CN105646730B - A method of extracting β -1,3-D- glucan from the cell wall of fungi - Google Patents
A method of extracting β -1,3-D- glucan from the cell wall of fungi Download PDFInfo
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Abstract
The invention discloses β -1 is extracted in a kind of cell wall from fungi, the method of 3-D- glucan, this method comprises: in the presence of the solvent, cell wall is mixed with alkali metal hydroxide and carries out alkali process, the precipitating that alkali process obtains is β -1, the crude product of 3-D- glucan, wherein, relative to every kilogram of cell wall, the dosage of the alkali metal hydroxide is 65-900g, and the temperature of the alkali process is 105-115 DEG C.Through the above technical solutions, β -1 that the present invention obtains, 3-D- glucan recovery rate and purity is high；Alkali consumption is few, and environmental pollution is smaller, and the used time is shorter, and extraction efficiency is higher, and specific energy consumption is less, and cost can be greatly lowered.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of β -1, the preparation method of 3-D- glucan, and in particular, to one
The method that kind extracts β -1,3-D- glucan from the cell wall of fungi.
Background technique
Brewer's yeast (Saccharomyces cerevisiae) belongs to Eumycota saccharomyces, and cell wall is more tough and tensile, about
The 25% of dry cell weight is accounted for, main component has glucan, mannosan, protein, chitin and lipid etc..Wherein, β -1,3-
D- glucan has anticancer, antibacterial, antiviral, reducing blood lipid and increasing as being difficult to by the dietary fiber of human consumption in body
The important physiological functions such as strong immunocompetence.Therefore, the exploitation of domestic and international β -1,3-D- glucan functional food has become hot spot,
β -1,3-D- glucan is in Japan, Southeast Asia, European & American Market as Medicines and Health Product more than ten year prevailing.According to Chinese drug and food
Product association statistics, China β -1 in 2007, the export volume of 3-D- glucan just have reached 500 tons, but belong to the product of low content.
As the improvement of people's living standards, β -1,3-D- glucan are increasingly accepted by people, the market demand constantly expands, price
Also it is always maintained at higher price.The country is used for β -1 of health product raw material, and the price of 3-D- glucan is 300-800 member/kg,
There is very high profit margin.However, current yeast cell wall is confined on feed using more, become food industry one is big
Loss.
Currently, common β -1,3-D- glucan preparation method have acid system, alkaline process, acid-base method, organic solvent-enzyme process and height
Warm water extract method etc..Traditional acid system, alkaline process (use concentration to mix for the NaOH solution of 3-4% with cell wall, it is every kilogram thin
The NaOH dosage of cell wall is 1.1kg or more, and the temperature of processing is 90 DEG C, and the time of processing is 2-4h) and acid-base method acid consumption or
Alkali consumption is higher, and environmental pollution is larger.And the processing time of these methods is longer, and total energy consumption is larger, to need higher
Cost input.The post-processing of organic solvent used in organic solvent-enzyme process is more difficult, and environmental pollution is larger, to operation
Safety requirements it is high.Though high-temperature-hot-water extraction avoids some disadvantages of soda acid and organic solvent, this method time-consuming is more
It is long, and obtained β -1,3-D- glucan purity are lower, are only about 40%.
Summary of the invention
The purpose of the present invention is overcoming existing alkaline process alkali consumption big and the defect of low efficiency, provide a kind of improved from true
The method of β -1,3-D- glucan is extracted in the cell wall of bacterium.
