CN104072632A - Method for efficiently extracting maitake mycelia polysaccharides through submerged fermentation production - Google Patents
Method for efficiently extracting maitake mycelia polysaccharides through submerged fermentation production Download PDFInfo
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Abstract
The invention discloses a method for efficiently extracting maitake mycelia polysaccharides through submerged fermentation production. The method is that mycelia separated from a maitake fermentation liquor is processed and separated through ultrasonic waves and composite enzymatic hydrolysis to obtain maitake polysaccharides. According to the invention, the conventional 'water extraction and alcohol precipitation' extraction method of polysaccharides is changed, the extraction yield and the product quality of maitake polysaccharides are improved, the consumption of purified water and energy consumption are reduced during production, and the economic benefits are increased.
Description
Technical field
The invention belongs to microbial technology field, specifically the method for maitake mushroom mycelia polysaccharide is produced in a kind of high efficiency extraction deep layer liquid state fermentation.
Background technology
Grifola frondosa [Grifola frondosa (Dicks.Fr.) S.F.Gray] is subordinate to Basidiomycotina, Hymenomycetes, Aphyllophorales, polyporaceae, tree Pseudomonas, have another name called polyporus frondosus, thousand Buddhist bacterium, chestnut bacterium, cloud gill fungus, lotus flower bacterium etc., it is fine and soft that Japan is referred to as dance, and the U.S. is referred to as jungle fowl.Its meat is tender and crisp, and taste, as shredded chicken, can be cooked into multiple delicious food, and contains several physiological active substances, is a kind of food, medicine dual-purpose gill fungus bacterium that R and D are worth that have.
Grifolan is a topmost active component in the numerous biologically active substances of Grifola frondosa, and it is to be rich in β-1,6-and β-1, the fungus polysaccharide of 3-glycosidic link from the sporophore of Grifola frondosa or mycelium with the separated class obtaining fermented liquid.Research shows, grifolan has biologic activity very widely, at antitumor, hypoglycemic, anti-hepatitis, anti-HIV (hiv virus) and improve the aspects such as function of immune system and all have good effect, and (as antitumor action) in some aspects, grifolan has apparently higher than the medical effect of other edible and medicinal fungi polysaccharide, also there is the medication superiority of convenient (as oral effective) simultaneously, for this reason, people are more and more dense to the research interest of grifolan, constantly experimentation on animals and the clinical study report relevant for its biological activity and pharmacological function produces, numerous researchers are also all actively put into its preparation, structural analysis, in the research of the relevant nature such as structure activity relationship and biological activity, go, make the relevant nature research of grifolan become one of the study hotspot in current this field.
China carries out artificial culture domestication since the beginning of the eighties to it, starts commercialization the nineties and produces, and growth momentum is powerful.But impact that artificial culture cycle of Grifola Frondosa sporophore is long, floor space is large, be subject to natural climate condition is large and be exposed in environment easily by living contaminants, large-scale artificial culture is difficult to promote.And natural Grifola Frondosa sporophore output is limited, be difficult to meet the market requirement of Grifola frondosa and series product thereof, therefore tended at present utilize, be not subject to liquid submerged fermentation technology natural condition restriction, can large-scale industrial production to cultivate maitake mushroom mycelia.
At present domestic existing universities and colleges launch the research of deep layer liquid state fermentation production Grifola frondosa, be all the mycelium that obtains Grifola frondosa be that major objective is launched research, and the relevant report of the domestic functional component polysaccharide about liquid state fermentation production Grifola frondosa is less at present.Extraction process about Mycophyta raw material polysaccharide mostly is traditional water extraction and alcohol precipitation method at present, to dried mycelium add 8 ?the water of 20 times of weight, control temperature 80 ?100 ℃ soak 2 ?4h, static 1 ?2h, then repeatedly lixiviate 2 ?3 times, then merge supernatant liquor and concentrate, then with 50 ?85% alcohol carry out alcohol precipitation, final oven dry can obtain Crude polysaccharides.The shortcoming of this technique is to add large water gaging to carry out for a long time lixiviate, and needs repeatedly lixiviate 2 ?3 times for improving yield, and the operational cycle is long, labour intensity is bigger than normal, and the extract yield of polysaccharide is many at 2 ?3%, and product cost is more expensive.And separated supernatant liquor is directly abandoned liquid as waste liquid and is drained, and has increased pollution treatment cost.
