A (en) * | 2023-07-13 | 2023-08-15 | 天津永续新材料有限公司 | Preparation method of phosphorylated nano-chitin based on mechanochemical method and phosphorylated nano-chitin |
| CN116589609B (en) * | 2023-07-13 | 2023-10-20 | 天津永续新材料有限公司 | Preparation method of phosphorylated nano-chitin based on mechanochemical method and phosphorylated nano-chitin |
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