CN102217487B - Method for producing mycelia of Antrodia camphorata through deep liquid state fermentation - Google Patents
Method for producing mycelia of Antrodia camphorata through deep liquid state fermentation Download PDFInfo
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Abstract
The invention relates to the field of fermentation engineering, in particular to a method for producing mycelia of Antrodia camphorata through deep liquid state fermentation, which comprises the following steps of strain slant culture, seed strain culture and fermentation culture; during the step of seed strain culture, the stains obtained through the slant culture in step one are further cultured to obtain secondary seed strains by liquid shaking culture, enlarging culture after liquid shaking culture, primary seed strain culture and secondary seed strain culture; during the fermentation culture, the secondary seed strains obtained in step two are transferred to a fermentation tank filled with a fermentation culture medium to implement deep liquid fermentation to obtain the mycelia of Antrodia camphorate. The method for producing mycelia of Antrodia camphorata through deep liquid state fermentation is different from common laboratory preparation methods, has the advantages of short period, high yield, high content of biological active substances, simple technique, controllable process and low cost, and is suitable for industrially producing high quality mycelia of Antrodia camphorata in large scale.
Description
Technical field
The present invention relates to field of fermentation engineering, relate in particular to a kind of method of producing mycelia of Antrodia camphorata through deep liquid state fermentation.
Background technology
Camphor tree sesame (Antrodia cinnamomea) claims again mushroom, blood glossy ganoderma etc. in the Antrodia camphorata, camphor tree mushroom, camphor tree, belongs to Basidiomycotina, Hymenomycetes, Aphyllophorales, many Cordycepps, Antrodia and belongs to rare medicinal fungi.Only be grown in the rotten hollow inwall of Cinnamomum kanahirai hay tree between the height above sea level 450-2000 rice of TaiWan, China mountain area, have strong yellow camphor tree smell.The camphor tree sesame contains several physiological active substances, such as polyose, triterpene compound, superoxide-dismutase, adenosine, small protein (containing immune protein), VITAMIN, steroid, xylogen, unsaturated longer chain fatty acid, ergosterol, trace element germanium etc., lectin, amino acid, Antrodia acid etc., wherein polysaccharide and triterpenoid are its main physiologically active substances.The camphor tree sesame has immunomodulatory, and is anticancer, detoxify and promote the subsdence of swelling, tranquilizing and allaying excitement, the promoting blood circulation and removing blood stasis and gentleness function such as long-pending that disappears.Taiwan stomachache, cancer, the hepatitis etc. of also being used for the treatment of among the people.
Camphor tree sesame natural growthing condition is harsh, and poor growth also only limits to annual June vegetative period to October, and natural production is rare.In addition illegal businessman's coyoting denudation causes soaring all the way of wild camphor tree sesame resource critical shortage and price.For remedying the scarcity of wild camphor tree sesame resource, protect forest resources, need research as how manual type cultivation camphor tree sesame and then a large amount of production badly.At present, the cultural method of Antrodia Camphorata mycelium comprises linden cultivation, solid culture and liquid culture etc.Linden is cultivated and solid culture all exists culture cycle long, easily pollutes, and is difficult for obtaining camphor tree sesame sporophore, and the camphor tree sesame of acquisition is difficult to be separated, and cost is high, is difficult for the problems such as scale operation.Adopt biotechnological means to utilize the liquid fermentation and culture Antrodia Camphorata mycelium, and to utilize mycelium and broth extraction bioactive ingredients be the present method of most economical, environmental protection.For addressing the above problem, the liquid fermentation culturing method of demanding providing a kind of suitable large-scale industrialized production urgently and guaranteeing Antrodia Camphorata mycelium bioactive ingredients content.
Summary of the invention
For addressing the above problem, the object of the present invention is to provide a kind of method of producing mycelia of Antrodia camphorata through deep liquid state fermentation, the method can be produced Antrodia Camphorata mycelium fast, in a large number.