Summary of the invention
The object of the invention is in order to solve the technical bottleneck of the water extract-alcohol precipitation of traditional polysaccharide, adopt this invention can be fast, High-efficient Production goes out grifolan.
To achieve these goals, the present invention mainly carries out according to following step:
The method that maitake mushroom mycelia polysaccharide is produced in high efficiency extraction deep layer liquid state fermentation of the present invention comprises the steps:
1) separation: Grifola gigantea (Pers.) Piat. Fermented liquid, through separation, is obtained to Grifola frondosa supernatant liquor and wet mycelium;
2) supersound process: to step 1) in the wet mycelium of gained, adding step 1) the Grifola frondosa supernatant liquor of 5 times of quality of gained 2 ?dilutes, and carries out supersound process 10 ?40min after stirring, and obtains supersound process liquid; The interpolation of supernatant liquor is mainly to extract the polysaccharide in mycelium as water-soluble solution, and addition is on the low side, extracts not exclusively, and yield is on the low side, and in ultrasonic procedure easy temperature drift, the equipment that affects is used; If add too much, post-processed need consume a large amount of energy, high expensive;
3) complex enzyme hydrolysis: by step 2) gained supersound process liquid regulate pH value be 4 ?7, control temperature 30 ?70 ℃, then to its add be equivalent to dry mycelium weight 0.2 ?3% cellulase and 0.2 ?3% proteolytic enzyme, carry out enzymolysis 1 ?4h, obtain enzymolysis solution;
4) separation, concentrated: by step 3) enzymolysis solution of gained carries out separation, and by the supernatant liquor of resulting separation, through cryoconcentration, obtaining concentrated proportion is the thickened pulp of 1.25～1.40 (50 ℃ of heat are surveyed);
5) alcohol precipitation, oven dry: 95% alcohol that adds thickened pulp weight 3-5 doubly to measure, with Ebullioscope, detecting mixed solution alcoholic strength is 65-80% (v/v), standing 2-6h, carries out vacuum drying by throw out; Drying to being dried thing moisture content is 3～8%, obtains mycelium polysaccharides extract.
Wherein, step 1), 4) described be separated into centrifugation, adopt the centrifugation apparatus such as tripod pendulum type batch centrifugal or supercentrifuge to carry out, rotating speed be 1500 ?5000rpm.
Step 2) ultrasonic equipment used is ultrasonic cell disruptor, and other equal ultrasonic devices all can; Ultrasonic power be 0.5 ?2.5kw.
Step 3) the unit enzyme work of described cellulase is 15000 μ/g～25000 μ/g, and the unit enzyme work of described proteolytic enzyme is 47000 μ/g～53000 μ/g.
Described cryoconcentration process all requires to control temperature not higher than 75 ℃, and vacuum tightness is not less than 0.085Mpa.
The temperature 50-75 ℃ of described vacuum drying, vacuum tightness is not less than 0.085Mpa.
In the present invention, alcohol precipitation number of times is more, and the content of polysaccharide is higher.The vacuum drying oven that vacuum drying adopts, the polysaccharide content that the present invention extracts 30 ?60%.
The present invention has advantages of following outstanding:
Traditional technique is at least by the purified water of 20 times of mycelium dry weight, to dilute, and this process using supernatant liquor dilutes, and addition be only mycelium dry weight 2 ?5 times; Have the following advantages: 1, the present invention utilizes the alternative purified water of supernatant liquor to dilute, and has reduced discharging of waste liquid, has reduced pollution treatment cost; 2, because containing part exocellular polysaccharide in supernatant liquor, during as solution dilution, wherein contained polysaccharide also can be recovered utilization.3, with supernatant liquor instead of pure water, saved purified water, reduced production costs.
