CN105950688A - Method for preparing plant-base hyaluronic acid from white fungus sporophores - Google Patents

Abstract

The invention discloses a method for preparing plant-base hyaluronic acid from white fungus sporophores, belonging to the technical field of hyaluronic acid preparation. The method comprises the following steps: washing white fungus sporophores, immersing in deionized water, grinding, carrying out microwave heating to obtain a pretreated slurry, pulverizing grape peel, culturing the pulverized grape peel in a potato sucrose culture medium, eluting with deionized water to obtain a mixed microbial eluent, mixing the mixed microbial eluent with the pretreated slurry and sucrose, carrying out fermentation, adding a cellulase, a potassium dihydrogen phosphate solution and the like, heating to react, filtering to obtain a filtrate, concentrating to obtain a concentrated solution, mixing the concentrated solution with anhydrous ethanol, refrigerating, centrifuging to obtain a precipitate, and carrying out vacuum freeze drying and grinding on the precipitate to obtain the plant-base hyaluronic acid. The method has the advantages of simple preparation steps, higher extraction rate (than other processes by 31.2% or above) and short preparation period; and the product has the advantages of high storability, favorable light transmittance and higher relative molecular weight (by 25-31%).

Description

A kind of method preparing plant base hyaluronic acid with Tremella fructification

Technical field

The present invention relates to a kind of method preparing plant base hyaluronic acid with Tremella fructification, belong to hyaluronic acid preparing technical field.

Background technology

Hyaluronic acid is a kind of acid mucopolysaccharide, first isolates this material from bovine vitreous body.Hyaluronic acid demonstrates multiple important physiological function with molecular structure and the physicochemical property of its uniqueness in body, such as lubricating joint, the permeability of regulation blood vessel wall, regulates protein, Water-Electrolyte diffusion and operating, promotes wound healing etc..Particular, it is important that hyaluronic acid has special water retention, it is the material that in the nature having now been found that, moisture retention is best, is referred to as preferable nature moisturizing factor.It is cockscomb and bovine vitreous body etc. that animal tissue extracts primary raw material, with acetone or alcohol by raw material defat, dehydration, with distilled water immersion, filtration, then process with sodium-chloride water solution and chloroformic solution, obtain mixed liquor after adding trypsin insulation afterwards, finally with ion-exchanger carry out processing, purification obtains the hyaluronic acid that refines.This method extraction ratio is extremely low, and only about 1%, separation process is complicated, causes hyaluronic acid expensive.Fermentable extracts using glucose as carbon source through fermentation liquid, and fermentation 48 hours, after fermentation ends, are filtered to remove mycelium and impurity, then i.e. obtain highly purified product with simple operationss such as the alcohol sedimentation method in the medium.Using the hyaluronic acid that fermentation method manufactures, advantage is can be by commodity design to set molecular size range.The selection that it is critical only that strain of fermentation method, selects streptococcus, Lactococcus class etc. more.Though purity is high, but fermentation period length, product relative molecular weight are low, product printing opacity rate variance, protein content high, glucuronic acid content is low.Chemosynthesis is extracted and is used native enzyme polyreaction, synthesize " hyaluronic acid oxaza amylene derivant " first by polysaccharide polymer, then add water decomposition enzyme, produce the complex of derivant and enzyme, finally in 90 degree of reactant liquors Celsius, remove enzyme therein, just synthesize hyaluronic acid.Use synthetic method can be substantially reduced the manufacturing cost of hyaluronic acid, but structural purity is low.

Summary of the invention

The technical problem to be solved: extract hyaluronic acid for animal tissue, the raw material resting period is shorter, extraction ratio is low, cause hyaluronic acid cost high, and microbe fermentation method extraction fermentation period is long, product relative molecular weight is low, cause the drawback of its printing opacity rate variance, provide one take Tremella fructification washing after, soak with deionized water, mill, microwave heating obtains pretreatment slurry, after taking Pericarpium Vitis viniferae pulverizing again, put into potato sucrose culture medium culturing, use deionized water drip washing, obtain mixed microorganism leacheate, with pretreatment slurry, after sucrose mixed fermentation, add cellulase, potassium dihydrogen phosphates etc. mix, reacting by heating filters to get filtrate, it is concentrated to give concentrated solution, mix with dehydrated alcohol, through cold preservation, it is centrifuged to obtain precipitate, its vacuum lyophilization is milled the method for prepared plant base hyaluronic acid.Preparation process of the present invention is simple, and extraction ratio is high, and manufacturing cycle is short, and relative molecular weight is high, efficiently solves that products material is the most easy to store, extraction ratio is low, products obtained therefrom printing opacity rate variance problem.