To achieve the goals above, the present inventor has carried out many experiments, as a result, it has been found that, it reduces base amount and fits
It can further improve β -1 when increasing temperature, the recovery rate and purity of 3-D- glucan, therefore, the present invention provides one kind from true
Extraction β -1 in the cell wall of bacterium, the method for 3-D- glucan, this method comprises: in the presence of the solvent, by cell wall and alkali gold
Belong to hydroxide mixing and carry out alkali process, the precipitating that alkali process obtains is β -1, the crude product of 3-D- glucan, wherein opposite
In every kilogram of cell wall, the dosage of the alkali metal hydroxide is 65-900g, and the temperature of the alkali process is 105-115
℃。
Through the above technical solutions, alkali-insoluble β -1 that the present invention obtains, the recovery rate and purity is high of 3-D- glucan,
Alkali consumption is lower, and environmental pollution is smaller, and the used time is shorter, and extraction efficiency is higher, and specific energy consumption is less, can significantly drop
Low cost improves value-added content of product.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The method that β -1,3-D- glucan is extracted in cell wall provided by the invention from fungi includes: the presence in solvent
Under, cell wall is mixed with alkali metal hydroxide and carries out alkali process, the precipitating that alkali process obtains is β -1,3-D- glucan
Crude product, wherein relative to every kilogram of cell wall, the dosage of the alkali metal hydroxide be 65-900g (preferably
175-335g, more preferably 185-240g), the temperature of the alkali process is 105-115 DEG C (preferably 105-110 DEG C).
In the present invention, relative to the solvent of 100mL, the dosage of the alkali metal hydroxide is preferably 0.6-2g, more excellent
It is selected as 0.8-1g.Based on the considerations of safety and save the cost, the solvent is usually water.
In the present invention, the alkali metal hydroxide can be for the common various alkali metal hydroxides in this field (usually
For highly basic), under preferable case, the alkali metal hydroxide is sodium hydroxide and/or potassium hydroxide.
In the present invention, the mode mixed to cell wall with alkali metal hydroxide is not particularly limited, can be by alkali gold
Belong to after hydroxide is configured to solution and being mixed again with cell wall, cell wall can also be separately added into alkali metal hydroxide molten
It is mixed in agent, based on easy to operate consideration, preferably is configured to mix with cell wall again after solution by alkali metal hydroxide
It closes.
In the present invention, the time of the alkali process is not required particularly, can be 5-60min, preferably 5-
10min.As can be seen that extracting method of the invention advantageously reduces the time of alkali process.
According to the preferred embodiment of the present invention, the method can also include the precipitating that separation alkali process obtains, and will
Washing of precipitate to pH value is 6-7.Separation can be using conventional solid-liquid separating method, such as centrifuge separation is (for the ease of centrifugation
Operation, the material after alkali process can be cooled to after 45 DEG C or less and be centrifuged again).It, can be using normal for washing
The reagent of rule is washed, as long as precipitating can be made to be in neutrality (such as 6-7) substantially, for example, (can preferably be distilled using water
Water) it is washed, it can also be washed using acid solution.
It is highly preferred that the method also includes the precipitating after washing is dried.Dry mode can be hot air drying
Dry (temperature can be 50-160 DEG C), spray drying, negative pressure drying, freeze-drying etc..
In the present invention, β -1 of acquisition, 3-D- glucan is usually the alkali-insoluble β -1,3-D- that molecular weight is 20-30KDa
Glucan.
In the present invention, the fungi is to contain alkali-insoluble β -1 in the cell wall of this field, 3-D- glucan it is various true
Bacterium can be saccharomycete (such as brewer's yeast), or gill fungus bacterium (such as Clitocybe maxima (Clitocybe maxima)).It is preferred that
In the case of, the fungi is saccharomycete (such as brewer's yeast).
In the present invention, the cell wall of the fungi can be prepared according to the method for this field routine, for example, can adopt
With the method for mechanical breaking-wall method, self-dissolving method, enzyme solution, chemical method etc..
Most preferred embodiment mode according to the present invention, the method includes in the presence of the solvent, by cell wall and alkali
Metal hydroxides mixing carries out alkali process, and the precipitating that alkali process obtains is β -1, the crude product of 3-D- glucan, wherein phase
For every kilogram of cell wall, the dosage of the alkali metal hydroxide is 185-240g, described relative to the solvent of 100mL
The dosage of alkali metal hydroxide is 0.8-0.9g, and the temperature of the alkali process is 105-108 DEG C, time 5-10min.
The present invention will be described in detail by way of examples below.
In following embodiment, used experimental method is such as conventional method without particular/special requirement；Used material and
Reagent etc. obtains from commercial channels unless otherwise specified；The weight for the cell wall being related to is based on its dry weight；β-1,3-D-
The content of glucan uses Megazyme company K-YBGL kit " MUSHROOM AND YEAST BETA GLUCAN ASSAY
KIT " is quantified.
