CN104099384B - A kind of deep layer liquid fermentation produces the method for crow spirit ginseng extracellular polysaccharide - Google Patents
A kind of deep layer liquid fermentation produces the method for crow spirit ginseng extracellular polysaccharide Download PDFInfo
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Abstract
The invention discloses a kind of method that deep layer liquid fermentation produces crow spirit ginseng extracellular polysaccharide. The method is that the crow spirit ginseng strain activated is carried out expansion joined by being linked into seed tank, then is linked in the special liquid culture medium producing polysaccharide ferments expanding the liquid seeds prepared, and is then easily separated by fermentation liquid, extracts extracellular polysaccharide. The present invention is just for being amplified to what the technical barrier run in industrialized production carried out studying by lab scale, continue on the basis of optimization fermentation medium (especially carbon source), lifting Biomass and extracellular polysaccharide at lab scale and pilot scale, solve amplification process runs into Fermentation Process of Parameter control, extraction equipment type selecting, the difficult problem such as optimization, final success grafting carries out industrialization production to 10 20t fermentation tanks, and produce the crow spirit ginseng extracellular polysaccharide of high-quality smoothly, be truly realized continuously, automatization, industrialization production.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of method that deep layer liquid fermentation produces crow spirit ginseng extracellular polysaccharide.
Background technology
Wu Lingcan belongs to the Xylaria nigripes of Ascomycetes, Xylariaceae, it it is a traditional medicinal fungi, formal name used at school Xylaria nigripes, another name Wu Lingcan, vertical (clump) egg of chicken, have another name called shake thunder, it is grown on the ant nest that the black wing Soil termites of deep under ground is discarded, is accordingly to be regarded as rare, rare medicinal herbs by the research of world's medicinal fungi, application, there is medical value widely.
Wu Lingcan is grown on the discarded fungus garden of Coptotermes formosanus Shtrari. of massif hillside fields warm on the sunny side or river levee slight slope, is by the Xylaria nigripes mycelia gathered on fungus garden, at its Later growth, the sclerotium that part is intensive, is commonly called as " Wu Lingcan ", weighs about 5 30 grams, spherical, class is spherical or irregular flat strips is block, diameter 2 12 centimetres, appearance sepia or black smooth or wrinkle more, there is shoestring (rhizoid) one end or has umbilication, inner white, meat, hard or soft, sweet in the mouth is fragrant. Sclerotium upper end has handle poise in fungus garden upper wall and be connected with shoestring. June in next year to October, when around fungus garden, soil temperature is more than 20 (at a time when this thunderstorm and autumn rain season), sclerotial germination mycelia, form stroma on earth's surface, be commonly called as " charcoal rod ", be scattered or all living creatures on earth's surface. Wild crow spirit ginseng mainly originates in the ground such as Sichuan, Yunnan, Guangdong, and also there is growth in Zhejiang, Jiangxi, Jiangsu, and Wu Ling joins the place of production with Guanxian county in Sichuan, very the ground such as shoulder for top grade. Owing to Wu Lingcan is grown on the ant nest that the Soil termites of 2 meters-20 meters, underground is discarded, digging is extremely difficult, therefore natural resources is extremely rare. Wu Lingcan is medicinal fungi and edible fungi, has better nutritivity and medical value, is comparatively ideal health care food (medicine) product.
Wu Lingcan, warm in nature, sweet in the mouth, have QI invigorating reinforce the kidney, spleen invigorating dehumidifying, tranquillizing and allaying excitement effect, cure mainly insufficiency of the spleen few food, puerperal and postoperative severe loss of blood, puerperal breast less, gastroptosis, hernia, palpitation and insomnia, infantile convulsion and traumatic injury etc.Experimentation shows; the main composition of Wu Lingcan is polysaccharide; its crude polysaccharides not only has raising body's immunity and anti-aging effects; and affect test show through institute of materia medica of China Medical institute mice breaked end ischemia time lengthening of panting; Wu Ling joins crude polysaccharides obvious cerebral protection, and the dysmnesia that galvanic shock is caused also have protective effect. China Concord Medical Science University shows tested by basis, and Wu Ling joins crude polysaccharides can substantially suppress activity of monoamine oxidase in mouse brain, thus improving central neurotransmitter function.