It is succinct that the present invention extracts route, saved a large amount of energy consumptions: because having reduced the add-on of purified water, accordingly in the separation in later stage, the operation such as concentrated, reduced a large amount of electricity, the consumption of steam energy.
Having shortened the operational cycle, reduced labour intensity: the present invention has changed traditional extracted many times, adopted a short period of time to process, is the 1/3-1/6 of conventional process time, has alleviated workman's labour intensity.
Improved extract yield: the extract yield of traditional water extraction polysaccharide is at 2-3%, and this invention has aggravated the dissolution rate of mycelium intracellular product with ultrasonic and enzymolysis, and the present invention extracts polysaccharide yield at 4-7%, than traditional technology, has promoted more than 1 times.
Promoted product quality: because the present invention adopts ultrasonic and complex enzyme hydrolysis processing mycelium, the mycelial proteolytic degradation of part is become to the small-molecule substance of solubility, reduced the content of impurity albumen in alcohol precipitation process, promoted the range of product of polysaccharide, adopt the present invention to extract polysaccharide content many more than 40%, than traditional technology, promoted more than 30%.
Adopt the present invention to make the high-quality polysaccharide of different content according to the market requirement and product standard, polysaccharide content can 30 ?60%.
Embodiment
By specific embodiment, further illustrate the present invention below.But the detail of embodiment only, for explaining the present invention, should not be construed as limited overall technical solution.The present invention is used in general Grifolas frondosa germ, and its source is not subject to the restriction of embodiment, and the Grifola frondosa bacterial classification that the bacterium numbering using in embodiment is CGMCC NO.4179 is only technical scheme in order to demonstrate the invention.
Embodiment 1
High efficiency extraction deep layer liquid state fermentation of the present invention is produced the method for maitake mushroom mycelia polysaccharide and is carried out in the steps below:
1, the preparation of Grifola gigantea (Pers.) Piat. Fermented liquid: Grifola frondosa bacterial classification is bought in institute of microbiology of the Chinese Academy of Sciences (its bacterium numbering is CGMCC NO.4179).The Grifola frondosa shaking flask bacterial strain having activated is inoculated in to seed culture medium (10L) by inoculum size 10%, wherein seed culture medium consists of glucose 1.8%, peptone 0.5%, soybean cake powder 1%, potassium primary phosphate 0.3%, magnesium sulfate heptahydrate 0.15%, soya-bean oil 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～28 ℃ of shaking culture obtain Grifola frondosa seed liquor for 7 days; Seed liquor is accessed to fermention medium (50L) by inoculum size 15%, wherein fermention medium consists of glucose 2.5%, peptone 0.3%, soybean cake powder 1.5%, potassium primary phosphate 0.2%, magnesium sulfate heptahydrate 0.2%, soya-bean oil 0.03%, regulates pH value 5.5; Ventilating ratio is 1:0.5vvm, and tank pressure 0.03Mpa cultivates 6 days for 26～28 ℃, ferments to fermented liquid and has bacterium ball to roll up, and filtrate is muddy; Reducing sugar is down to minimum 0.3% also slightly fluctuation; Microscopy mycelia is in small, broken bits, without living contaminants, obtains Grifola gigantea (Pers.) Piat. Fermented liquid.Wherein " % " in above-mentioned substratum composition all represents g/100ml.
2, Grifola gigantea (Pers.) Piat. Fermented liquid is separated: get above-mentioned Grifola gigantea (Pers.) Piat. Fermented liquid 10Kg and carry out separation by supercentrifuge 2000rpm, obtain the wet mycelium (water content is 80%) of Grifola frondosa supernatant liquor liquid 9.35Kg and 0.65Kg.
3, supersound process: add the Grifola frondosa supernatant liquor of 2.35Kg to dilute in the 0.65Kg wet mycelium of step 2 gained, carry out 1.5kw supersound process 20min after stirring, obtain supersound process liquid 3Kg.
4, complex enzyme hydrolysis: it is 5.5 that step 3 gained supersound process liquid is regulated to pH value, in water-bath, control temperature 45 C, then to it, slowly add cellulase 2.1g (purchased from Zaozhuang Jie Nuo biological enzyme company limited, unit enzyme 20000 μ/g alive, lower with), proteolytic enzyme 1.9g (purchased from Zaozhuang Jie Nuo biological enzyme company limited, the unit enzyme 50000 μ/g that lives, lower with) and constantly stir, after stirring, enzymolysis 2h is carried out in timing, obtains enzymolysis solution.
5, separated, concentrated: the enzymolysis solution of step 4 gained is carried out to separation with supercentrifuge 4000rpm, by the supernatant liquor 2.5Kg Rotary Evaporators cryoconcentration of resulting separation, concentration process all requires to control temperature not higher than 75 ℃, vacuum tightness is not less than 0.085MPa, and obtaining concentrated proportion is the thickened pulp 65g of 1.3 (50 ℃ of heat are surveyed).
6, alcohol precipitation, oven dry: to 95% alcohol that adds 250ml in 65g thickened pulp, with Ebullioscope, detect mixed solution alcoholic strength 72% (v/v), standing 2h after slowly stirring, dries the separation of bottom settlings thing with vacuum drying oven, temperature 60 C, vacuum tightness 0.09MPa; Dry 1h and obtain maitake mushroom mycelia polyoses extract 16.5g.Detecting moisture content is 3.7%, and polysaccharide content is 44%, and polysaccharide extract yield is 5.58%.
Polysaccharide extract yield in the present invention calculates by dry bacterial powder, i.e. polysaccharide extract yield=maitake mushroom mycelia polyoses extract quality * polysaccharide content/[wet mycelium quality * (1 ?wet mycelium water content)] * 100%
Embodiment 2
High efficiency extraction deep layer liquid state fermentation of the present invention is produced the method for maitake mushroom mycelia polysaccharide and is carried out in the steps below:
1, the preparation of Grifola gigantea (Pers.) Piat. Fermented liquid: Grifola frondosa bacterial classification is bought in institute of microbiology of the Chinese Academy of Sciences (its bacterium numbering is CGMCC NO.4179).The Grifola frondosa shaking flask bacterial strain having activated is inoculated in to seed culture medium (20L) by inoculum size 14% (v/v), wherein seed culture medium consists of glucose 2.0%, peptone 0.3%, soybean cake powder 1%, potassium primary phosphate 0.3%, magnesium sulfate heptahydrate 0.15%, soya-bean oil 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～28 ℃ of shaking culture obtain Grifola frondosa seed liquor for 7 days; Seed liquor is accessed to fermention medium (100L) by inoculum size 18% (v/v), wherein fermention medium consists of glucose 2.5%, peptone 0.5%, soybean cake powder 1.5%, potassium primary phosphate 0.2%, magnesium sulfate heptahydrate 0.2%, soya-bean oil 0.03%, regulates pH value 5.5; Ventilating ratio is 1:0.5vvm, and tank pressure 0.03Mpa cultivates 5 days for 26～28 ℃, ferments to fermented liquid and has bacterium ball to roll up, and filtrate is muddy; Reducing sugar is down to minimum 0.3% also slightly fluctuation; Microscopy mycelia is in small, broken bits, without living contaminants, obtains Grifola gigantea (Pers.) Piat. Fermented liquid.Wherein " % " in above-mentioned substratum composition all represents g/100ml.
2, Grifola gigantea (Pers.) Piat. Fermented liquid is separated: get above-mentioned Grifola gigantea (Pers.) Piat. Fermented liquid 20Kg and carry out separation by supercentrifuge 2000rpm, obtain the wet mycelium (water content is 78%) of Grifola frondosa supernatant liquor liquid 18.7Kg and 1.3Kg.
3, supersound process: add the Grifola frondosa supernatant liquor of 3.7Kg to dilute in the 1.3Kg wet mycelium of step 2 gained, carry out 2.0kw after stirring
Supersound process 30min, obtains supersound process liquid 5Kg.
4, complex enzyme hydrolysis: it is 5.0 that step 3 gained supersound process liquid is regulated to pH value, in water-bath, control 55 ℃ of temperature, then to it, slowly add cellulase 4.5g (purchased from Zaozhuang Jie Nuo biological enzyme company limited, unit enzyme 20000 μ/g alive, lower with), proteolytic enzyme 4.2g (purchased from Zaozhuang Jie Nuo biological enzyme company limited, the unit enzyme 50000 μ/g that lives, lower with) and constantly stir, after stirring, enzymolysis 2h is carried out in timing, obtains enzymolysis solution.
5, separated, concentrated: the enzymolysis solution of step 4 gained is carried out to separation with supercentrifuge 4000rpm, by the supernatant liquor 3.8Kg Rotary Evaporators cryoconcentration of resulting separation, concentration process all requires to control temperature not higher than 75 ℃, vacuum tightness is not less than 0.085MPa, and obtaining concentrated proportion is the thickened pulp 120g of 1.4 (50 ℃ of heat are surveyed).
6, alcohol precipitation, oven dry: to 95% alcohol that adds 400ml in 120g thickened pulp, with Ebullioscope, detect mixed solution alcoholic strength 73% (v/v), standing 2.5h after slowly stirring, dries the separation of bottom settlings thing with vacuum drying oven, temperature 60 C, vacuum tightness 0.09MPa; Dry 1h and obtain maitake mushroom mycelia polyoses extract 38.5g.Detecting moisture content is 3.3%, and polysaccharide content is 41%, and polysaccharide extract yield is 5.52%.
Claims (5)
1. an extracting method for maitake mushroom mycelia polysaccharide, is characterized in that the method comprises the steps:
1) separation: Grifola gigantea (Pers.) Piat. Fermented liquid, through separation, is obtained to Grifola frondosa supernatant liquor and wet mycelium;
2) supersound process: to step 1) in the wet mycelium of gained, adding step 1) the Grifola frondosa supernatant liquor of gained 2-5 times quality dilutes, and carries out supersound process 10-40min after stirring, and obtains supersound process liquid;
3) complex enzyme hydrolysis: by step 2) gained supersound process liquid adjusting pH value is 4-7, control temperature 30-70 ℃, then to it, add and be equivalent to the cellulase of dry mycelium weight 0.2-3% and the proteolytic enzyme of 0.2-3%, carry out enzymolysis 1-4h, obtain enzymolysis solution;
4) separation, concentrated: by step 3) enzymolysis solution of gained carries out separation, and the supernatant liquor of resulting separation, through cryoconcentration, is obtained to the thickened pulp that concentrated proportion is 1.25～1.40;
5) alcohol precipitation, oven dry: 95% alcohol that adds thickened pulp weight 3-5 doubly to measure, with Ebullioscope, detecting mixed solution alcoholic strength is 65-80% (v/v), standing 2-6h, carries out vacuum drying by throw out; Drying to being dried thing moisture content is 3～8%, obtains mycelium polysaccharides extract.
2. the extracting method of maitake mushroom mycelia polysaccharide according to claim 1, is characterized in that the described centrifugation that is separated into, and rotating speed is 1500-5000rpm.
3. the extracting method of maitake mushroom mycelia polysaccharide according to claim 1, is characterized in that the unit enzyme work of described cellulase is 15000 μ/g～25000 μ/g, and the unit enzyme work of described proteolytic enzyme is 47000 μ/g～53000 μ/g.
4. the extracting method of maitake mushroom mycelia polysaccharide according to claim 1, is characterized in that described cryoconcentration process all requires to control temperature not higher than 75 ℃, and vacuum tightness is not less than 0.085Mpa.
5. the extracting method of maitake mushroom mycelia polysaccharide according to claim 1, is characterized in that the temperature 50-75 ℃ of described vacuum drying, and vacuum tightness is not less than 0.085Mpa.
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Application publication date: 20141001 |