Fungus culture method for high-yield biological polysaccharide

文档序号：1350612 发布日期：2020-07-24 浏览：38次中文

阅读说明：本技术 一种高产生物多糖的真菌培养方法 (Fungus culture method for high-yield biological polysaccharide ) 是由郭宏亮林檬庄秀园王轩朱勤健叶榛于 2019-01-17 设计创作，主要内容包括：本发明提供了一种高产生物多糖的真菌培养方法。具体地,本发明方法采用改良PDA固体培养基活化裂褶菌,通过改进PDA培养基的配方及制备方法,裂褶菌在该改良PDA平板上的生长状态和活性发生显著改变,不仅便于平板件传代、缩短了传代时间,而且显著提高了发酵培养的β-葡聚糖产量。本发明的方法操作简单、成本低,β-葡聚糖产量非常高,并且不含有纤维素类杂质,后期分离纯化简单、纯度高。(The invention provides a fungus culture method for high-yield biological polysaccharide, and particularly relates to a method for activating schizophyllum commune by adopting an improved PDA solid culture medium, wherein the growth state and activity of the schizophyllum commune on an improved PDA flat plate are obviously changed by improving the formula and the preparation method of the PDA culture medium, so that the passage of the flat plate piece is facilitated, the passage time is shortened, and the yield of β -glucan cultured by fermentation is obviously improved.)

1. A method of producing β -glucan, the method comprising the steps of:

(1) providing an improved PDA solid culture medium, and culturing Schizophyllum commune on the improved PDA solid culture medium;

(2) inoculating the Schizophyllum commune obtained from the previous step in a seed culture medium and culturing to obtain a bacterial suspension; and

(3) transferring the bacterial suspension obtained by the culture in the previous step into a fermentation culture medium and culturing to obtain β -glucan;

wherein, in the step (1), the modified PDA solid culture medium is a PDA solid culture medium containing solid particles, wherein the solid particles comprise: potato meal, corn meal, soybean meal, corncob meal, or a combination thereof.

2. The method as claimed in claim 1, wherein the modified PDA solid culture medium contains 5-10 wt% of solid particles, based on the total weight of the modified PDA solid culture medium.

3. The method according to claim 1, wherein the solid particles have an average particle size d of 0.15-5mm, preferably 0.18-3 mm.

4. The method of claim 1, wherein the seed medium and/or fermentation medium contains solid insoluble matter.

5. The process of claim 1, wherein said solid insolubles are selected from the group consisting of: perlite, soybean meal, corn meal, soybean meal, crushed corn grits, crushed corncob, or a combination thereof.

6. The method of claim 1, wherein the seed medium and/or the fermentation medium is free of cellulose or a derivative thereof.

7. The method of claim 1, wherein the seed medium isContains glucose 20-90 g/L, yeast extract 0.5-5 g/L, casein peptone 0.5-5 g/L, and NH 0.5-5 g/L₄Cl、0.2-2g/L KH₂PO₄、0.2-2g/L MgSO₄·7H₂O、0.2-2g/L MnSO₄·H₂O, corn flour 0.5-5 g/L g, CaCO 0.1-2 g/L g₃Based on the total volume of the seed culture medium; or

The fermentation medium is an improved MRS medium, wherein the improved MRS medium contains 60-200 g/L glucose, 1-10 g/L yeast extract, 1-10 g/L casein peptone and 1-20 g/L NH₄Cl、0.5-5g/L KH₂PO₄、0.2-2g/L MgSO₄·7H₂O、0.2-2g/L MnSO₄·H₂O, 1-20 g/L perlite, 1-20 g/L corn flour, 1-20 g/L soybean flour and 0.1-3 g/L CaCO₃Based on the total volume of the fermentation medium.

8. An β -glucan, wherein the β -glucan is prepared by the method of claim 1 for producing β -glucan.

9. A formulation comprising (a) the β -glucan of claim 8 and (b) a pharmaceutically, cosmetically or dietetically acceptable carrier or excipient.

10. An improved PDA solid culture medium, which is characterized in that the improved PDA solid culture medium is a PDA solid culture medium containing solid particles, wherein the solid particles comprise: potato meal, corn meal, soybean meal, corncob meal, or a combination thereof.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fungus culture method for high-yield biological polysaccharide.