Owing to Wu Lingcan is grown on the ant nest that the Soil termites of 2 meters-20 meters, underground is discarded, digging is extremely difficult, therefore natural resources is extremely rare. domestic only minority universities and colleges launch the research of deep layer liquid fermentation production crow spirit ginseng at present, and the overwhelming majority is all confined to lab scale level, it is all launch research with the mycelium obtaining crow spirit ginseng for main target, and the domestic functional component polysaccharide about liquid fermentation production crow spirit ginseng is almost without relevant report at present, more do not research and solve and how to be amplified to industrialization produces, from lab scale grafting, the culture medium prescription run into and choose, Fermentation Operating Parameters controls, the technical barriers such as the research of Polysaccharide from Bee, therefore at present domestic but without real industrialization liquid fermentation produce crow spirit gracilis polysaccharide producer.
Summary of the invention
The invention aims to solve the technical bottleneck of industrial-scale production crow spirit gracilis polysaccharide, adopt this invention can continuously, automatization, industrialization the clever gracilis polysaccharide of crow producing high-quality.
To achieve these goals, the present invention carries out essentially according to following step:
The industrialization deep layer liquid fermentation of the present invention produces the method for crow spirit ginseng extracellular polysaccharide and comprises the steps:
A kind of method preparing crow spirit ginseng fermentation liquid, comprise the steps: that the liquid fermentation medium after crow spirit ginseng seed culture fluid is accessed sterilizing carries out deep layer liquid fermentation and obtains Wu Ling ginseng fermentation liquid, the inoculum concentration of described deep layer liquid fermentation is 10～20%, ventilating ratio is 1:0.3-1.0vvm, tank pressure 0.02～0.05Mpa, cultivate 3～5 days at 22～30 DEG C; Described liquid fermentation medium formula is: glucose 1-3%, starch 1-3%, peptone 0.1-0.5% (v/v), soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, zinc sulfate 0.1-0.5%, defoamer 0.01-0.05%, 20000u/ml Thermostable α-Amylase 0.001-0.007% (v/v), regulate pH value 5.0-6.5. In above culture medium composition, " % " except Thermostable α-Amylase all represents g/100ml.
Described defoamer can be vegetable oil (Oleum Glycines, the Oleum Brassicae campestris) material such as class, organic ethers.
Described crow spirit ginseng seed culture fluid is cultivated by two grades and is obtained, and concrete grammar is as follows:
1) first order seed is cultivated: activated shaking flask strain accesses the liquid seed culture medium after sterilizing, inoculum concentration is 0.1～2% (v/v), ventilating ratio is 1:0.3 0.7vvm, tank pressure 0.02～0.05Mpa, cultivate 3～5 days at 22～30 DEG C, obtain Wu Ling and join first order seed culture fluid, standby;
2) secondary seed is cultivated: by step 1) the first order seed culture fluid of gained accesses the liquid seed culture medium after sterilizing, inoculum concentration is 10～20% (v/v), ventilating ratio is 1:0.3 1.0vvm, tank pressure 0.02～0.05Mpa, cultivate 2～4 days at 22～30 DEG C, obtain Wu Ling and join secondary seed culture fluid;
Wherein said liquid seed culture medium culture medium is: glucose 2-4%, peptone 0.1-0.5%, soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, zinc sulfate 0.1-0.5%, defoamer 0.01-0.05%, regulates pH value 5.0-6.5.The volume of first class seed pot is 100-500L, and the volume of secondary seed tank is 1000-5000L, and loadings is 70-90% (v/v). " % " in above culture medium composition all represents g/100ml.
The fermenter volume of described deep layer liquid fermentation is 10000-20000L, and loadings is 70-90% (v/v).