Recovery rate is calculated as follows:
Wherein: X is recovery rate；
M is gained β -1, the quality of 3-D- glucan, g；
C is gained β -1, the purity of 3-D- glucan, weight %；
m0For the quality of cell wall used, g；
c0For β -1 in cell wall, the content of 3-D- glucan, weight %.
Preparation example 1
This preparation example is used to prepare cell wall used in each embodiment.
Brewer's yeast pretreatment: use 200 mesh screen cloths to beer waste yeast (dry matter content for 16 weight %, by Tianjin
Snowflake Beer Co., Ltd provides) it carries out after sieving removing impurity, 2000 × g is centrifuged 10min, precipitates clear with cold physiological saline
2 times are washed to get clean beer yeast cells.
The preparation of the cell wall of brewer's yeast: distilled water is added to be made into 50 weights pretreated clean beer yeast cells
The cell suspension for measuring % is crushed twice under the conditions of 1700bar using high-pressure homogeneous crusher；8341 × g is centrifuged 20min, sinks
Shallow lake is cleaned 3 times with cold deionized water, and up to the cell wall of brewer's yeast, (water content is 82 weights after centrifugation removal supernatant
Measure %).
Clitocybe maxima (purchased from China Forest Microbiological Culture Collection administrative center, CFCC87186) is taken, is made in accordance with the following steps
The cell wall of standby Clitocybe maxima: (1) transferring test tube stock on the inclined-plane PDA (eggplant-shape bottle), cultivated in 25 DEG C of insulating boxs 6 days it is standby
With；(2) shaking flask culture, culture medium prescription: 2 weight % of soluble starch, 0.6 weight % of Dried Corn Steep Liquor Powder, biphosphate are carried out
0.1 weight % of potassium；Liquid amount: 150ml/500ml triangular flask；Inoculum concentration: each shaking flask is inoculated with about 2cm2Lawn；Condition of culture:
25 DEG C, 150rpm, 3.5 days or so；(3) cultured bacterium solution collects mycelium through being separated by solid-liquid separation, using high-speed homogenizer
Mycelium is broken up, pretreatment and the preparation of cell wall is carried out according still further to above-mentioned method identical with beer yeast cells wall, obtains
The cell wall of Clitocybe maxima (water content is 85 weight %).
Embodiment 1
The NaOH solution that 48mL concentration is 0.9g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 108 DEG C of processing 5min, is cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 6.5,105 DEG C of dry 2h.
β -1 in gained β -1,3-D- glucan product, the content of 3-D- glucan are 77.11%, recovery rate 77.38%.
Comparative example 1
The NaOH solution that 100mL concentration is 2g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 130 DEG C of processing 5min, is cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 6.5,105 DEG C of dry 2h.
β -1 in gained β -1,3-D- glucan product, the content of 3-D- glucan are 67.97%, recovery rate 37.74%.
Comparative example 2
The NaOH solution that 100mL concentration is 4g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 90 DEG C of processing 150min, is subsequently cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 6.5,105 DEG C dry
Dry 2h.β -1 in gained β -1,3-D- glucan product, the content of 3-D- glucan are 50.5%, recovery rate 35.54%.
Embodiment 2
The NaOH solution that 48mL concentration is 0.8g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 105 DEG C of processing 5min, is cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 7,80 DEG C of dry 4h.Institute
β -1, β -1 in 3-D- glucan product are obtained, the content of 3-D- glucan is 76.95%, recovery rate 78.31%.
Embodiment 3
The NaOH solution that 72mL concentration is 1g/100mL is added in the cell wall of 12g brewer's yeast, is made into cell wall suspension,
110 DEG C of processing 10min are placed in, are cooled to 45 DEG C, 9690 × g is centrifuged 20min, and being washed to pH value is 6.8, using spray drying side
Method is dried, and control inlet temperature is 150 DEG C, and outlet temperature is 80 DEG C.β -1,3- in gained β -1,3-D- glucan product
The content of D- glucan is 78.24%, recovery rate 77.88%.