Background

The polysaccharide is a natural high molecular compound polymerized by more than 10 monosaccharide molecules, and is widely distributed in animals, plants, microorganisms (bacteria and fungi) and algae lichens, the polysaccharide has the effects of regulating immunity, resisting tumors, reducing glycolipids, delaying senescence and the like as an important bioactive substance, and has wide application prospects in the fields of medical care, food, animal breeding and the like, β -glucan is natural polysaccharide, mushroom glucan taking β -1, 3-glycosidic bonds as a framework has the functions of enhancing immunity, resisting tumors, resisting bacteria, resisting viruses and the like, and also has remarkable effects in the aspects of moisturizing, resisting wrinkles, resisting oxidation, resisting ultraviolet, promoting wound healing and the like, and in addition, compared with the traditional yeast or cereal glucan, the mushroom glucan has a more uniform and stable structure, higher molecular mass and more excellent water solubility, so the mushroom β -glucan solution with high viscosity and high light transmittance is widely applied to the fields of food, cosmetics and pharmacy, and the preparation of the mushroom β -glucan solution with high viscosity and high light transmittance has important significance.

At present, β -glucan is extracted mainly by two methods, one is directly extracted from cereals such as oat or fruiting body fungi such as mushroom, and the other is extracted from fermentation liquor by liquid fermentation of fungi or bacteria to obtain β -glucan, the premise of preparing β -glucan by fermentation is to obtain fermentation liquor with high β -glucan content, the traditional fermentation method β -glucan yield is usually low, or cellulose or derivatives thereof are used in culture medium, so that cellulose impurities which are difficult to separate are mixed in the final product.

Therefore, there is a strong need in the art for a method for producing β -glucan in high yield.

Disclosure of Invention

The invention aims to provide a method for obtaining fermentation liquor with high β -glucan content by liquid fermentation.

In a first aspect of the invention, there is provided a method of producing β -glucan, the method comprising the steps of:

(1) providing an improved PDA solid culture medium, and culturing Schizophyllum commune on the improved PDA solid culture medium;

(2) inoculating the Schizophyllum commune obtained from the previous step in a seed culture medium and culturing to obtain a bacterial suspension; and

(3) transferring the bacterial suspension obtained by the culture in the previous step into a fermentation culture medium and culturing to obtain β -glucan;

In another preferred example, the potato meal is solid particles obtained by crushing potatoes.

In another preferred example, the potatoes are uncooked or cooked potatoes.

In another preferred example, the corn meal is solid particles obtained by crushing corn (grains).

In another preferred example, the soybean meal coarse powder is solid particles obtained by crushing soybean meal.

In another preferred example, the corncob meal is solid particles obtained by crushing corncobs.

In another preferred embodiment, the modified PDA solid culture medium contains 5-10 wt% of solid particles, based on the total weight of the modified PDA solid culture medium.

In another preferred embodiment, the solid particles have an average particle diameter d of 0.15mm to 5mm (100 mesh to 4 mesh), preferably 0.18mm to 3mm (80 mesh to 6 mesh), more preferably 0.18mm to 1mm (80 mesh to 16 mesh).

In another preferred embodiment, the particle size of the corn meal, the soybean meal or the corn cob meal is 0.15mm-0.85mm, preferably 0.18mm-0.425 mm.

In another preferred embodiment, the average particle diameter d of the potato coarse powder is 0.15mm to 5mm, preferably 0.18mm to 3mm, and more preferably 0.18mm to 1 mm.

In another preferred example, the potato coarse powder is solid particles obtained by crushing cooked potatoes.

In another preferred embodiment, the cooking is performed in water at a temperature above 80 ℃, preferably 90-100 ℃.

In another preferred embodiment, the cooking time is 0.5 to 2.5 hours, preferably 0.5 to 1.5 hours.

In another preferred example, the PDA solid culture medium formula comprises 950-1000m L/L potato juice, 18-22 g/L glucose and 15-20 g/L agar, and the PH of the PDA solid culture medium is preferably 5.5-6.5 based on the total volume of the PDA culture medium.

In another preferred embodiment, the potato juice is prepared by peeling potatoes, adding water and boiling, and filtering (eight layers of gauze) to obtain the juice, wherein the mass volume ratio of the peeled potatoes is 180-220 g/L based on the total volume of the potato juice.

In another preferred embodiment, the seed culture medium and/or the fermentation medium contains solid-type insoluble substances.

In another preferred embodiment, the solid insolubles are selected from the group consisting of: perlite, soybean meal, corn meal, soybean meal, crushed corn grits, crushed corncob, or a combination thereof.