The endpoint standard of described deep layer liquid fermentation: fermentation liquid is peat-reek, fungus ball substantial increase, and filtrate is muddy; Reducing sugar is down to minimum and slightly fluctuates, and reducing sugar controls at 0.5% (g/100g) below; Microscopy mycelia is in small, broken bits, without living contaminants.
The extracting method of a kind of crow spirit ginseng extracellular polysaccharide, comprises the steps:
1) separate: by the crow spirit ginseng separation of fermentative broth of above-mentioned gained, obtain Wu Ling and join filtrate;
2) concentration: by step 1) filtrate of gained carries out adopting two grade low-temps concentrations, obtains the concentrated pulp that concentration proportion is 1.25～1.40 (50 DEG C of heat are surveyed);
3) precipitate with ethanol, drying: add concentrated pulp weight 3-5 times amount 95% ethanol, detect mixed liquor alcoholic strength with alcohol meter and be about 65-80% (v/v), stand 8-16h, precipitate is carried out vacuum drying, temperature 50-75 DEG C, vacuum is not less than 0.085MPa; Drying to dried object moisture is 3～8% (g/100g), obtains the outer sugar of born of the same parents.
Wherein, step 1) described in separation adopt horizontal screw centrifuge to carry out, rotating speed controls at 2500 3500r/min.
Step 2) described in two grade low-temps concentration be: one-level adopt 1 3t double effect evaporator concentration, it is 1.15 1.20 (50 DEG C of heat are surveyed) that fermentation liquid is concentrated to proportion, two grades adopt 1 3t haplo-effect concentrator concentration, continue to be concentrated to the concentrated pulp that proportion is 1.25～1.40 (50 DEG C of heat are surveyed) by the concentrated solution that proportion is 1.1 1.20 (50 DEG C of heat are surveyed); Concentration process all requires to control temperature not higher than 75 DEG C, and vacuum is not less than 0.085MPa.
In the present invention, precipitate with ethanol is to adopt 1 3t Alcohol-settling tank, and precipitate with ethanol number of times is more many, and the content of the outer sugar of born of the same parents is more high. The vacuum drying oven that vacuum drying adopts, the born of the same parents that the present invention extracts outer sugar content is 20 50%.
The present invention has following prominent advantage:
The present invention is just for being amplified to what the technical barrier run in industrialized production carried out studying by lab scale, continue on the basis of optimization fermentation medium (especially carbon source), lifting Biomass and extracellular polysaccharide at lab scale and pilot scale, solve amplification process runs into Fermentation Process of Parameter control, extraction equipment type selecting, the difficult problem such as optimization, final success grafting carries out industrialization production to 10 20t fermentation tanks, and produce the crow spirit ginseng extracellular polysaccharide of high-quality smoothly, be truly realized continuously, automatization, industrialization production.
In order to improve the yield of purpose product extracellular polysaccharide, the present invention is optimized in the carbon source of fermentation medium and combines: adopt the compound prescription of fugitive glucose and long-acting starch. Earlier fermentation is to grow mycelium, and it is main for improving biological content, therefore with the addition of the glucose carbon source that thalline is preferentially selected in culture medium, breeds rapidly; The fermentation later stage to improve the content of purpose product, and therefore carbon source provides thalline relatively to utilize slower starch, and adds appropriate amount of starch enzyme in the medium, controls thalline quantity at proper size, not the outer sugar of the raw metabolite born of the same parents of pregnancy ceased.
There is provided practicable technical scheme for industrial-scale production, solve and be amplified to the technical barrier run in industrialized production by lab scale.
In order to provide born of the same parents the yield of outer sugar, the present invention optimizes the formula of fermentation medium, have employed compound prescription for carbon source part, both ensure that the Biomass of early stage thalline, ensures again the yield of later stage metabolite;
The employing present invention according to the market demand and product standard, can prepare the polysaccharide of the high-quality of different content, and polyoses content can 20 50%.
Detailed description of the invention
The present invention is further illustrated below by way of specific embodiment. But the detail of embodiment is only used for explaining the present invention, should not be construed as limited overall technical solution.
