CN102603916B - Refining method of (1-3)-beta-D-glucan - Google Patents

Abstract

The invention relates to a refining method of (1-3)-beta-D-glucan. The method comprises the following steps of: degreasing Indian buead powder, adding an alkali for reaction and roughing out, and adding dimethylsulfoxide for refining. According to the refining method disclosed by the invention, fruiting bodies are directly adopted as raw materials, the sources are extensive, and the cost is low; and the process disclosed by the invention has the advantages of low cost, simplicity in operation, higher yield, high product purity and relatively stable performance.

Description

The process for purification of (1-3)-callose

Technical field

The present invention relates to a kind of process for purification of (1-3)-callose.

Background technology

Invasive infections with fungi more and more is subject to clinician's attention, and in the patient of the immune function depressions such as chemotherapy of tumors, marrow/organ transplantation, AIDS and critical illness, its morbidity and mortality ratio obviously rise.Broad spectrum antibiotics is the Major Risk Factors that causes invasive infections with fungi to occur with wound property operation of diagnosis and treatment is arranged.Because invasive infections with fungi lacks the specificity clinical manifestation, the microbiological examination such as traditional experiment chamber smear, cultivation are consuming time and positive rate is low, histopathological examination has seriously limited clinical application because there being wound to increase the weight of conditions of patients, these complicated factors make to be subjected to the diagnosis of invasive infections with fungi extremely difficult and lag behind, and are also the major reasons that mortality ratio increases.In fungi infestation patient blood plasma, containing (1-3)-callose is to pay close attention at present more a kind of diagnostic method, (1-3)-callose exists only in the cell walls of fungi, there are not (1-3)-callose in bacterium and human cell's wall, so blood plasma (1-3)-callose positive has the value of specific diagnosis deep fungal infection.

The accepted standard of fungi dextran detection at present product are (1-3)-callose ,(1-3)-callose of existing city dealer, purity is lower or price is more expensive.

Summary of the invention

The object of the present invention is to provide a kind of process for purification of (1-3)-callose, (1-3)-callose purity of preparation is higher, cost is lower.

The technical scheme that the present invention takes is as follows:

(1) degreasing: Indian Bread is used respectively to dehydrated alcohol, 80% ethanol and water extraction, filtration, and filter residue obtains the degreasing Poria cocos through dehydration, drying;

(2) rough: degreasing Poria cocos water is mixed with the suspension that weight ratio is 0.8%-1.2%, be cooled to 0-4 ℃, add while stirring 5N NaOH to dissolve, then 4 ℃, 10000rpm are centrifugal, and 15-30min removes insolubles, and supernatant liquor is placed in 0-4 ℃ of ice-water bath, and to add while stirring volume ratio be that 10% acetic acid neutralizes; 4 ℃, the centrifugal 15-30min collection of 10000rpm gelatinous precipitate; Above-mentioned gelatinous precipitate is water, alcohol, ether washing, dehydration successively; Then put into sulphuric acid desiccator and carry out drying under reduced pressure, obtain rough (1-3)-callose;

(3) refining: as to get rough (1-3)-callose and add methyl-sulphoxide, at room temperature stirring and dissolving; 4 ℃, the centrifugal 15-30min of 10000rpm, remove precipitation; Supernatant liquor slowly adds 0-4 ℃ of frozen water while stirring, until precipitation no longer increases, 4 ℃, the centrifugal 15-30min collecting precipitation of 10000rpm, precipitate water, alcohol, ether washing, dehydration successively, then put into sulphuric acid desiccator and carry out drying under reduced pressure, made with extra care (1-3)-callose.

Remarkable advantage of the present invention: at present, domestic polysaccharide raw materials for production are mainly the mycelium that obtains of fungus sporophore fermentation and fermented liquid etc., raw material of the present invention directly adopts sporophore (the seed of Poria cocos entity, originally as bulk, dries rear crushing grinding powdered), and wide material sources and cost are low; And technique is by contrast, the hot water extraction is more time-consuming and recovery rate is low, enzyme process and supersonic method again the space structure of destructible polysaccharide and activity and cost higher, this process costs is low, simple to operate, recovery rate is also higher, its product purity is high, performance is also more stable.

The accompanying drawing explanation

Fig. 1 is the high-efficient liquid phase chromatogram of Dextran-40.

Fig. 2 is the high-efficient liquid phase chromatogram of refining BG.

Fig. 3 is the typical curve of Dextran-40 sterling.

Fig. 4 is the typical curve of refining BG.

Fig. 5 is the typical curve at the 0--4 ℃ of refining BG of Refrigerator store after 7 days.

Embodiment

Embodiment 1

(1) degreasing

1. the preparation of raw material, reagent and utensil

2. get the beaker that Indian Bread 500g puts into 3L, add 2L alcohol, cover with masking foil, stir 30min under room temperature.Then with 63 purpose sand core funnels, carry out suction filtration.

3. more than, 2. operation repeats twice.

4. the Indian Bread of leaching adds 80% alcohol, by magnetic stirrer 30min(room temperature).

5. use G3 sand core funnel suction filtration.

6. by 4. 5. operation, repeat twice.

7. the Indian Bread of leaching adds 4L water magnetic stirrer 30min.

8. use G3 sand core funnel suction filtration.

9. by 7. 8. operation, repeat twice.

The Indian Bread that 10. will obtain is put into the G3 sand core funnel, the medication spoon stir, and meanwhile add dehydration of alcohol,

Use afterwards 40 ℃ of vacuum-dryings, the 500g Poria cocos can obtain 400g degreasing Poria cocos like this.

(2) rough (1-3)-β-glucan (BG)

1. extracting degreasing Poria cocos 12g puts into the 2L Erlenmeyer flask, adds water 1080ml, uses magnetic stirrer, makes its dispersion, is suspension.

2. under cooling conditions (0-4 ℃), add 5N NaOH 120ml while stirring, dissolves.

3. move into centrifuge tube, 4 ℃, 10000rpm, 20min, centrifugally remove a small amount of insolubles.

4. supernatant liquor is poured into to the 2L beaker, be placed in ice-water bath (0-4 ℃) and add while stirring 10% acetic acid, make the solution neutralization.

5. 4 ℃, 10000rpm, 20min, centrifugal collection gelatinous precipitate.

6. washing of precipitate: above-mentioned gelatinous precipitate adds 400ml water, and the medication spoon stirs, and with 5. centrifugal collecting precipitation of step, in precipitation, adds 250ml alcohol again, operates equally twice.Finally add ether 200ml, same twice operated wash, dehydration.

7. the precipitation after cleaning is put into sulphuric acid desiccator and is carried out drying under reduced pressure.Starting material 12g like this, rough rear approximately 11g.

(3) refining BG preparation

1. get rough BG10g and add the 2L Erlenmeyer flask, add 1.2L methyl-sulphoxide (DMSO), cover masking foil, at room temperature stirring and dissolving.

2. 4 ℃, the centrifugal 20min of 10000rpm, remove precipitation.

3. pour centrifugal supernatant liquor into the 5L beaker, slowly add while stirring 0-4 ℃ of frozen water, until precipitation no longer increases.

4. centrifugal, 4 ℃, 10000rpm, 20min, collecting precipitation.

5. precipitation adds the water of 400ml successively, the alcohol of 200ml, and the ether of 150ml, wash in order, dewater.

6. precipitation is put into sulphuric acid desiccator, carries out drying under reduced pressure, utilizes so the rough BG of 10g to obtain the refining BG of 9g, and recovery rate is about 85%.The high performance liquid chromatography fingerprint image shows that peak 1 is beta-glucan, as shown in Figure 2.

(4) refining BG titration

1. titration

1) MT-1B type detection kit: Associates of CAPE COD Incorporated (U.S.), Kit Number:GD11007,55Test Endpoint Assay Kit.

2) Dextran-40(is as the dextran sterling): Wako (Japanese Wako Pure Chemical Industries, Ltd.), and light one-level, molecular-weight average=32000-45000, purity is 99%, and loading amount is 50g, and Lot is DCK2746.

3) making of dextran sterling curve:

The preparation of a, 1mg/ml dextran-40: accurately take 0.5g dextran-40 (MW40000) and join in the beaker that 500ml water is housed, use magnetic stirrer, at room temperature make it to dissolve fully, then add the 10g gac, at room temperature stir 30min.By above solution, every 100ml D-GS(0.22 μ m) the strainer filtration.The rear merging 500ml 4 ℃ of preservations in the 500ml bottle of packing into.

B, dilution: the solution of getting above-mentioned a is a small amount of, and water accurately is diluted to 40pg/ml, 20pg/ml, 10pg/ml, 5pg/ml, every kind of about 1ml of concentration.

The redissolution of c, main reagent and packing: MT-1B type detection reagent master reagent (Contains freeze-dried chromogenic glucan detection reagent) first uses 0.2M Tris-Hcl buffer reagent/PH8.0 to redissolve by loading amount, on enzyme plate, choose afterwards 10 holes, cloth becomes 2 row's 5 row, adds 50 ul master's reagent toward every hole.Remaining main reagent is put into 0--4 ℃ of Refrigerator store.

D, application of sample: two holes toward the 1st row respectively add the water of 50ul as blank, since the 2nd row, by concentration, toward two parallel holes, respectively add from low to high the dextran sterling solution of 50ul, after adding well, 30s vibrates on micro-shaker, put into again in the thermostat water bath of 37 ± 1 ℃ and react 30min, with diazo reagent, develop the color afterwards.

E, detection: above-mentioned enzyme plate is put into to microplate reader, under the 545nm wavelength, detect record data.

4) making of refining BG curve:

Preparation and the dilution of a, refining BG solution: will make with extra care BG3-4mg and move in test tube, with 0.1N NaOH, being made into concentration is 1.0mg/ml.And with 10 times of dilution methods, progressively be diluted to 100pg/ml with 0.1N NaOH, then with 0.01N NaOH, to dilute respectively be 40pg/ml, 20pg/ml, 10pg/ml, 5pg/ml.

The packing of b, main reagent: from taking out remaining main reagent in refrigerator, with mode packing master reagent identical in step 3) c.Remaining sample is put into 0--4 ℃ of Refrigerator store.

C, application of sample: the 0.01N NaOH that parallel two holes of past the 1st row respectively add 50ul is as blank, and all the other operations are constant.Remaining sample is put into 0--4 ℃ of Refrigerator store, to operate equally, demarcates once after the week again.

D, detection: equally above-mentioned enzyme plate is put into to microplate reader, at the 545nm wavelength, detect record data.

2. result

1）Dextran-40

Table 1 is the detected result of Dextran-40 sterling

2) refining BG

Table 2 is the detected result for the first time of refining BG

Table 3 is for making with extra care BG in the detected result of 0--4 ℃ of Refrigerator store after 7 days

3. analyze

By data analysis, contrast, the relation conefficient of Dextran-40 sterling is 0.9995, and the relation conefficient of refining BG is 0.9992, near the result of sterling, that is to say impurity or interfering substance seldom, the purity of Dextran-40 is 99%, the purity made from extra care BG that converts is greater than 98%, so the purity of refining BG is very near Dextran-40, and reflect its solution at 0--4 ℃ of Refrigerator store after one week by table 3, relation conefficient still is greater than 0.98, with the numerical value of Dextran-40 sterling, be more or less the same, namely tire substantially constant, so stability is better, can be used as standard substance, for the fungi dextran, detect.

Claims (1)

1. the process for purification of (1-3)-callose, it is characterized in that: described process for purification is following steps:

(1) degreasing: Indian Bread is used respectively to dehydrated alcohol, 80% ethanol and water extraction, filtration, and filter residue obtains the degreasing Poria cocos through dehydration, drying; Described Indian Bread directly adopts sporophore, and the seed of Poria cocos entity, originally as bulk, dries rear crushing grinding powdered;

(2) rough: degreasing Poria cocos water is mixed with the suspension that weight ratio is 0.8%-1.2%, be cooled to 0-4 ℃, add while stirring 5N NaOH to dissolve, then 4 ℃, 10000rpm are centrifugal, and 15-30min removes insolubles, and supernatant liquor is placed in 0-4 ℃ of ice-water bath, and to add while stirring volume ratio be that 10% acetic acid neutralizes; 4 ℃, the centrifugal 15-30min collection of 10000rpm gelatinous precipitate; Above-mentioned gelatinous precipitate is water, alcohol, ether washing, dehydration successively; Then put into sulphuric acid desiccator and carry out drying under reduced pressure, obtain rough (1-3)-callose;

(3) refining: as to get rough (1-3)-callose and add methyl-sulphoxide, at room temperature stirring and dissolving; 4 ℃, the centrifugal 15-30min of 10000rpm, remove precipitation; Supernatant liquor slowly adds 0-4 ℃ of frozen water while stirring, until precipitation no longer increases, 4 ℃, the centrifugal 15-30min collecting precipitation of 10000rpm, precipitate water, alcohol, ether washing, dehydration successively, then put into sulphuric acid desiccator and carry out drying under reduced pressure, made with extra care (1-3)-callose.

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