In another preferred embodiment, the content of the solid insoluble matter is 0.1 to 5 wt% based on the total mass of the medium.

In another preferred embodiment, the seed medium and/or the fermentation medium does not contain cellulose or derivatives thereof.

In another preferred embodiment, the seed culture medium comprises 20-90 g/L glucose, 0.5-5 g/L yeast extract, 0.5-5 g/L casein peptone, and 0.5-5 g/L NH₄Cl、0.2-2g/L KH₂PO₄、0.2-2g/L MgSO₄·7H₂O、0.2-2g/LMnSO₄·H₂O, corn flour 0.5-5 g/L g, CaCO 0.1-2 g/L g₃Based on the total volume of the seed culture medium.

In another preferred embodiment, the fermentation medium is selected from the group consisting of: MRS medium or modified MRS medium.

In another preferred embodiment, the fermentation medium is selected from one or more of the following:

culture medium A containing 20-150 g/L glucose, 1-3 g/L yeast extract, and 1-3 g/L KH₂PO₄、0.5-1.5g/L MgSO₄·7H₂O；

Culture medium B, tryptone 10-40 g/L g, Ca 2-10 g/L g (NO)₃)₂·4H₂O、0.5-3g/L KH₂PO₄、0.5-1.5g/L MgSO₄·7H₂O, 0.5-3m L/L microelement I (1.6 g/L MnSO)₄·H₂O、2.4g/L ZnSO₄·7H₂O、2g/LCaCl₂·6H₂O), 0.5-3m L/L microelement II (5 g/L FeSO)₄·7H₂O)；

Culture medium C containing 25-30 g/L glucose, 10-15 g/L sorbitol, 0.2-0.8 g/L KH₂PO₄、3-5g/L MgSO₄·7H₂O、0.5-1g/L(NH₄)₂SO₄0.1-0.5 g/L triethylamine hydrochloride, 0.5-3m L/L microelement (16-20 g/L Na)₂EDTA、2.5-3.0g/L ZnSO₄·7H₂O、2.2-2.6g/L FeSO₄·7H₂O、0.06-0.12g/L CoSO₄·7H₂O、0.1-0.3g/L CuCl₂·7H₂O、12-16g/L NaBO₂·4H₂O、0.06-0.12g/L Na₂MoO₄·2H₂O、0.22-0.26g/L NiCl₂·2H₂O)；

Culture medium D comprising 10-200 g/L glucose, 1-50 g/L yeast extract, 1-50 g/L beef extract, and 1-10 g/L (NH)₄)₂SO₄、1-10g/L KH₂PO₄、0.1-5g/L MgSO₄·7H₂O, 1-100 g/L adsorption resin HS 1.

In another preferred example, the fermentation medium is a modified MRS medium.

In another preferred example, the modified MRS medium is a medium modified from an MRS medium.

In another preferred example, the modified MRS culture medium contains 60-200 g/L glucose, 1-10 g/L yeast extract, 1-10 g/L casein peptone and 1-20 g/L NH₄Cl、0.5-5g/L KH₂PO₄、0.2-2g/L MgSO₄·7H₂O、0.2-2g/LMnSO₄·H₂O, 1-20 g/L perlite, 1-20 g/L corn flour, 1-20 g/L soybean flour and 0.1-3 g/L CaCO₃Based on the total volume of the culture medium.

In another preferred example, in step (1), Schizophyllum commune is cultured in the modified PDA solid medium and passaged n times, wherein n is a positive integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6 times.

In another preferred embodiment, the passaging is performed on modified PDA solid medium or ordinary PDA solid medium.

In another preferred embodiment, said passaging comprises passaging at least 1 time on modified PDA solid medium.

In another preferred embodiment, the temperature of the culture is 25-30 ℃.

In another preferred embodiment, in step (1) and/or step (2), the culturing period is 4 to 8 days, preferably 5 to 7 days.

In another preferred embodiment, in step (2), the rotation speed of the cultivation is 120-180rpm, preferably 140-160 rpm.

In another preferred embodiment, the volume of the seed culture medium is 100-300m L, preferably 150-250m L, the culture temperature is 25-30 ℃, the culture time is 4-8 days, preferably 5-7 days, and the rotation speed of the shaking table is 120-180rpm, preferably 140-160 rpm.