The crow spirit ginseng bacterium that following example use is bought in institute of microbiology of Chinese Academy of Sciences common micro-organisms center, and strain coding CGMCCNO.1038, for this strain bacterium, describes the concrete scheme of the present invention in detail. But the inventive method is common to all of crow spirit ginseng bacterium, is not limited in this bacterial strain.
Embodiment 1
Industrialization deep layer liquid fermentation of the present invention produces the method for crow spirit ginseng extracellular polysaccharide and carries out in the steps below:
1, Wu Ling joins the preparation of fermentation liquid: by inoculum concentration 1%, the crow spirit ginseng shaking flask bacterial strain activated is inoculated in seed culture medium (300L), wherein seed culture medium consists of glucose 2%, peptone 0.2%, soybean cake powder 1%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, zinc sulfate 0.10%, Oleum Glycines 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～28 DEG C of shaken cultivation 4 days are that get Wu Ling joins primary seed solution; Primary seed solution is accessed secondary seed medium (3000L) by inoculum concentration 15%, wherein secondary seed medium consists of glucose 2%, peptone 0.2%, soybean cake powder 1%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, zinc sulfate 0.10%, defoamer 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, cultivates 3 days i.e. get Wu Ling for 26～28 DEG C and joins secondary seed solution; Secondary seed solution is accessed fermentation medium (7500L) by inoculum concentration 18%, wherein fermentation medium consists of glucose 2%, starch 2%, peptone 0.3%, soybean cake powder 1.5%, potassium dihydrogen phosphate 0.2%, Magnesium sulfate heptahydrate 0.2%, zinc sulfate 0.10%, Oleum Glycines 0.03%, thermostable α-amylase (Zaozhuang outstanding person promise enzyme biology company limited, 20000u/ml) 0.002% (v/v), regulates pH value 5.5; Ventilating ratio is 1:0.5vvm, tank pressure 0.03Mpa, cultivates 4 days for 26～28 DEG C, ferments to fermentation liquid and has peat-reek, fungus ball to roll up, and filtrate is muddy; Reducing sugar is down to minimum 0.3% and slightly fluctuates; Microscopy mycelia is in small, broken bits, and without living contaminants, namely get Wu Ling joins fermentation liquid 8850Kg.
2, Wu Ling joins separation of fermentative broth: be easily separated by horizontal screw centrifuge by above-mentioned crow spirit ginseng fermentation liquid 8850Kg, rotating speed controls at 3000r/min, disengaging time is at 2h, obtaining Wu Ling and join the wet mycelium (water content is 80%) of filtrate 8000Kg and 660Kg, wet mycelium directly cold drying can pulverize prepared fermentation crow spirit ginseng mycopowder.
4, two grade low-temp concentration: the crow spirit ginseng filtrate 8000Kg vacuum of previous step gained is transferred to 3t double effect evaporator, it is 0.085MPa in vacuum, thickening temperature is be concentrated into proportion (the 50 DEG C of heat are surveyed) concentrated solution that is 1.20 under 65 DEG C of conditions, concentrated solution vacuum is transferred to 1.5t single-action ball-type concentrator again, being 0.09MPa in vacuum, thickening temperature is continue under 60 DEG C of conditions to be concentrated into the concentrated pulp 280kg that proportion is 1.30 (50 DEG C of heat are surveyed).
5, precipitate with ethanol, drying: in the Alcohol-settling tank fill 280kg concentrated pulp add weight 1000kg 95% ethanol, detect mixed liquor alcoholic strength 72% (v/v) with alcohol meter, stand 12h, bottom sediment separation vacuum drying oven is dried, temperature 60 C, vacuum 0.09MPa;Dry 15h and obtain born of the same parents outer sugar 100kg. Detection moisture is 3.5%, and polyoses content is 33%.