Embodiment 4
The KOH solution that 60mL concentration is 0.6g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 115 DEG C of processing 5min, is cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 6.7, using freeze-drying
Method (vacuum degree 0.1mbar, condenser temperature be -50 DEG C) be dried, drying time 10h.The Portugal gained β -1,3-D-
The content of β -1 in glycan product, 3-D- glucan is 76.43%, recovery rate 69.23%.
Embodiment 5
The NaOH solution that 48mL concentration is 0.85g/100mL is added in the cell wall of 12g brewer's yeast, it is outstanding to be made into cell wall
Liquid is placed in 108 DEG C of processing 5min, is cooled to 45 DEG C, and 9690 × g is centrifuged 20min, and being washed to pH value is 6.7,105 DEG C of dry 2h.
β -1 in gained β -1,3-D- glucan product, the content of 3-D- glucan are 78.11%, recovery rate 78.38%.
Embodiment 6
β -1,3-D- glucan, the Portugal gained β -1,3-D- are extracted from the cell wall of Clitocybe maxima according to the method for embodiment 5
The content of β -1 in glycan product, 3-D- glucan is 34.2%, recovery rate 46.23%.
Comparative example 3
The NaOH solution that 100mL concentration is 4g/100mL is added in the cell wall of 12g Clitocybe maxima, is made into cell wall suspension,
90 DEG C of processing 150min are placed in, are subsequently cooled to 45 DEG C, 9690 × g is centrifuged 20min, and being washed to pH value is 6.7,105 DEG C of dryings
2h.β -1 in gained β -1,3-D- glucan product, the content of 3-D- glucan are 21.2%, recovery rate 22.34%.
From above embodiments as it can be seen that method of the invention can improve β -1 under the conditions of base amount and lower temperature,
The recovery rate and purity of 3-D- glucan, more friendly to environment, process treatment time is short, and β -1, the Portugal 3-D- is greatly improved
The extraction efficiency of glycan.
Embodiment 1 is compared with comparative example 1-2, embodiment 6 is compared with comparative example 3, it can be seen that use alkali
In the method within the scope of the present invention such as dosage of metal hydroxides, β -1, the purity and recovery rate of 3-D- glucan is significantly mentioned
It is high.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. extracting β -1, the method for 3-D- glucan, which is characterized in that this method comprises: molten in a kind of cell wall from fungi
In the presence of agent, cell wall is mixed with alkali metal hydroxide and carries out alkali process, the precipitating that alkali process obtains is β -1,3-
The crude product of D- glucan, wherein relative to every kilogram of cell wall, the dosage of the alkali metal hydroxide is 175-
335g, the temperature of the alkali process are 105-115 DEG C, and the time of the alkali process is 5-10min.
2. according to the method described in claim 1, wherein, the dosage of the alkali metal hydroxide is 185-240g.
3. according to the method described in claim 1, wherein, relative to the solvent of 100mL, the dosage of the alkali metal hydroxide
For 0.6-2g.
4. according to the method described in claim 1, wherein, relative to the solvent of 100mL, the dosage of the alkali metal hydroxide
For 0.8-1g.
5. method described in any one of -4 according to claim 1, wherein the alkali metal hydroxide is sodium hydroxide
And/or potassium hydroxide.
6. according to the method described in claim 1, wherein, the temperature of the alkali process is 105-110 DEG C.
7. according to the method described in claim 1, wherein, the method also includes the precipitatings that separation alkali process obtains, and will sink
Washing to pH value of forming sediment is 6-7.
8. according to the method described in claim 7, wherein, the method also includes the precipitating after washing is dried.
9. according to the method described in claim 1, wherein, the fungi is saccharomycete and/or gill fungus bacterium.
10. according to claim 1 or method described in 9, wherein the fungi is brewer's yeast (Saccharomyces
) and/or Clitocybe maxima (Clitocybe maxima) cerevisiae.
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| CN101161686B (en) * | 2007-11-23 | 2010-04-14 | 广东省微生物研究所 | Method for preparing immunopotentiator beta-dextran |
| CN101407559B (en) * | 2008-11-25 | 2011-01-26 | 天津科技大学 | Method for rapidly preparing yeast beta-1,3-dextran |
| CN101560269B (en) * | 2009-05-22 | 2012-01-11 | 广东药学院 | Method for preparing yeast beta-1, 3-glucan |
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