In another preferred embodiment, the volume of the seed culture medium is 2-4L, preferably 2.5-3.5L, the temperature of the culture is 25-30 ℃, the time of the culture is 3-6 days, preferably 3-4 days, the stirring speed of the culture is 100-400rpm, preferably 200-300rpm, and the aeration is 0.2-0.6m³H, preferably 0.3 to 0.4m³/h。

In another advantageIn an alternative embodiment, the volume of the seed culture medium is 20-40L, preferably 25-35L, the temperature of the culture is 25-30 ℃, the time of the culture is 3-7 days, preferably 3-5 days, the stirring speed of the culture is 120-300rpm, preferably 200-240rpm, and the aeration is 2-5m³H, preferably 2 to 3m³/h。

In another preferred embodiment, the volume of the seed culture medium is 200-400L, preferably 250-350L, the culture temperature is 25-30 ℃, the culture time is 3-7 days, preferably 3-5 days, the culture stirring speed is 50-150rpm, preferably 80-120rpm, and the aeration is 15-30m³H, preferably 20 to 25m³/h。

In another preferred example, in step (3), the culturing period is 5 to 12 days, preferably 6 to 10 days.

In another preferred embodiment, in step (3), the bacterial suspension is inoculated into seeds or fermentation medium in an inoculation amount of 0.5-20%.

In another preferred example, in step (3), the volume of the fermentation medium is 200-.

In another preferred embodiment, the volume of the fermentation medium is 100-300m L, preferably 150-250m L, the culture temperature is 25-30 ℃, the culture time is 5-9 days, preferably 6-8 days, and the rotation speed of the shaking table is 120-180rpm, preferably 140-160 rpm.

In another preferred embodiment, the volume of the fermentation medium is 2-4L, preferably 2.5-3.5L, the temperature of the culture is 25-30 ℃, the time of the culture is 6-12 days, preferably 8-10 days, the stirring speed of the culture is 100-400rpm, preferably 200-300rpm, and the aeration is 0.2-0.6m³H, preferably 0.3 to 0.4m³/h。

In another preferred embodiment, the volume of the fermentation medium is 20-40L, preferably 25-35L, the temperature of the culture is 25-30 ℃, the time of the culture is 6-12 days, preferably 8-10 days, the stirring speed of the culture is 120-300rpm, preferably 200-240rpm, and the aeration is 2-5m³H, preferably 3 to 4m³/h。

In another preferred embodimentThe volume of the fermentation medium is 200-400L, preferably 250-350L, the culture temperature is 25-30 ℃, the culture time is 8-14 days, preferably 10-12 days, the culture stirring speed is 50-150rpm, preferably 80-120rpm, and the aeration is 15-30m³H, preferably 20 to 25m³/h。

In another preferred embodiment, the volume of the fermentation medium is 2000-4000L, preferably 2500-3500L, the culture temperature is 25-30 ℃, the culture time is 6-12 days, preferably 8-10 days, the culture stirring speed is 20-120rpm, preferably 40-80rpm, and the aeration is 60-200m³H, preferably 100-³/h。

In another preferred example, after the step (2) and before the step (3), the method further comprises the step of performing seed expansion culture on the bacterial suspension. The seed amplification culture refers to transferring thalli cultured in a seed culture medium into a next-stage seed culture medium for amplification culture so as to obtain thalli with more quantity or better quality.

In another preferred example, the method further comprises the step of (4) extracting β -glucan from the fermentation liquid obtained by the culture in the step (3).

In another preferred example, the extracting includes:

(i) mixing the fermentation liquor obtained by the culture in the step (3) with water, wherein the ratio V2/V1 of the volume V2 of the water to the volume V1 of the fermentation liquor is 1-3;

(ii) heating to 60-80 deg.C, maintaining for 1-10h, and separating to obtain supernatant;

(iii) (iii) mixing the supernatant of step (ii) with alcohols (including methanol, ethanol, propanol, isopropanol, polyalcohol, etc., preferably ethanol), precipitating β -dextran, and

(iv) optionally (iv) drying said β -glucan.

In another preferred embodiment, the method further comprises the step of (5) purifying the β -glucan.

In another preferred embodiment, the method produces β -glucan in a yield of 7-100 g/L.

In another preferred embodiment, the molecular weight of the β -glucan produced by the method is more than or equal to 2kD, preferably 2kD to 40000kD, and more preferably 20kD to 20000 kD.