Embodiment 2
1, Wu Ling joins the preparation of fermentation liquid: by inoculum concentration 1.5%, the crow spirit ginseng shaking flask bacterial strain activated is inoculated in seed culture medium (300L), wherein seed culture medium consists of glucose 2%, peptone 0.2%, yeast powder 1%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, zinc sulfate 0.10%, Oleum Brassicae campestris 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, 26～28 DEG C of shaken cultivation 3 days are that get Wu Ling joins primary seed solution; Primary seed solution is accessed secondary seed medium (3000L) by inoculum concentration 15%, wherein secondary seed medium consists of glucose 2%, peptone 0.2%, soybean cake powder 1%, potassium dihydrogen phosphate 0.3%, Magnesium sulfate heptahydrate 0.15%, zinc sulfate 0.10%, defoamer 0.02%, regulate pH value 5.5, ventilating ratio is 1:0.3vvm, tank pressure 0.03Mpa, cultivates 3 days i.e. get Wu Ling for 26～28 DEG C and joins secondary seed solution; Secondary seed solution is accessed fermentation medium (15000L) by inoculum concentration 18%, wherein fermentation medium consists of glucose 1.8%, starch 2.2%, peptone 0.3%, soybean cake powder 2%, potassium dihydrogen phosphate 0.2%, Magnesium sulfate heptahydrate 0.2%, zinc sulfate 0.10%, Oleum Brassicae campestris 0.03%, thermostable α-amylase (Zaozhuang outstanding person promise enzyme biology company limited, 20000u/ml) 0.002% (v/v), regulates pH value 5.8; Ventilating ratio is 1:0.3vvm, tank pressure 0.04Mpa, cultivates 4 days for 26～28 DEG C, ferments to fermentation liquid perfume (or spice) and has peat-reek, fungus ball to roll up, and filtrate is muddy; Reducing sugar is down to minimum 0.4% and slightly fluctuates; Microscopy mycelia is in small, broken bits, and without living contaminants, both get Wu Ling joined fermentation liquid 17700Kg.
2, Wu Ling joins the separation of fermentation liquid: be easily separated by horizontal screw centrifuge by above-mentioned crow spirit ginseng fermentation liquid 17700Kg, obtaining Wu Ling and join the wet mycelium (water content is 77%) of filtrate 16250Kg and 1180Kg, wet mycelium directly cold drying can pulverize prepared fermentation crow spirit ginseng mycopowder. Concrete technology condition is: rotating speed controls at 3200r/min, and disengaging time is at 4.5h.
4, two grade low-temp concentration: the crow spirit ginseng filtrate 16250Kg vacuum of previous step gained is transferred to 3t double effect evaporator, it is 0.09MPa in vacuum, thickening temperature is be concentrated into proportion (the 50 DEG C of heat are surveyed) concentrated solution that is 1.18 under 63 DEG C of conditions, concentrated solution vacuum is transferred to 1.5t single-action ball-type concentrator again, being 0.09MPa in vacuum, thickening temperature is be concentrated into the concentrated pulp 500kg that proportion is 1.35 (50 DEG C of heat are surveyed) under 65 DEG C of conditions.
5, precipitate with ethanol, drying: in the Alcohol-settling tank fill 500kg concentrated pulp add weight 2200kg 95% ethanol, detect mixed liquor alcoholic strength 70% (v/v) with alcohol meter, stand 10h, bottom sediment separation vacuum drying oven is dried, temperature 65 DEG C, vacuum 0.09MPa; Dry 18h and obtain born of the same parents outer sugar 210kg. Detection moisture is 4.5%, and polyoses content is 30%.