For solving above-mentioned technical problem, the present invention uses the technical scheme as described below to be:

(1) weigh 80～100g Tremella fructifications, after being washed with deionized 3～5 times, put in the beaker filling 500～600mL deionized waters, after soaking 1～2h, it is placed on stone grinder mill grinding, process of lapping is mixed into 120～180mL deionized waters, obtain Tremella slurry, proceeded to again in microwave applicator, with 500～600W power, microwave heating 15～25min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；

(2) 10～20g Pericarpium Vitis viniferaes are weighed, put in tissue pulverizer and pulverize, Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 35～45 DEG C of constant incubators again, cultivate 2～3 days, by 60～80mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 10～20mL gained mixed microorganism leacheates subsequently, proceeded to fill in the fermentation tank of 100～150mL above-mentioned gained pretreatment slurry, added 6～8g sucrose, after sterilizing waddy stirring 10～15min, fermentation process 6～8h, obtains fermentation slurry；

(3) being sequentially added into 20～30mL above-mentioned gained fermentation slurries in reactor, 100～200mL mass concentrations are 8～10% potassium dihydrogen phosphate, 2～4g cellulase, 1～3g pectase and 1～3g neutral protease, starting agitator, setting speed is 400～600r/min, is heated to 45～50 DEG C, constant temperature stirring reaction 60～80min, subsequently with 80～85 DEG C of heat treated 5～10min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；

(4) above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 70～80 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 200～300mL dehydrated alcohol again by concentrated solution, stir 3～5min with Glass rod, proceeded to cold preservation 10～15h in 2～4 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded to-35～-25 DEG C of vacuum freeze driers in, after being dried 2～4h, become powder with mortar grinder, obtain plant base hyaluronic acid.

The application process of the present invention: weigh plant base hyaluronic acid prepared by the 20～30g present invention and put in glass drying oven, it is added thereto to 25～28mL Fructus Citri Limoniae juice, 8～10mL Mel and 160～200mL pure water simultaneously, after stirring makes it be sufficiently mixed uniformly, bottling can be prepared by astringent.This plant base hyaluronic acid can improve skin-nourishing metabolism; skin smooth is made to moisten; gained astringent can compact skin; permanent moisturizing; and the intrusion of alien bacteria, dust, ultraviolet can be intercepted, protect the skin from infringement, prevent aging; and be again good Transdermal absorption while moisturizing to, be worthy to be popularized and use.

The present invention is compared with additive method, and Advantageous Effects is:

(1) preparation process of the present invention is simple, and extraction ratio is higher than additive method more than 31.2%, and manufacturing cycle is short；

(2) product is easy to store, light transmittance good, and relative molecular weight improves 25～31%.

Detailed description of the invention

First weigh 80～100g Tremella fructifications, after being washed with deionized 3～5 times, put in the beaker filling 500～600mL deionized waters, after soaking 1～2h, it is placed on stone grinder mill grinding, process of lapping is mixed into 120～180mL deionized waters, obtain Tremella slurry, proceeded to again in microwave applicator, with 500～600W power, microwave heating 15～25min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；Then 10～20g Pericarpium Vitis viniferaes are weighed, put in tissue pulverizer and pulverize, Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 35～45 DEG C of constant incubators again, cultivate 2～3 days, by 60～80mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 10～20mL gained mixed microorganism leacheates subsequently, proceeded to fill in the fermentation tank of 100～150mL above-mentioned gained pretreatment slurry, added 6～8g sucrose, after sterilizing waddy stirring 10～15min, fermentation process 6～8h, obtains fermentation slurry；Being sequentially added into 20～30mL above-mentioned gained fermentation slurries again in reactor, 100～200mL mass concentrations are 8～10% potassium dihydrogen phosphate, 2～4g cellulase, 1～3g pectase and 1～3g neutral protease, starting agitator, setting speed is 400～600r/min, is heated to 45～50 DEG C, constant temperature stirring reaction 60～80min, subsequently with 80～85 DEG C of heat treated 5～10min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；Finally above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 70～80 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 200～300mL dehydrated alcohol again by concentrated solution, stir 3～5min with Glass rod, proceeded to cold preservation 10～15h in 2～4 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded to-35～-25 DEG C of vacuum freeze driers in, after being dried 2～4h, become powder with mortar grinder, obtain plant base hyaluronic acid.

Example 1

First weigh 80g Tremella fructification, after being washed with deionized 3 times, put in the beaker filling 500mL deionized water, after soaking 1h, it is placed on stone grinder mill grinding, process of lapping is mixed into 120mL deionized water, obtain Tremella slurry, proceeded to again in microwave applicator, with 500W power, microwave heating 15min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；Then weighing 10g Pericarpium Vitis viniferae, put in tissue pulverizer and pulverize, the Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 35 DEG C of constant incubators again, cultivate 2 days, by 60mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 10mL gained mixed microorganism leacheate subsequently, proceeded to fill in the fermentation tank of 100mL above-mentioned gained pretreatment slurry, added 6g sucrose, after sterilizing waddy stirring 10min, fermentation process 6h, obtains fermentation slurry；Being sequentially added into 20mL above-mentioned gained fermentation slurry again in reactor, 100mL mass concentration is 8% potassium dihydrogen phosphate, 2g cellulase, 1g pectase and 1g neutral protease, starting agitator, setting speed is 400r/min, is heated to 45 DEG C, constant temperature stirring reaction 60min, subsequently with 80 DEG C of heat treated 5min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；Finally above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 70 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 200mL dehydrated alcohol again by concentrated solution, stir 3min with Glass rod, proceeded to cold preservation 10h in 2 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded in-35 DEG C of vacuum freeze driers, after being dried 2h, become powder with mortar grinder, obtain plant base hyaluronic acid.

Weighing plant base hyaluronic acid prepared by the 20g present invention and put in glass drying oven, be added thereto to 25mL Fructus Citri Limoniae juice, 8mL Mel and 160mL pure water simultaneously, after stirring makes it be sufficiently mixed uniformly, bottling can be prepared by astringent.This plant base hyaluronic acid can improve skin-nourishing metabolism; skin smooth is made to moisten; gained astringent can compact skin; permanent moisturizing; and the intrusion of alien bacteria, dust, ultraviolet can be intercepted, protect the skin from infringement, prevent aging; and be again good Transdermal absorption while moisturizing to, be worthy to be popularized and use.

Example 2

First weigh 90g Tremella fructification, after being washed with deionized 4 times, put in the beaker filling 550mL deionized water, after soaking 2h, it is placed on stone grinder mill grinding, process of lapping is mixed into 150mL deionized water, obtain Tremella slurry, proceeded to again in microwave applicator, with 550W power, microwave heating 20min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；Then weighing 15g Pericarpium Vitis viniferae, put in tissue pulverizer and pulverize, the Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 40 DEG C of constant incubators again, cultivate 3 days, by 70mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 15mL gained mixed microorganism leacheate subsequently, proceeded to fill in the fermentation tank of 125mL above-mentioned gained pretreatment slurry, added 7g sucrose, after sterilizing waddy stirring 13min, fermentation process 7h, obtains fermentation slurry；Being sequentially added into 25mL above-mentioned gained fermentation slurry again in reactor, 150mL mass concentration is 9% potassium dihydrogen phosphate, 3g cellulase, 2g pectase and 2g neutral protease, starting agitator, setting speed is 500r/min, is heated to 48 DEG C, constant temperature stirring reaction 70min, subsequently with 83 DEG C of heat treated 8min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；Finally above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 75 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 250mL dehydrated alcohol again by concentrated solution, stir 4min with Glass rod, proceeded to cold preservation 13h in 3 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded in-30 DEG C of vacuum freeze driers, after being dried 3h, become powder with mortar grinder, obtain plant base hyaluronic acid.

Weighing plant base hyaluronic acid prepared by the 25g present invention and put in glass drying oven, be added thereto to 26mL Fructus Citri Limoniae juice, 9mL Mel and 180mL pure water simultaneously, after stirring makes it be sufficiently mixed uniformly, bottling can be prepared by astringent.This plant base hyaluronic acid can improve skin-nourishing metabolism; skin smooth is made to moisten; gained astringent can compact skin; permanent moisturizing; and the intrusion of alien bacteria, dust, ultraviolet can be intercepted, protect the skin from infringement, prevent aging; and be again good Transdermal absorption while moisturizing to, be worthy to be popularized and use.

Example 3

First weigh 100g Tremella fructification, after being washed with deionized 5 times, put in the beaker filling 600mL deionized water, after soaking 2h, it is placed on stone grinder mill grinding, process of lapping is mixed into 180mL deionized water, obtain Tremella slurry, proceeded to again in microwave applicator, with 600W power, microwave heating 25min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；Then weighing 20g Pericarpium Vitis viniferae, put in tissue pulverizer and pulverize, the Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 45 DEG C of constant incubators again, cultivate 3 days, by 80mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 20mL gained mixed microorganism leacheate subsequently, proceeded to fill in the fermentation tank of 150mL above-mentioned gained pretreatment slurry, added 8g sucrose, after sterilizing waddy stirring 15min, fermentation process 8h, obtains fermentation slurry；Being sequentially added into 30mL above-mentioned gained fermentation slurry again in reactor, 200mL mass concentration is 10% potassium dihydrogen phosphate, 4g cellulase, 3g pectase and 3g neutral protease, starting agitator, setting speed is 600r/min, is heated to 50 DEG C, constant temperature stirring reaction 80min, subsequently with 85 DEG C of heat treated 10min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；Finally above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 80 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 300mL dehydrated alcohol again by concentrated solution, stir 5min with Glass rod, proceeded to cold preservation 15h in 4 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded in-25 DEG C of vacuum freeze driers, after being dried 4h, become powder with mortar grinder, obtain plant base hyaluronic acid.

Weighing plant base hyaluronic acid prepared by the 30g present invention and put in glass drying oven, be added thereto to 28mL Fructus Citri Limoniae juice, 10mL Mel and 200mL pure water simultaneously, after stirring makes it be sufficiently mixed uniformly, bottling can be prepared by astringent.This plant base hyaluronic acid can improve skin-nourishing metabolism; skin smooth is made to moisten; gained astringent can compact skin; permanent moisturizing; and the intrusion of alien bacteria, dust, ultraviolet can be intercepted, protect the skin from infringement, prevent aging; and be again good Transdermal absorption while moisturizing to, be worthy to be popularized and use.

Claims (1)

1. the method preparing plant base hyaluronic acid with Tremella fructification, it is characterised in that concrete preparation process is:

(1) weigh 80～100g Tremella fructifications, after being washed with deionized 3～5 times, put in the beaker filling 500～600mL deionized waters, after soaking 1～2h, it is placed on stone grinder mill grinding, process of lapping is mixed into 120～180mL deionized waters, obtain Tremella slurry, proceeded to again in microwave applicator, with 500～600W power, microwave heating 15～25min, removal, stand so that it is naturally cool to room temperature, obtain pretreatment slurry；

(2) 10～20g Pericarpium Vitis viniferaes are weighed, put in tissue pulverizer and pulverize, Pericarpium Vitis viniferae after pulverizing is placed in potato sucrose culture medium, move in 35～45 DEG C of constant incubators again, cultivate 2～3 days, by 60～80mL deionized water drip washing media surface, obtain mixed microorganism leacheate, measure 10～20mL gained mixed microorganism leacheates subsequently, proceeded to fill in the fermentation tank of 100～150mL above-mentioned gained pretreatment slurry, added 6～8g sucrose, after sterilizing waddy stirring 10～15min, fermentation process 6～8h, obtains fermentation slurry；

(3) being sequentially added into 20～30mL above-mentioned gained fermentation slurries in reactor, 100～200mL mass concentrations are 8～10% potassium dihydrogen phosphate, 2～4g cellulase, 1～3g pectase and 1～3g neutral protease, starting agitator, setting speed is 400～600r/min, is heated to 45～50 DEG C, constant temperature stirring reaction 60～80min, subsequently with 80～85 DEG C of heat treated 5～10min, stand, after naturally cooling to room temperature, it is filtered to remove filtering residue, obtains filtrate；

(4) above-mentioned gained filtrate is proceeded in rotary evaporator, under the conditions of 70～80 DEG C, concentrated by rotary evaporation is to the 1/3 of original volume, proceed to fill in the beaker of 200～300mL dehydrated alcohol again by concentrated solution, stir 3～5min with Glass rod, proceeded to cold preservation 10～15h in 2～4 DEG C of refrigerators after stirring again, be centrifuged off supernatant, gained precipitate is proceeded to-35～-25 DEG C of vacuum freeze driers in, after being dried 2～4h, become powder with mortar grinder, obtain plant base hyaluronic acid.