In another preferred example, the molecular weight of the β -glucan produced by the method is 5kD-35000kD, 10kD-30000kD, 50kD-25000kD, 100kD-20000kD, 200kD-18000kD, 400kD-16000kD, 500kD-14000kD, 1000kD-12000kD, 2000kD-4000kD, 3000kD-5000kD, 4000kD-6000kD, 5000kD-7000kD, 6000kD-8000kD, 7000kD-9000kD or 8000kD-10000 kD.

In another preferred embodiment, the β -glucan solution produced by the method has a viscosity (25 ℃) of greater than or equal to 40 mPas, preferably 100-10000 mPas, preferably 500-2000 mPas, preferably 800-1500 mPas, more preferably 1000-1200 mPas in a 0.5% solution.

In another preferred embodiment, the method produces a 0.5% solution of β -glucan having a light transmittance of 88% -100%, preferably 92% -100%.

In a second aspect of the invention, there is provided β -glucan prepared from β -glucan by the method of the first aspect of the invention for producing β -glucan.

In a third aspect of the invention, there is provided a formulation comprising (a) the β -glucan of the second aspect of the invention, and (b) a pharmaceutically, cosmetically or dietetically acceptable carrier or excipient.

In a fourth aspect of the present invention, there is provided an improved PDA solid medium, which is a PDA solid medium containing solid particles, wherein the solid particles comprise: potato meal, corn meal, soybean meal, corncob meal, or a combination thereof.

In another preferred example, the potato meal is solid particles obtained by crushing potatoes.

In another preferred example, the potatoes are uncooked or cooked potatoes.

In another preferred example, the corn meal is solid particles obtained by crushing corn (grains).

In another preferred example, the soybean meal coarse powder is solid particles obtained by crushing soybean meal.

In another preferred example, the corncob meal is solid particles obtained by crushing corncobs.

In another preferred embodiment, the modified PDA solid culture medium contains 5-10 wt% of solid particles, based on the total weight of the modified PDA solid culture medium.

In another preferred embodiment, the solid particles have an average particle size d of 0.15mm to 5mm, preferably 0.18mm to 3mm, more preferably 0.18mm to 1 mm.

In another preferred embodiment, the particle size of the corn meal, the soybean meal or the corn cob meal is 0.15mm-0.85mm, preferably 0.18mm-0.425 mm.

In another preferred embodiment, the average particle diameter d of the potato coarse powder is 0.15mm to 5mm, preferably 0.18mm to 3mm, and more preferably 0.18mm to 1 mm.

In another preferred example, the potato coarse powder is solid particles obtained by crushing cooked potatoes.

In another preferred embodiment, the cooking is performed in water at a temperature above 80 ℃, preferably 90-100 ℃.

In another preferred embodiment, the cooking time is 0.5 to 2.5 hours, preferably 0.5 to 1.5 hours.

In another preferred embodiment, the modified PDA solid culture medium is prepared by adding solid particles to the PDA solid culture medium, wherein the solid particles comprise: potato meal, corn meal, soybean meal, corncob meal, or a combination thereof.

In another preferred embodiment, the modified PDA solid culture medium is prepared by the following method:

(1) providing potatoes (preferably 180-220g potato pieces per liter of culture medium), mixing the potatoes with water (preferably 400-600m L water per liter of culture medium) and boiling (preferably boiling for 0.5-5 hours) to obtain a cooking liquor containing the cooked potatoes;

(2) crushing the cooking liquor containing the cooked potatoes, filtering the crushed cooking liquor by using 8 layers of gauze to obtain filtrate, and adding solid particles into the filtrate, wherein the solid particles comprise potato coarse powder, corn coarse powder, bean pulp coarse powder, corncob coarse powder or a combination of the potato coarse powder, the corn coarse powder, the bean pulp coarse powder and the corncob coarse powder which are obtained by crushing corresponding solid matters;

(3) adding 18-22 g/L glucose, 15-20 g/L agar and water into the filtrate containing solid particles, and optionally adding PDA culture medium

(4) Sterilizing the modified PDA solid culture medium.

In another preferred embodiment, the modified PDA solid culture medium is prepared by the following method:

(2) pulverizing the decoction containing potato, and filtering with 2-4 layers of gauze to obtain filtrate containing potato coarse powder;

(3) adding 18-22 g/L glucose, 15-20 g/L agar and water into the filtrate containing potato coarse powder to obtain improved PDA culture medium, and optionally adding water and agar to obtain PDA culture medium

(4) Sterilizing the modified PDA solid culture medium.