Embodiment 3
For better verifying that the compound prescription of fugitive glucose and long-acting starch in the present invention produces the superiority of crow spirit gracilis polysaccharide in industrialization, do again several contrast test. Test only have adjusted carbon source glucose in liquid fermentation medium or (with) consumption of starch, comparative example 1 does not contain starch, concentration of glucose is 1.5%; Not containing glucose in comparative example 2, starch concentration is 2.5%, and in comparative example 3, glucose content is 2.5%, and content of starch is 0.5%;The all same present invention of all the other conditions, result of the test is in Table 1:
Table 1
Claims (6)
1. the method preparing crow spirit ginseng fermentation liquid, it is characterized in that the method comprises the steps: that the liquid fermentation medium after crow spirit ginseng seed culture fluid is accessed sterilizing carries out deep layer liquid fermentation and obtains Wu Ling ginseng fermentation liquid, the inoculum concentration of described deep layer liquid fermentation is 10 ~ 20%v/v, ventilating ratio is 1:0.3-1.0vvm, tank pressure 0.02～0.05Mpa, cultivates 3 ~ 5 days at 22 ~ 30 DEG C; Described liquid fermentation medium formula is: glucose 1-3%, starch 1-3%, peptone 0.1-0.5%, soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, zinc sulfate 0.1-0.5%, defoamer 0.01-0.05%, 20000u/ml Thermostable α-Amylase 0.001-0.007%v/v, regulate pH value 5.0-6.5.
2. the method for preparation crow spirit ginseng fermentation liquid according to claim 1, it is characterised in that described crow spirit ginseng seed culture fluid is cultivated by two grades and obtained, and concrete grammar is as follows:
1) first order seed is cultivated: activated shaking flask strain accesses the liquid seed culture medium after sterilizing, inoculum concentration is 0.1 ~ 3%v/v, and ventilating ratio is 1:0.3-0.7vvm, tank pressure 0.02～0.05Mpa, cultivate 3 ~ 5 days at 20 ~ 30 DEG C, obtain Wu Ling and join first order seed culture fluid;
2) secondary seed is cultivated: the first order seed culture fluid of step 1) gained accesses the liquid seed culture medium after sterilizing, inoculum concentration is 10 ~ 20%v/v, and ventilating ratio is 1:0.3-1.0vvm, tank pressure 0.02～0.05Mpa, cultivate 2 ~ 4 days at 22 ~ 30 DEG C, obtain Wu Ling and join secondary seed culture fluid.
3. the method for preparation crow spirit ginseng fermentation liquid according to claim 2, it is characterized in that described liquid seed culture medium is: glucose 2-4%, peptone 0.1-0.5%, soybean cake powder 1-3%, potassium dihydrogen phosphate 0.1-0.5%, Magnesium sulfate heptahydrate 0.1-0.5%, zinc sulfate 0.1-0.5%, defoamer 0.01-0.05%, regulate pH value 5.0-6.5.
4. the extracting method of a crow spirit ginseng extracellular polysaccharide, it is characterised in that the method comprises the steps:
1) separate: the crow spirit ginseng fermentation liquor of claim 1 gained is separated, obtains Wu Ling and join supernatant;
2) concentration: being undertaken the filtrate of step 1) gained adopting two grade low-temp concentrations, obtaining concentration proportion is the concentrated pulp of 1.25～1.40;
3) precipitate with ethanol, drying: add concentrated pulp weight 3-5 times amount 95% ethanol, with alcohol meter detect mixed liquor alcoholic strength be 65-80%v/v, stand 8-16h, precipitate is carried out vacuum drying, temperature 50-75 DEG C, vacuum is not less than 0.085MPa; Drying to dried object moisture is 3～8%, and wherein moisture unit is g/100g, obtains the outer sugar of born of the same parents.
5. the extracting method of crow spirit ginseng extracellular polysaccharide according to claim 4, is characterized in that the separation described in step 1) adopts horizontal screw centrifuge to carry out, and rotating speed controls at 2500-3500r/min.
6. the extracting method of crow spirit ginseng extracellular polysaccharide according to claim 4, it is characterized in that step 2) described in two grade low-temps concentration be: one-level adopt double effect evaporator concentration, it is 1.15-1.20 that fermentation liquid is concentrated to proportion, two grades adopt haplo-effect concentrator concentration, continue to be concentrated to the concentrated pulp that proportion is 1.25～1.40 by the concentrated solution that proportion is 1.15-1.20; Concentration process all requires to control temperature not higher than 75 DEG C, and vacuum is not less than 0.085MPa.
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