阅读说明：本技术 一种白芨葡甘聚糖酯的制备方法及其应用 (Preparation method and application of bletilla striata glucomannan ester ) 是由 邓辉 陈存武 韩邦兴 张卫明 谷仿丽 刘�东 马世宏 宋程 卫培培 于 2019-11-07 设计创作，主要内容包括：本发明公开一种白芨葡甘聚糖酯的制备方法,本发明包括以下步骤：(1)将白芨葡甘聚糖分散于蒸馏水中,用磷酸缓冲液调节pH至6.0,加入β-甘露聚糖酶,于50-60℃保温振荡后,煮沸灭酶；(2)白芨葡甘聚糖的酯化：将步骤(1)中的产物溶于有机溶剂或有机溶剂与水的混合物中,然后加入C6～C20饱和脂肪酸、脂肪酶搅拌反应,将获得的反应液进行纯化,制得白芨葡甘聚糖酯。本发明还提供上述制备方法制得的白芨葡甘聚糖酯在制备乳化剂和微囊粉中的应用,本发明的有益效果在于：本发明制备的疏水性白芨葡甘聚糖通过自组装成纳米胶团,增强包裹物在水溶液中的分散性,延长释放时间,且制备方法简单。(The invention discloses a preparation method of bletilla striata glucomannan ester, which comprises the following steps: (1) dispersing bletilla striata glucomannan in distilled water, adjusting pH to 6.0 with phosphate buffer, adding beta-mannase, keeping temperature at 50-60 deg.C, shaking, boiling to inactivate enzyme; (2) esterification of bletilla striata glucomannan: dissolving the product obtained in the step (1) in an organic solvent or a mixture of the organic solvent and water, adding C6-C20 saturated fatty acid and lipase, stirring for reaction, and purifying the obtained reaction liquid to obtain the bletilla glucomannan ester. The invention also provides the application of the bletilla striata glucomannan ester prepared by the preparation method in preparing the emulsifier and the microcapsule powder, and the invention has the beneficial effects that: the hydrophobic bletilla striata glucomannan prepared by the invention is self-assembled into a nano micelle, the dispersibility of the wrappage in aqueous solution is enhanced, the release time is prolonged, and the preparation method is simple.)

1. A preparation method of bletilla striata glucomannan ester is characterized in that: the method comprises the following steps:

(1) taking bletilla striata glucomannan solution, adjusting pH to 6.0 with phosphate buffer, adding beta-mannase, keeping temperature at 50-60 deg.C, shaking, boiling to inactivate enzyme;

(2) esterification of bletilla striata glucomannan: dissolving the product obtained in the step (1) in an organic solvent or a mixture of the organic solvent and water, adding C6-C20 saturated fatty acid and lipase, adjusting the water activity of the mixture to 0.4-0.8, stirring and reacting at the reaction temperature of 42-50 ℃ and the enzyme dosage of 300-800U/mL, and purifying the obtained reaction liquid to obtain the bletilla glucomannan ester.

2. The preparation method of bletilla striata glucomannan ester according to claim 1, wherein: the preparation method of the bletilla striata glucomannan solution comprises the following steps: weighing bletilla striata glucomannan, and dispersing in distilled water.

3. The preparation method of bletilla striata glucomannan ester according to claim 1, wherein: in the step (1), the temperature is kept and the oscillation is carried out for 2 to 5 hours, and the boiling is carried out for 10 to 15 min.

4. The preparation method of bletilla striata glucomannan ester according to claim 1, wherein: the organic solvent is one of dimethyl sulfoxide, N-dimethylformamide and formamide.

5. The preparation method of bletilla striata glucomannan ester according to claim 1, wherein: the C6-C20 saturated fatty acid comprises one of lauric acid, myristic acid, palmitic acid and stearic acid.

6. The preparation method of bletilla striata glucomannan ester according to claim 1, wherein: the purification method of the bletilla striata glucomannan ester comprises the following steps: putting the obtained reaction liquid into a dialysis bag, dialyzing in 10 times of volume of distilled water, changing water every 4-6h, dialyzing for 48h, collecting the solution in the dialysis bag, heating, centrifuging to obtain supernatant, and drying the supernatant under negative pressure to obtain bletilla glucomannan ester.

7. The preparation method of bletilla striata glucomannan ester according to claim 6, wherein: the heating temperature in the purification step is 100 ℃, and the heating time is 10-20 min.

8. The preparation method of bletilla striata glucomannan ester according to claim 7, wherein: the centrifugal speed is 8000-10000 rpm.

9. The use of bletilla striata glucomannan ester prepared by the preparation method of any one of claims 1-8 in preparing an emulsifier, wherein: the method comprises the following steps: putting bletilla striata glucomannan ester into distilled water, homogenizing at 10000r/min, performing ultrasonic treatment, filtering the solution after ultrasonic treatment with a 0.45 μm filter membrane, filtering with a 0.22 μm filter membrane, and collecting the filtrate to obtain the emulsifier.

10. The application of bletilla striata glucomannan ester prepared by the preparation method of any one of claims 1-8 in preparing microcapsule powder is characterized in that: the method comprises the following steps:

(1) placing bletilla striata glucomannan ester in distilled water, homogenizing at 10000r/min, performing ultrasound treatment, filtering the solution after ultrasound treatment with a 0.45 μm filter membrane, filtering with a 0.22 μm filter membrane, and collecting the filtrate to obtain an emulsifier;

(2) mixing the emulsifier prepared in the step (1) with ganoderma lucidum spore oil, homogenizing at 10000r/min, and spray drying the homogenized emulsion to obtain microcapsule powder.

Technical Field

The invention relates to the technical field of functional biomaterials, in particular to a preparation method and application of bletilla striata glucomannan ester.

Background

Bletilla striata glucomannan (KGM) is the main component in bletilla striata gum. In Japan, USA and China, the application research of bletilla striata in the fields of medicine, food, chemical industry and textile has made an important progress, but natural long-chain bletilla striata glucomannan is extremely difficult to dissolve in water and is not soluble in other organic solvents, so the research is greatly limited.

In recent years, natural polysaccharides have been reported as emulsifiers and emulsion stabilizers in the fields of foods, cosmetics, medicines, and the like. The use of biologically synthesized or purified oligo-and exopolysaccharide esters as emulsifiers or surfactants in industry has also advanced.

Patent CN201010270592.0 provides a method for esterification of konjac glucomannan, which adopts a mechanochemical method, comprising the following steps: the first step is as follows: drying the konjac glucomannan until the water content is lower than 4%, and then adding an esterification modifier with the mass of 0.5-5% of the mass of the konjac glucomannan; the second step is that: preparing a grinding medium according to the volume filling rate of 50-90% of a grinding tank of a vibration mill; the third step: uniformly mixing the konjac glucomannan and the grinding medium, adding the mixture into a grinding tank of the vibration mill, sealing the grinding tank, and starting the vibration mill for grinding for 20-60 minutes; the fourth step: separating the grinding medium from the sample by screening to obtain the esterified konjac glucomannan.

Disclosure of Invention

One of the technical problems to be solved by the invention is a preparation method of bletilla striata glucomannan ester with stable emulsifying property.

The invention solves the technical problems through the following technical means:

the invention provides a preparation method of bletilla striata glucomannan ester, which comprises the following steps:

(1) taking bletilla striata glucomannan solution, adjusting pH to 6.0 with phosphate buffer, adding beta-mannase, keeping temperature at 50-60 deg.C, shaking, boiling to inactivate enzyme;

(2) esterification of bletilla striata glucomannan: dissolving the product obtained in the step (1) in an organic solvent or a mixture of the organic solvent and water, adding C6-C20 saturated fatty acid and lipase, adjusting the water activity of the mixture to 0.4-0.8, stirring and reacting at the reaction temperature of 42-50 ℃ and the enzyme dosage of 300-800U/mL, and purifying the obtained reaction liquid to obtain the bletilla glucomannan ester.

Has the advantages that: the hydrophobic bletilla striata glucomannan ester is prepared by taking bletilla striata glucomannan as a raw material, the hydrophobic bletilla striata glucomannan prepared by the method forms a core-shell structure by self-assembling into a nano micelle, the nano micelle wraps a substance which is hydrophobic, easy to oxidize and needs slow release, the dispersibility of the wrapping substance in an aqueous solution is enhanced, the release time is prolonged, the prepared bletilla striata glucomannan ester is easy to emulsify to form the micelle, the application field of the bletilla striata glucomannan is expanded, and the preparation method is simple and efficient.

Preferably, the preparation method of the bletilla striata glucomannan solution comprises the following steps: weighing 1-2g bletilla striata glucomannan, and dispersing in 100ml distilled water.

Preferably, in the step (1), the temperature is kept and the shaking is carried out for 2-5h, and the boiling is carried out for 10-15 min.

Preferably, the organic solvent is one of dimethyl sulfoxide, N-dimethylformamide and formamide.

Preferably, the beta-mannanase is derived from bacillus subtilis.

Preferably, 0.2-0.5% w/v β -mannanase is added to the glucomannan solution.

Preferably, the C6-C20 saturated fatty acid includes one of lauric acid, myristic acid, palmitic acid and stearic acid.

Preferably, the rotation speed of the stirring reaction in the step (2) is 150-300 r/min.

Preferably, the lipase is derived from bacillus subtilis.

Preferably, the purification method of bletilla striata glucomannan ester comprises the following steps: putting the obtained reaction liquid into a dialysis bag, dialyzing in 10 times of volume of distilled water, changing water every 4-6h, dialyzing for 48h, collecting the solution in the dialysis bag, heating, centrifuging to obtain supernatant, and drying the supernatant under negative pressure to obtain bletilla glucomannan ester.

Has the advantages that: and (3) replacing the organic solvent by distilled water, heating the denatured enzyme, centrifuging to remove, and purifying to obtain the target product.

Preferably, the heating temperature in the purification step is 100 ℃ and the heating time is 10-20 min.

Preferably, the centrifugal rotation speed is 8000-10000 rpm.

Preferably, the preparation method of the bletilla striata glucomannan solution comprises the following steps: weighing 1-2g bletilla striata glucomannan, and dispersing in 100ml distilled water.

Preferably, the preparation method of the bletilla striata glucomannan solution comprises the following steps:

(1) mixing rhizoma Bletillae with water, and homogenizing in a homogenizer to obtain rhizoma Bletillae slurry;

(2) centrifuging the bletilla striata serous fluid in the step (1) to obtain a supernatant;

(3) establishing an ethanol-dipotassium hydrogen phosphate double-water-phase system: the volume fraction of ethanol in the ethanol aqueous solution is 70%, the weight fraction of dipotassium hydrogen phosphate in the dipotassium hydrogen phosphate aqueous solution is 55%, and the volume ratio of the ethanol aqueous solution to the dipotassium hydrogen phosphate aqueous solution is 3: 7;

(4) placing the supernatant and the ethanol-dipotassium hydrogen phosphate aqueous two-phase system in the step (2) into an ultrasonic countercurrent extraction device for extraction, wherein the ratio of the supernatant to the ethanol-dipotassium hydrogen phosphate aqueous two-phase system is 1:45(g: mL);

(5) standing the extracting solution in the step (4), taking down the phase solution, filtering, and respectively carrying out ultrafiltration separation on the filtrate by a hollow fiber ultrafiltration device with molecular weight cut-off of 100K and 10K to obtain bletilla striata glucomannan solution with molecular weight of 10-100K and more than 100K.

Has the advantages that: the bletilla striata glucomannan aqueous solution is not a product prepared by redissolving dry powder, so that the intermolecular action is less damaged, and the film-forming property and the emulsibility are better.

Preferably, the water in step (1) is deionized water.

Preferably, the mass ratio of the bletilla striata to the deionized water is 1: 4.

Preferably, in the step (1), the mixture of bletilla striata and deionized water is placed in a homogenizer, and is homogenized for 5min at 13000-15000rpm at 4-10 ℃, with 1min pause, and the total homogenizing time is 25-30 min.

Preferably, the bletilla striata slurry is centrifuged for 15-20min at 5000-8000rpm and 4-10 ℃ in the step (2).

Has the advantages that: breaking coarse fibers, reducing the viscosity of bletilla striata glucomannan, separating unswollen starch granules and other insoluble impurities from plant tissues, dissociating the fibers, starch and glucomannan in bletilla striata by using low-temperature high-speed homogenate, and precipitating unswollen starch granules and other insoluble impurities by using low-temperature centrifugation, wherein other preparations are not added, so that the exogenous impurity pollution of the product is reduced; the method does not adopt high-temperature treatment, and is carried out at normal temperature or low temperature, so that the damage and reaction of the temperature to the bletilla striata glucomannan are reduced.

Preferably, the extraction in the step (4) is carried out at 25 ℃ and 500W for 20 min.

Preferably, the lower phase solution in the step (5) is filtered by a 400-mesh nylon filter cloth.

Preferably, in the step (5), under the conditions that the pressure is 0.20MPa, the ultrafiltration temperature is 30 ℃ and the volume flow rate of the feed liquid is 0.06-0.08L/min, the bletilla striata glucomannan concentrated solution with the molecular weight of more than 100K is obtained by interception.

Preferably, the filtrate after ultrafiltration by the hollow fiber ultrafiltration device with the molecular weight cut-off of 100K is subjected to ultrafiltration separation by the hollow fiber ultrafiltration device with the molecular weight cut-off of 100K, and in the step (5), the bletilla striata glucomannan concentrated solution with the molecular weight of 10-100K is obtained by cutting off under the conditions that the pressure is 0.20MPa, the ultrafiltration temperature is 25 ℃, and the volume flow of the feed liquid is 0.04-0.05L/min.

The second technical problem to be solved by the invention is to provide the application of the bletilla striata glucomannan ester prepared by the preparation method in preparing the emulsifier.

The invention provides an application of bletilla striata glucomannan ester prepared by the preparation method in preparing an emulsifier, which comprises the following steps:

putting bletilla striata glucomannan ester into distilled water, homogenizing at 10000r/min, performing ultrasonic treatment, filtering the solution after ultrasonic treatment with a 0.45 μm filter membrane, filtering with a 0.22 μm filter membrane, and collecting the filtrate to obtain the emulsifier.

Preferably, bletilla striata glucomannan ester with the degree of substitution of 0.20 is selected to prepare the emulsifier.

Has the advantages that: the hydrophobic bletilla striata glucomannan ester prepared by the method can be used for packaging, protection and slow release of fat-soluble bioactive components and essential oil.

The third technical problem to be solved by the invention is to provide the application of the bletilla striata glucomannan ester prepared by the preparation method in preparing the microcapsule powder.

The invention provides an application of bletilla striata glucomannan ester prepared by the preparation method in preparation of microcapsule powder, which comprises the following steps:

(1) placing bletilla striata glucomannan ester in distilled water, homogenizing at 10000r/min, performing ultrasound treatment, filtering the solution after ultrasound treatment with a 0.45 μm filter membrane, filtering with a 0.22 μm filter membrane, and collecting the filtrate to obtain an emulsifier;

(2) mixing the emulsifier prepared in the step (1) with ganoderma lucidum spore oil, homogenizing at 10000r/min, and spray drying the homogenized emulsion to obtain microcapsule powder.

Has the advantages that: the microcapsule powder is prepared by drying, and can be used for packaging and storing bioactive substances.

Preferably, the volume ratio of the ganoderma lucidum spore oil to the emulsifier is 1: 2.

Preferably, bletilla striata glucomannan ester with the degree of substitution of 0.35 is selected to prepare the micro-capsule powder.

The invention has the advantages that:

(1) the hydrophobic bletilla striata glucomannan ester is prepared by taking bletilla striata glucomannan as a raw material, the hydrophobic bletilla striata glucomannan prepared by the method forms a core-shell structure by self-assembling into a nano micelle, the nano micelle wraps a substance which is hydrophobic, easy to oxidize and needs slow release, the dispersibility of the wrapping substance in an aqueous solution is enhanced, the release time is prolonged, the prepared bletilla striata glucomannan ester is easy to emulsify to form the micelle, the application field of the bletilla striata glucomannan is expanded, and the preparation method is simple;

(2) the beta-mannase enzymatic method is adopted to remove the branch macromolecule bletilla striata glucomannan into a smaller molecular product, the reaction condition is mild, and the water solubility of the product is better;

(3) the emulsifier prepared from the bletilla striata glucomannan ester has good stability and shows good salt resistance and acid resistance;

(4) the preparation process of the hydrophobic bletilla striata glucomannan ester is milder, quicker and more efficient through the optimized enzymatic reaction conditions;

(5) the hydrophobic bletilla striata glucomannan ester prepared by the method can be used for packaging, protection and slow release of fat-soluble bioactive components and essential oil;

(6) the microcapsule powder is prepared by drying, and can be used for packaging and storing bioactive substances.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but 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.

Test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.