The present invention also provides a method of preparing a culture medium, the method comprising the steps of:

(2) pulverizing the decoction containing potato, and filtering with 2-4 layers of gauze to obtain filtrate containing potato coarse powder; or crushing the cooking liquor containing the cooked potatoes, filtering the crushed cooking liquor by using 8 layers of gauze to obtain filtrate, and adding solid particles into the filtrate, wherein the solid particles comprise potato coarse powder, corn coarse powder, bean pulp coarse powder, corncob coarse powder or a combination of the potato coarse powder, the corn coarse powder, the bean pulp coarse powder and the corncob coarse powder which are obtained by crushing corresponding solid matters;

(3) adding 18-22 g/L glucose, 15-20 g/L agar and water into the filtrate containing solid particles, and optionally adding PDA culture medium

(4) Sterilizing the modified PDA solid culture medium.

The invention also provides the application of the potato coarse powder in preparing the culture medium, wherein the potato coarse powder is solid particles obtained by crushing soaked or un-soaked potatoes.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Detailed Description

The present inventors have made extensive and intensive studies to develop a fermentation method for producing β -glucan in high yield for the first time, which activates Schizophyllum commune by using an improved PDA solid medium, and have unexpectedly found that the growth state, growth rate and activity of Schizophyllum commune on the improved PDA plate are significantly changed (for example, the growth rate and growth density of Schizophyllum commune mycelia are significantly increased) by improving the formulation and preparation method of the PDA solid medium, thereby facilitating plate passage, shortening passage time, and significantly increasing β -glucan yield of fermentation culture.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Schizophyllum commune (Fr.) Quel

Schizophyllum commune Fr., namely white ginseng, tree flower, white flower and the like, is edible mushroom fungus of Schizophyllaceae of Agaricales of Basidiomycetes, and is distributed in Shanxi, Shaanxi, Heilongjiang, Sichuan, Guizhou, Yunnan and other areas of China.Schizophyllum commune fruiting body contains 7 essential amino acids except tryptophan, mineral elements such as zinc, iron, potassium, calcium, phosphorus, selenium, germanium and the like, and active substances such as nicotinic acid, flavone and the like, and has β -glucan content of 30-60%, so that the shizophyllum commune has the effects of clearing liver and improving eyesight, nourishing and strengthening the body, and is a rare mushroom fungus used as both medicine and food.

β dextran

β -dextran is a natural polysaccharide, in the natural environment, a great variety of β -dextran can be found, usually existing in cell walls of special kinds of bacteria, yeasts and fungi (lucid ganoderma) and also existing in coatings of higher plant seeds, wherein, the mushroom dextran taking β -1, 3-glycosidic bond as the skeleton has the functions of enhancing immunity, resisting tumor, bacteria and virus, and has obvious effects on moisture preservation, anti-wrinkle, antioxidation, uvioresistance, wound healing promotion and the like, and compared with the traditional yeast or cereal dextran, the mushroom dextran has more uniform and stable structure, higher molecular weight and more excellent water solubility, therefore, the mushroom β -dextran solution with high viscosity and high light transmittance is widely applied to the fields of food, cosmetics and pharmacy, and has important significance for preparing the mushroom β -dextran solution with high viscosity and high light transmittance.

The molecular weight of β -glucan (Schizophyllum commune polysaccharide) in the Schizophyllum commune fermentation liquid prepared by the method is high, the refined β -glucan solution is high in viscosity and light transmittance, has better biological activity and is suitable for various applications, and the Schizophyllum commune fermentation liquid does not contain cellulose or derivatives thereof, the structure of the cellulose and the derivatives thereof is similar to that of β -glucan, so that the determination of β -glucan content is influenced, and the separation and purification of β -glucan are not facilitated.

Improved PDA solid culture medium

The invention also provides an improved PDA solid culture medium, which is a PDA solid culture medium containing solid particles, wherein the solid particles comprise: potato meal, corn meal, soybean meal, corncob meal, or a combination thereof.

In another preferred example, the potato meal is solid particles obtained by crushing potatoes.

In another preferred example, the potatoes are uncooked or cooked potatoes.

In another preferred example, the corn meal is solid particles obtained by crushing corn (grains).

In another preferred example, the soybean meal coarse powder is solid particles obtained by crushing soybean meal.

