CN106008739A - Preparation method of oyster polysaccharide - Google Patents

Abstract

The invention discloses a preparation method of oyster polysaccharide, and the preparation method is free of ethanol precipitation, safe and reliable in operation, low in cost and high in purity. The method comprises the following steps: homogenizing oyster meat, leaching for 10min with volume/mass 10-15 times of boiling water, carrying out intermittent mixing, and filtering to obtain filtrate; adjusting the pH of the filtrate to 4-5, standing for 12h, centrifuging for 20min at the speed of 6000rpm, and collecting supernatant; adjusting the pH of the supernatant to 7-8, and carrying out enzymolysis at the temperature of 20-30 DEG C for 6h by using compound enzyme in an amount being 0.5-1% of the mass of the supernatant, wherein the compound enzyme is a mixture formed by mixing marine alkaline protease, papain, flavourzyme and trypsin according to an activity unit ratio of 3: 3: 2: 2; centrifuging enzymatic hydrolysate for 20min at the speed of 6000rpm, and collecting supernatant of the enzymatic hydrolysate; filtering the supernatant of the enzymatic hydrolysate with an ultrafiltration membrane with molecular cut off being 50kDa, and drying trapped fluid which passes the membrane to obtain the oyster polysaccharide.

Description

The preparation method of Concha Ostreae polysaccharide

Technical field

The present invention relates to a kind of method preparing Concha Ostreae polysaccharide from Concha Ostreae, especially a kind of without ethanol precipitation, operation The preparation method of the Concha Ostreae polysaccharide safe and reliable, low cost, purity are high.

Background technology

Concha Ostreae is one of Important Economic cultivated shellfish of China, has about seed oyster kind more than 20 in China's Coastal Areas, As Pacific oyster (Crassostrea gigas), Crassostrea rivularis (Crassostrea rivularis), pleat Concha Ostreae (Crassostrea plicatula), Dalian Bay Concha Ostreae (Crassostrea talienwhanensis) and close squama Concha Ostreae (Ostrea denselamellosa) etc. kind.Concha Ostreae contains abundant nutrient substance, in its dry product protein account for 45%～ 57%, fat accounts for 7%～11%, and polysaccharide accounts for 19%～38%.Research shows that Concha Ostreae polysaccharide has promotion cell metabolism, changes Mercy is dirty, liver and sanguimotor function, is active component important in Concha Ostreae.At present, the preparation method of Concha Ostreae polysaccharide has Multiple, such as the application for a patent for invention of Chinese Patent Application No. 201510322757.7, disclose " a kind of ocean Concha Ostreae biological polyoses Extract preparation method ", comprise the concrete steps that: " rub after Concha Ostreae fresh meat is cleaned up, add the water homogenate of 20-30 times of quality volume, Regulation pH, adds 0.5%(mass fraction) protease, 50-60 DEG C of insulation reaction 3-5h；Again by centrifuge, it is heavy to collect Form sediment, obtain Concha Ostreae crude extract；It is sufficiently stirred for Concha Ostreae crude extract dissolving in 15-25 times of water, stands, removed not by centrifuge Molten thing；Clear liquid is filtered by cardboard filter device, then filtrate is slowly added to 2-4 times of volume 95% ethanol, at 3-5 DEG C Under the conditions of static 12-16 hour, then be centrifuged, precipitation taken out, vacuum dried, obtain Concha Ostreae polysaccharide ".Although not open tool The protease of body, but it can be seen that its enzymolysis process need to be carried out under 50-60 DEG C of hot conditions, need to expend the energy (heat energy), Strengthen production cost.While it is true, or it can not be made to separate thorough with the pilotaxitic texture of protein bound for polysaccharide enzymolysis, only High concentration (95%) ethanol can be used to make the polysaccharide precipitation in liquid, and ethanol exists safety during large-scale industrial production Hidden danger.

Summary of the invention

The present invention is to solve the above-mentioned technical problem existing for prior art, it is provided that a kind of without ethanol precipitation, behaviour Make the preparation method of Concha Ostreae polysaccharide safe and reliable, that low cost, purity are high.

The technical solution of the present invention is: the preparation method of a kind of Concha Ostreae polysaccharide, it is characterised in that successively according to as follows Step is carried out:

A. carry out homogenized after fresh Concha Ostreae of shelling being removed internal organs, extract with the boiling water of volume/mass 10 ~ 15 times 10min, intermittent stirring, filtration, obtain filtrate；

B. regulation filtrate pH is 4 ~ 5, stands 12h, 6000rpm and is centrifuged 20 min, collects supernatant；

C. regulation supernatant pH is 7 ~ 8, uses supernatant matter with the compound enzyme of supernatant mass percent 0.5% ~ 1% at 20 ~ 30 DEG C Amount percentage ratio 0.5% ~ 1% compound enzyme enzymolysis 6 h under the conditions of 20 ~ 30 DEG C, under the conditions of enzymolysis 6 h, described compound enzyme is 6000u/ The marine alkaline proteinase of mg, the papain of 6000u/mg, the flavor protease of 15000 u/mg and 6000u/mg pancreas egg White enzyme is mixed than for 3:3:2:2 ratio in unit of activity；

D. enzymolysis solution 6000rpm is centrifuged 20min, collects enzymolysis supernatant；

E. by enzymolysis supernatant through the ultrafiltration membrance filter of molecular cut off 50kDa, trapped fluid after dried film, Concha Ostreae is obtained Polysaccharide.

The present invention is to use four kinds of protease to be configured to compound protease in proportion Concha Ostreae homogenate supernatant is carried out enzymolysis, Various enzyme interactings, can make polysaccharide in Concha Ostreae separate with protein pilotaxitic texture (20 ~ 30 DEG C) enzymolysis at normal temperatures, make collection Supernatant in containing the higher Concha Ostreae polysaccharide of concentration, it is not necessary to ethanol precipitates, only need to be through the ultrafilter membrane of molecular cut off 50kDa Filter the enzyme and enzymatic hydrolysate removed in enzymolysis solution, i.e. can get the Concha Ostreae polysaccharide of purity >=96%, have operation safe and reliable, Low cost, purity advantages of higher.

The pilotaxitic texture of the polysaccharide in Concha Ostreae with protein bound can be separated well, it is not necessary to ethanol precipitate, only need through Cross the ultrafiltration membrance filter of molecular cut off 50kDa, can with removing the enzyme of molecular weight and enzymatic hydrolysate, obtain purity >= The Concha Ostreae polysaccharide of 96%, has operation safe and reliable, low cost, purity advantages of higher.

Detailed description of the invention

The freshest Concha Ostreae of shelling carries out homogenized after removing internal organs, extracts 10min with 15 times of (volume/mass) boiling water, Intermittent stirring, filtration, obtain filtrate, and filtering residue same method is reprocessed 2 times, and 3 filtrates are merged；

B. it is 5 by the filtrate second acid for adjusting pH of collection, stands 12h, 6000rpm and be centrifuged 20 min, collect supernatant；

C. regulating supernatant pH with sodium hydroxide is 7, is added by the compound enzyme of supernatant mass percent 1% in supernatant, often Temperature (20 ~ 30 DEG C) enzymolysis 6 h, described compound enzyme is by marine alkaline proteinase (6000u/mg), papain (6000u/ Mg), flavor protease (15000 u/mg) and trypsin 6000u/mg) mix than for 3:3:2:2 ratio in unit of activity；

D. obtained enzymolysis solution 6000rpm again is centrifuged 20min, obtains enzymolysis supernatant；

E. the enzymolysis supernatant obtained is sprayed through ultrafilter membrane (molecular cut off 50kDa), the trapped fluid after crossing film It is dried, obtains Concha Ostreae polysaccharide.Concha Ostreae polysaccharide purity >=96% is measured through Phenol sulfuric acid procedure.

Claims (1)

1. the preparation method of a Concha Ostreae polysaccharide, it is characterised in that carry out the most in accordance with the following steps:

A. carry out homogenized after fresh Concha Ostreae of shelling being removed internal organs, extract with the boiling water of volume/mass 10 ~ 15 times 10min, intermittent stirring, filtration, obtain filtrate；

B. regulation filtrate pH is 4 ~ 5, stands 12h, 6000rpm and is centrifuged 20 min, collects supernatant；

C. regulation supernatant pH is 7 ~ 8, with the compound enzyme of supernatant mass percent 0.5% ~ 1% enzymolysis under the conditions of 20 ~ 30 DEG C 6h, described compound enzyme is the marine alkaline proteinase of 6000u/mg, the papain of 6000u/mg, the local flavor of 15000 u/mg Protease and 6000u/mg trypsin are mixed than for 3:3:2:2 ratio in unit of activity；

D. enzymolysis solution 6000rpm is centrifuged 20min, collects enzymolysis supernatant；

E. by enzymolysis supernatant through the ultrafiltration membrance filter of molecular cut off 50kDa, trapped fluid after dried film, Concha Ostreae is obtained Polysaccharide.