For achieving the above object, technical scheme of the present invention is:
A kind of method of producing mycelia of Antrodia camphorata through deep liquid state fermentation may further comprise the steps:
1) slant culture of bacterial classification: bacterial classification accessed in the slant medium cultivate, culture temperature 22-33 ℃, cultivate 1-4 week, obtain slant strains;
2) seed spawn culture, the bacterial classification that the step 1) slant culture is obtained further carries out seed culture, comprising: liquid shaking bottle cultivation, liquid shaking bottle be enlarged culturing, first order seed spawn culture and secondary seed spawn culture again, obtains the secondary seed bacterial classification;
3) fermentation culture is with step 2) the secondary seed bacterial classification that obtains is transferred to and carries out deep layer liquid state fermentation in the fermentor tank that fermention medium is housed, and obtains Antrodia Camphorata mycelium;
Described step 2) be specially:
A) slant strains that step 1) is obtained is transferred in the shake-flask culture base, 22-33 ℃ of shake-flask culture, and rotating speed 80-180rpm cultivated 3-9 days, obtained the first shaking flask bacterial classification;
B) the shaking flask strain transfer that step a) is obtained carries out again enlarged culturing in the shaking flask that seed culture medium is housed, inoculum size is 8-12%; Culture temperature 22-33 ℃, rotating speed 80-180rpm cultivated 3-9 days, obtained the second shaking flask bacterial classification;
C) the shaking flask strain transfer that step b) is obtained carries out the first order seed cultivation in the first class seed pot that seed culture medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 cultivated 3-9 days, obtained the first order seed bacterial classification;
D) the first order seed strain transfer that step c) is obtained carries out the secondary seed cultivation in the secondary seed tank that seed culture medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 cultivated 3-9 days, obtained the secondary seed bacterial classification;
Described step 3) is specially: with step 2) the secondary seed bacterial classification that obtains is transferred to and carries out deep layer liquid state fermentation in the fermentor tank that fermention medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 fermented 3-12 days, obtained Antrodia Camphorata mycelium;
The prescription of described fermention medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
Further, the ratio of culture volume and shaking flask volume is 2:5 in the described shaking flask.
Further, the volume of described first class seed pot is 10-500L, and the fermention medium that is equipped with in described first class seed pot should be 5L-400L mutually; The volume of described secondary seed tank is 100-5000L, and the fermention medium that is equipped with in described secondary seed tank should be 50L-4000L mutually.
Further, the prescription of described slant medium is: 1%-4% glucose, and the 1%-4% malt extract, the 0.01%-0.2% peptone, agar 1%-4%, in g/ml, pH3.0-7.0.
Further, the prescription of described shake-flask culture base is: 1%-4% glucose, and the 1%-4% malt extract, the 0.01%-0.2% peptone, in g/ml, pH3.0-7.0.
Further, the prescription of described seed culture medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
Further, the volume of fermentor tank is 1000-50000L, and the fermention medium that is equipped with in described fermentor tank should be 500L-40000L mutually.
The method of producing mycelia of Antrodia camphorata through deep liquid state fermentation of the present invention has following advantage:
1) cycle short, need 16-52 days, the shortlyest only need 16 days, compare with solid culture, culture cycle shortens greatly;
2) output is high, and yield is 2%, in 40 tons of fermention mediums, once can the production dry weight is 800 kilograms mycelium;
3) after tested, polysaccharide content is at 5%-8% in the mycelium, and triterpenes content is about 5%, and with the solid-phase ratio, bioactive substance content is suitable;
4) technique is simple, and process is controlled;
5) cost is low;
The method of the invention is different from the common laboratory preparation method, is suitable for large-scale industrialization and produces high-quality Antrodia Camphorata mycelium.
Embodiment
The method of 1 one kinds of producing mycelia of Antrodia camphorata through deep liquid state fermentations of embodiment
Substratum:
The prescription of slant medium is: 1%-4% glucose, and the 1%-4% malt extract, the 0.01%-0.2% peptone, agar 1%-4%, in g/ml, pH3.0-7.0.
Screening formulation is: 2% glucose, and 2% malt extract, 0.1% peptone, agar 2%, in g/ml, pH5.0.
The prescription of shake-flask culture base is: 1%-4% glucose, and the 1%-4% malt extract, the 0.01%-0.2% peptone, in g/ml, pH3.0-7.0.
Screening formulation is: 2% glucose, and 2% malt extract, 0.1% peptone, in g/ml, pH5.0.
The prescription of seed culture medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
Screening formulation is: 4% wheat bran juice, and 4% W-Gum, 0.1% sal epsom, in g/ml, pH5.0.
The prescription of fermention medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
Screening formulation is: 4% wheat bran juice, and 4% W-Gum, 0.1% sal epsom, in g/ml, pH5.0.
The whole substratum of the present embodiment adopt screening formulation.
The present embodiment adopts camphor tree sesame bacterium bacterial classification identical with Chinese invention patent 01115869.7 described CGMCCNo.0575.
Specifically may further comprise the steps:
1) slant culture of bacterial classification: bacterial classification accessed in the slant medium cultivate, culture temperature 22-33 ℃, cultivated for 1 week, obtain slant strains;
2) seed spawn culture, the bacterial classification that the step 1) slant culture is obtained further carries out seed culture, comprising: liquid shaking bottle cultivation, liquid shaking bottle be enlarged culturing, first order seed spawn culture and secondary seed spawn culture again, obtains the secondary seed bacterial classification;
Described step 2) be specially:
A) slant strains that step 1) is obtained is transferred in the shake-flask culture base, and the ratio of shake-flask culture base and shaking flask volume is 2:5,26 ℃ of shake-flask culture, and rotating speed 120rpm cultivated 5 days, obtained the first shaking flask bacterial classification;
B) the shaking flask strain transfer that step a) is obtained carries out again enlarged culturing in the shaking flask that seed culture medium is housed, inoculum size is 10%; The ratio of seed culture medium and shaking flask volume is 2:5; 26 ℃ of culture temperature, rotating speed 120rpm cultivated 5 days, obtained the second shaking flask bacterial classification;
C) the shaking flask strain transfer that step b) is obtained carries out the first order seed cultivation in the first class seed pot that seed culture medium is housed, inoculum size is 10%, 26 ℃ of culture temperature, tank pressure 0.03Mpa, stir speed (S.S.) 170rpm, air flow 1:1, cultivated 5 days, and obtained the first order seed bacterial classification;
D) the first order seed strain transfer that step c) is obtained carries out the secondary seed cultivation in the secondary seed tank that seed culture medium is housed, inoculum size is 10%, 26 ℃ of culture temperature, tank pressure 0.03Mpa, stir speed (S.S.) 170rpm, air flow 1:1, cultivated 5 days, and obtained the secondary seed bacterial classification;
The volume of described first class seed pot is 500L, and the fermention medium that is equipped with in described first class seed pot should be 400L mutually; The volume of described secondary seed tank is 5000L, and the fermention medium that is equipped with in described secondary seed tank should be 4000L mutually.
3) fermentation culture, with step 2) the secondary seed bacterial classification that obtains is transferred to and carries out deep layer liquid state fermentation in the fermentor tank that fermention medium is housed, inoculum size is 10%, and the volume of fermentor tank is 50000L, and the fermention medium that is equipped with in described fermentor tank should be 40000L mutually.
26 ℃ of culture temperature, tank pressure 0.03Mpa, stir speed (S.S.) 170rpm, air flow 1:1 fermented 6 days, obtained Antrodia Camphorata mycelium.
The method of embodiment 1 described producing mycelia of Antrodia camphorata through deep liquid state fermentation,
1) cycle short, need 16-52 days, the shortlyest only need 33 days, compare with solid culture, culture cycle shortens greatly;
2) output is high, and yield is 2%, in 40 tons of fermention mediums, once can the production dry weight is 800 kilograms mycelium;
3) after tested, polysaccharide content is at 5%-8% in the mycelium, and triterpenes content is about 5%, and with the solid-phase ratio, bioactive substance content is suitable;
4) technique is simple, and process is controlled;
5) cost is low;
The method of the invention is different from the common laboratory preparation method, is suitable for large-scale industrialization and produces high-quality Antrodia Camphorata mycelium.
Claims (7)
1. the method for a producing mycelia of Antrodia camphorata through deep liquid state fermentation may further comprise the steps:
1) slant culture of bacterial classification: bacterial classification accessed in the slant medium cultivate, culture temperature 22-33 ℃, cultivate 1-4 week, obtain slant strains;
2) seed spawn culture, the bacterial classification that the step 1) slant culture is obtained further carries out seed culture, comprising: liquid shaking bottle cultivation, liquid shaking bottle be enlarged culturing, first order seed spawn culture and secondary seed spawn culture again, obtains the secondary seed bacterial classification;
3) fermentation culture is with step 2) the secondary seed bacterial classification that obtains is transferred to and carries out deep layer liquid state fermentation in the fermentor tank that fermention medium is housed, and obtains Antrodia Camphorata mycelium;
Described step 2) be specially:
A) slant strains that step 1) is obtained is transferred in the shake-flask culture base, 22-33 ℃ of shake-flask culture, and rotating speed 80-180rpm cultivated 3-9 days, obtained the first shaking flask bacterial classification;
B) the shaking flask strain transfer that step a) is obtained carries out again enlarged culturing in the shaking flask that seed culture medium is housed, inoculum size is 8-12%; Culture temperature 22-33 ℃, rotating speed 80-180rpm cultivated 3-9 days, obtained the second shaking flask bacterial classification;
C) the shaking flask strain transfer that step b) is obtained carries out the first order seed cultivation in the first class seed pot that seed culture medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 cultivated 3-9 days, obtained the first order seed bacterial classification;
D) the first order seed strain transfer that step c) is obtained carries out the secondary seed cultivation in the secondary seed tank that seed culture medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 cultivated 3-9 days, obtained the secondary seed bacterial classification;
Described step 3) is specially: with step 2) the secondary seed bacterial classification that obtains is transferred to and carries out deep layer liquid state fermentation in the fermentor tank that fermention medium is housed, inoculum size is 8-12%, culture temperature 22-33 ℃, tank pressure 0.01-0.05Mpa, stir speed (S.S.) 120-220rpm, air flow 1:0.2-2.0 fermented 3-12 days, obtained Antrodia Camphorata mycelium;
The prescription of described fermention medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
2. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1 is characterized in that, the ratio of culture volume and shaking flask volume is 2:5 in the described shaking flask.
3. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1 is characterized in that, the volume of described first class seed pot is 10-500L, and the fermention medium that is equipped with in described first class seed pot should be 5L-400L mutually; The volume of described secondary seed tank is 100-5000L, and the fermention medium that is equipped with in described secondary seed tank should be 50L-4000L mutually.
4. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1 is characterized in that, the prescription of described slant medium is: 1%-4% glucose, the 1%-4% malt extract, 0.01%-0.2% peptone, agar 1%-4%, in g/ml, pH3.0-7.0.
5. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1 and 2 is characterized in that, the prescription of described shake-flask culture base is: 1%-4% glucose, and the 1%-4% malt extract, the 0.01%-0.2% peptone, in g/ml, pH3.0-7.0.
6. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1 is characterized in that, the prescription of described seed culture medium is: 1%-7% wheat bran juice, and the 1%-7% W-Gum, 0.01%-0.2% sal epsom, in g/ml, pH2.0-7.0.
7. the method for a kind of producing mycelia of Antrodia camphorata through deep liquid state fermentation according to claim 1, it is characterized in that: the volume of fermentor tank is 1000-50000L, and the fermention medium that is equipped with in described fermentor tank should be 500L-40000L mutually.
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| CN102687640A (en) * | 2012-04-23 | 2012-09-26 | 浙江省海洋开发研究院 | Antrodia camphorata fungi liquid submerged culture method and antrodia camphorata fungi polysaccharide extraction method |
| CN103125270A (en) * | 2013-02-28 | 2013-06-05 | 深圳市仁泰生物科技有限公司 | High-yield antrodia cinnamomea mycelium fermentation method for triterpenoids |
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