In another preferred example, the corncob meal is solid particles obtained by crushing corncobs.

In another preferred embodiment, the modified PDA solid culture medium contains 5-10 wt% of solid particles, based on the total weight of the modified PDA solid culture medium.

In another preferred embodiment, the solid particles have an average particle size d of 0.15mm to 5mm, preferably 0.18mm to 3mm, more preferably 0.18mm to 1 mm.

In another preferred embodiment, the particle size of the corn meal, the soybean meal or the corn cob meal is 0.15-0.85mm, preferably 0.18-0.425 mm.

In another preferred embodiment, the average particle diameter d of the potato coarse powder is 0.15mm to 5mm, preferably 0.18mm to 3mm, and more preferably 0.18mm to 1 mm.

In another preferred example, the potato coarse powder is solid particles obtained by crushing cooked potatoes.

In another preferred example, the corn coarse powder, the bean pulp coarse powder and the corncob coarse powder are retentates obtained after crushing and sieving corresponding solid matters.

The method adopts an improved PDA solid culture medium to activate the schizophyllum commune. The improved growth form, growth speed and activity of schizophyllum commune on the PDA plate are obviously different from those of the common PDA plate, which is beneficial to plate passage and inoculation from the plate to a seed culture medium, and influences the final fermentation level through seed culture.

Specifically, by improving the formula of the PDA culture medium, the growth state and the growth activity of the Schizophyllum commune on the improved PDA flat plate are obviously changed, for example, the growth speed and the growth density of Schizophyllum commune hyphae are obviously improved, the grown hyphae are flourishing and fluffy, the passage of the flat plate piece is facilitated, the passage time is shortened, and the yield of β -glucan produced by later-stage fermentation culture is obviously improved.

In the present invention, "PDA solid medium" or "ordinary PDA solid medium" are used interchangeably and refer to a conventional PDA solid medium without modification. The PDA solid medium of the present invention can be prepared by a conventional method.

In another preferred embodiment, the modified PDA solid culture medium is prepared by the following method:

taking a plurality of fresh potatoes, peeling the potatoes, cutting the potatoes into uniform small blocks, weighing 220g of 180-one, adding 500m L purified water, boiling for 1 hour, taking out the immersion liquid (containing the potato blocks) and beating the immersion liquid for 10-30 seconds by using a grinder, taking out the ground matter, filtering by using 2-4 layers of gauze, taking the filtrate which contains the coarse powder of the potatoes which can be filtered out, adding 18-22g of glucose and 15-20g of agar, using the constant volume purified water to 1000m L, and sterilizing to prepare the improved PDA solid culture medium.

In another preferred embodiment, the modified PDA solid culture medium is prepared by the following method:

weighing a plurality of fresh potatoes, peeling the potatoes, cutting the potatoes into small uniform blocks, weighing 200g of the potatoes, adding L m of purified water, boiling the potatoes until the potato blocks can be broken by glass rods, filtering the potatoes by eight layers of gauze, taking filtrate, adding 20g of glucose, 15-20g of agar and 5-10g of 80-20 meshes (0.18-0.85mm) of corn coarse powder, bean pulp coarse powder, corn cob coarse powder or a composition of the agar after sieving, fixing the volume to 1000m L by using the purified water, boiling the mixture in water until the agar is completely dissolved, sterilizing the mixture at the high pressure of 121 ℃ for 15min, cooling the culture medium to about 65 ℃, and then pouring the mixture into a flat plate to prepare the improved PDA plate culture medium.

In another preferred embodiment, the method for preparing the common PDA solid culture medium comprises the following steps:

weighing a plurality of fresh potatoes, peeling the potatoes, cutting the potatoes into small uniform blocks, weighing 200g of the potatoes, adding L g of purified water of 500m, boiling the potatoes until the potato blocks can be broken by a glass rod, filtering the potatoes by using eight layers of gauze, taking filtrate, adding 20g of glucose and 15-20g of agar into the filtrate, fixing the volume to L m of the purified water, boiling the mixture by insulating water until the agar is completely dissolved, sterilizing the mixture for 15min at 121 ℃ under high pressure, cooling the culture medium to about 65 ℃, and pouring the culture medium into a flat plate to prepare the common PDA plate culture medium.

Method of the invention

The present invention provides a method for obtaining a fermentation broth with a high content of β glucan by liquid fermentation, typically, the method comprises the steps of: