CN101822170A - Method for producing Anrodia camphorata mycelia based on solid-state surface culture - Google Patents
Method for producing Anrodia camphorata mycelia based on solid-state surface culture Download PDFInfo
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Abstract
The invention discloses a method for producing Anrodia camphorata mycelia based on solid-state surface culture, and belongs to the field of bioengineering. The method comprises the following steps of: screening components of a culture medium of the Anrodia camphorate and optimizing the proportion of the cereal raw material in the culture medium so that the culture medium is firmly adsorbed to the inner wall of a culture vessel or the culture surface to bring convenience to the surface culture of the Anrodia camphorata mycelia; inoculating the Anrodia camphorata mycelia in the culture surface of a reactor and culturing the Anrodia camphorata mycelia in the environment of certain temperature and moisture; feeding the culture medium every a certain time in batch during the surface culture so as to timely replenish the nutrient components required by the growth of the Anrodia camphorata mycelia and making the growth environment of the Anrodia camphorata mycelia close to the natural culture environment until a large amount of mycelia or large colony is formed; and quickly drying the Anrodia camphorata culture at a low temperature after the culture. The method of the invention makes the growth environment of the Anrodia camphorata mycelia close to that of the wild Anrodia camphorate and improves the productivity of the Anrodia camphorata culture by timely feeding the culture medium in batch, so that the obtained Anrodia camphorata culture has high content of active ingredient and closer efficacy to the wild fruit body.
Description
Technical field
The invention provides the method that Antrodia camphorata mycelium is cultivated on a kind of surface fast, belong to bioengineering field.
Background technology
Camphor tree sesame (Anrodia camphorata), wild rice or Antrodia camphorata, Cinnamomum kanahirai hay mushroom in another name camphor tree mushroom, camphor tree wild rice, the camphor tree cave.The camphor tree sesame has toxin expelling, the oil that purifies the blood, detoxifcation protect the liver, the whole intestines of tonifying kidneys, strengthening heart, beneficial stomach, analgesia are antibiotic, reinforced immunological, disallergization, anticancer disease, eliminate magical effect such as tumour.
The fungi of camphor tree sesame Shi Sheng Long on the distinctive Cinnamomum kanahirai hay tree in Taiwan belongs to aptychus Zoopagales, Polyporaceae.The growth of camphor tree sesame is very slow, also only limits to annual June vegetative period to October, and it is quite long to form the time that produces fruit body, so the natural production rareness.Camphor tree sesame fruit body is perennial, be close to and be born in wood surface, fruit body nascent cerise, gradually long white, the light red brown, filbert or khaki of becoming, aging back becomes mitriform, the cot yellowish-brown is to black, crust has tangible ring-shaped structure, and quality is extremely hard, changeableization of form, tabular, mitriform, horse-hof shape or tower shape are arranged, generally with tabular in the majority.This explanation camphor tree sesame bacterium is suitable for superficial growth.Fresh or dried camphor tree sesame fruit body has strong fragrant camphor tree flavor, is rich in special physiological activators such as triterpene compound in the camphor tree sesame fermentating metabolism product.
Wild camphor tree sesame is close to disappearance, and the fruit body artificial culture of camphor tree sesame need be felled the Cinnamomum kanahirai hay tree as culture matrix and carrier, so can not under the prerequisite of a large amount of felling Cinnamomum kanahirai hay trees, adopt artificial cultivation mode (linden cultivation) to produce camphor tree sesame fruit body, and owing to adopt the linden cultivation, the growth cycle of camphor tree sesame is long, is not suitable for suitability for industrialized production.In order to make the edible universalness of camphor tree sesame, protection Cinnamomum kanahirai hay tree resource needs existing camphor tree sesame artificial culture technology is reformed, and has the camphor tree sesame product that more approaches natural camphor tree sesame composition and effect with exploitation.At present, camphor tree sesame artificial cultivation method has liquid submerged-culture method and solid medium cultivation.
Liquid deep layer is cultivated Antrodia camphorata mycelium, though can shorten cultivation cycle greatly, its physiologically active and active ingredient and natural fruit body differ greatly; Especially because ventilation is cultivated, distinctive fragrance component loss totally in the camphor tree sesame culture.
Solid-state cultivation of camphor tree sesame bacterium and the growing environment of wild camphor tree sesame are comparatively approaching.The kind of triterpenoid is obviously more than liquid cultured products in the solid-state culture of camphor tree sesame.Present solid medium cultivation is to add the solid state fermentation raw material in space bag or triangular flask, the material bed thickness of piling up has several cm thicks, insert camphor tree sesame seed liquor (or spore), incubation time is generally some months or half a year, microbiological contamination easily, the very difficult fruit body of cultivating into, and functional components and wild fruit body still have tangible difference in this solid-state culture, and the feature of this solid medium cultivation is the thick-layer cultivation.
One of solid-state culturing purposes of the present invention is to wish a large amount of acquisition Antrodia camphorata myceliums and culture, and utilizes synthetic in a large number distinctive metabolite of camphor tree sesame of thalline and fragrance component.Wild camphor tree sesame belongs to aerobic growth, the thalline of camphor tree sesame is difficult to be penetrated into culture matrix inside, the camphor tree sesame is actually constantly extends its mycelium on the growth substrate surface, and forms its fruit body gradually, so the feature that the camphor tree sesame is cultivated is that surface area as much as possible need be provided.Therefore characteristic of the present invention just is to simulate the epontic mode of natural camphor tree sesame, in round, increase the attachment surface of culture as far as possible, make camphor tree sesame bacterium in the growth fast of the surface of medium, form abundant mycelium, thereby make Antrodia camphorata mycelium produce the distinctive metabolite of more camphor tree sesames.
Camphor tree sesame culture is under high temperature acts on for a long time, and its physiological activator and product appearance can be had a strong impact on.In the aftertreatment technology of the production method of camphor tree sesame culture, mainly adopt the large quantity of moisture in the desivac removal culture, at present to reach the purpose of long preservation.Though the desivac effect is good, energy consumption is very big, and needs expensive freeze drying equipment.Be starved of a kind of can dryly on a large scale the processing, can save the treatment process of the energy again.
Summary of the invention
The object of the invention is cultivated Antrodia camphorata mycelium for quick exhibiting high surface, and makes this mycelium and metabolite thereof approach wild Antrodia camphorata mycelium.
Technical scheme of the present invention: a kind of method of cultivating the production Antrodia camphorata mycelium based on solid state surface,
1. bacterial classification: the bacterial classification of employing is camphor tree sesame bacterium (Anrodia camphorata), is provided by Fumao Edible Mushroom Co., Ltd., Shanghai of Taiwan-owned enterprise.
The related bacterial classification of this patent can be with reference to following document:
[Hu Ou, etc.Medicinal health value of camphor tree sesame and Application and Development [J]. subtropical plant science, 2006,35 (4): 75-78];
[Chen Tiqiang, Fang Zhongyu.Taiwan rare medicinal fungus camphor tree sesame and parasitic seeds Cinnamomum kanahirai hay [J]. Fujian agriculture science and technology, 2003,1:41-42]
2. seed culture
Culture medium prescription g/L: glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, citric acid 0.1～5; Soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L;
The seed culture condition: 20～30 ℃ of cultivation temperature, shaking speed 70～1600r/min, pH 3.0～7.0, and incubation time 2～10d makes seed liquor;
3. solid-state cultivation
Solid medium prescription: adopt food-grade cereal: rice, millet, glutinous rice, one or more fermentations in the wheat bran, container loading amount 60～180g/L, with medium cereal dry weight basis, the weight ratio of cereal and nutrient solution is 6: 1～1: 1; Nutrient solution g/L: glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L, pH 1.0～7.0; 121 ℃ of sterilization 10～40min;
This medium is suitable for camphor tree sesame thalline and grows fast on the surface.And can keep camphor tree sesame culture on the inner wall surface of incubator or other culture surface, firmly to adhere to.
Solid state surface cultural method: 20～35 ℃ of cultivation temperature, humidity 60%～90%, fermentation time 15～120d;
4. quick low temperature drying: cultured material scattered to be put in 10～60 ℃ of culturing room, oven dry 2～24h, and moisture content: below 8%, promptly get Antrodia camphorata mycelium, so both can the flash baking material, can reduce the bacterium of mixing again material is polluted.
The special camphor tree sesame of the present invention solid state surface cultured method comprises following content:
(1), the solid state surface cultural method in blake bottle: solid medium sterilization: several different cereal are pressed mixing in maximum, minimum 1: 4～4: 1 the proportion of weight ratio, 121 ℃ of sterilization 10～40min;
Inoculation: seed liquor and the 0.05～4mL/g nutrient solution of 0.05～4mL/g are connected on the solid medium of gone out bacterium and cooling;
Medium is covered with the measure on blake bottle surface: the certain angle that after the inoculation blake bottle is inclined upwardly, and the rolling blake bottle makes solid medium be covered with the blake bottle wall;
Cultivate: blake bottle is put in 20～35 ℃ of temperature, the culturing room of humidity 60%～90%, fermentation time is 15～120d;
Feed supplement: during cultivating 5～120d, add the solid medium for preparing every 3～5d stream, and make medium be covered with the surface, continue to cultivate, make the Antrodia camphorata mycelium thickening or form big bacterium colony.
Or (2), cultivate the cultural method of cultivating in the reactor with the sprinkling solid medium on the surface:
Reactor and coupling devices sterilization are cultivated in the surface: reactor and coupling devices are cultivated at 121 ℃ of sterilization 10～120min in the surface;
Medium sterilization: with different cereal in 1: 4～4: 1 ratio mixing of maximum, minimum weight ratio, 121 ℃ the sterilization 10～40min;
Inoculation: seed liquor and the 0.05～4mL/g nutrient solution of 0.05～4mL/g are connected on the medium of gone out bacterium and cooling;
Medium is covered with the measure that reactor is cultivated on the surface: will inoculate the back medium is cultivated reactor on the surface by pipeline and nozzles spray surface;
Cultivate: 20～35 ℃ of temperature, 15～120d ferments in the culturing room of humidity 60%～90%;
Feed supplement: during cultivating 15～120d, intermittently spray fresh solid medium every 3～5d, and make solid medium be covered with the surface, continue to cultivate, make the Antrodia camphorata mycelium thickening or form big bacterium colony.
Beneficial effect of the present invention: innovative point of the present invention is
1. camphor tree sesame solid medium prescription is more suitable in Antrodia camphorata mycelium on the surface fast and grow with the medium of this formulated vigorously, and keeps the solid-state culture of camphor tree sesame firmly attached to cultivating on the supporter.
2. the method for camphor tree sesame solid state surface cultured method and incubation fed-batch medium, this method can keep camphor tree sesame bacterium near growing fast in the natural camphor tree sesame growing environment and forming a large amount of mycelium and spore.
3. the flash baking method of camphor tree sesame bacterium culture.
Camphor tree sesame cultural method provided by the present invention, the spontaneous growth that more approaches wild camphor tree sesame environment can be provided, in incubation, but stream adds fresh culture, thereby reach the purpose of the production Antrodia camphorata mycelium of mass efficient, can improve and keep the content of the physiologically active ingredient of camphor tree sesame bacterium, and shorten the camphor tree sesame production cycle greatly.
Description of drawings
Fig. 1 bottle formula fermentation schematic diagram that rolls.(a) revolving bottle is sandwiched between two rods, and wherein a rod (shown in the solid arrow) is to drive rod; Another rod (shown in the dotted arrow) is the subordinate rod.(b) fermentation flask with cover.Fermentation flask is placed between two rods, and wherein a rod is to drive rod, can drive the fermentation flask rotation, and the material in the bottle stirs thereupon.This is similar to small-sized rotating drum type reactor.
Fig. 2 cultivates reactor in the surface.1, inoculation mouthful (medium flow adds mouthful or the filtrated air inlet), 2, switch valve, 3, two-sided culture plate, 4, shut off valve, 5, air cushion, 6, air bleeding valve, 7, motor, 8, the dismounting valve, 9, axle, 10, tank body.
2-A cultivates the reactor front view in the surface, and 2-B cultivates the reactor-side view in the surface.
Embodiment
Embodiment 1:(is based on the solid state surface cultural method in blake bottle)
Bacterial classification: camphor tree sesame bacterium (Anrodia camphorata).
Seed culture medium (g/L): glucose 20, Na
2HPO
40.5, MgSO
40.5, citric acid 0.5; Corn steep liquor 40mL/L, soybean hydrolyzate 20mL/L.
Seed culture condition: 28 ℃ of cultivation temperature, shaking speed 120r/m, pH nature, incubation time 4d.
Solid medium (g/L): adopt food-grade cereal (rice, millet, glutinous rice etc.) fermentation.Charge 60～180g/L, with the dry weight basis that adds materials, rice and the ratio of nutrient solution 6: 1～1: 1, nutrient solution (g/L): glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L, pH 1.0～7.0.121 ℃ of sterilization 20min.
Solid-state condition of culture: inoculum concentration: 0.05～4mL/g is put in 20～35 ℃ of temperature, the culturing room of humidity 60%～90%, and fermentation time is 15～120d;
Artificial rolling bottle method: the certain angle that after the inoculation blake bottle is inclined upwardly, and the rolling blake bottle makes solid medium be covered with the blake bottle wall; In incubation, stream adds fresh solid medium at regular intervals, and makes solid medium be covered with the surface, continues to cultivate, and makes the Antrodia camphorata mycelium thickening or forms big bacterium colony.
Machinery rolling bottle method: postvaccinal blake bottle is put on the rolling rod, and solid medium is covered with the blake bottle wall; In incubation, stream adds fresh solid medium at regular intervals, and makes solid medium be covered with the surface, continues to cultivate, and makes the Antrodia camphorata mycelium thickening or forms big bacterium colony.
Product oven dry: after fermentation is finished, directly blake bottle is opened wide and be put in 10～60 ℃ of incubators oven dry 2～24h, moisture content: get final product below 8%.
Embodiment 2 (adding solid-state culture method) to cultivate reactor and stream on the surface:
Bacterial classification: camphor tree sesame bacterium (Anrodia camphorata).
Seed culture medium (g/L): glucose 10-30, Na
2HPO
40.5, MgSO
40.5, citric acid 0.5; Corn steep liquor 40mL/L, soybean hydrolyzate 20mL/L.
The seed culture condition: cultivation temperature is 28 ℃, shaking speed 120r/m, pH nature, incubation time 4d.
Solid medium (g/L): different cereal (rice, millet, glutinous rice etc.) are pressed mixing in maximum, minimum 1: 4～4: 1 the proportion of weight ratio.With the dry weight basis that adds materials, rice and the ratio of nutrient solution 6: 1～1: 1, nutrient solution (g/L): glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L, pH 1.0～7.0.
Solid-state condition of culture: seed liquor and the 0.05～4mL/g nutrient solution of 0.05～4mL/g are connected on the medium of gone out bacterium and cooling, are put in 20～35 ℃ of temperature, the culturing room of humidity 60%～90%, fermentation time 15～120d.
Medium is covered with the measure that reactor is cultivated on the surface: reactor inner surface culture plate along with the axle rotating process in, inoculation back medium is sprayed on the surface of culture plate; Stream adds fresh solid medium at regular intervals, makes solid medium be covered with the surface of culture plate by the nozzle in the reactor, continues to cultivate, and makes the Antrodia camphorata mycelium thickening or forms big bacterium colony.
Product oven dry: after fermentation is finished, directly the surface is cultivated reactor and place 10～60 ℃ of culturing room, oven dry 2～24h, moisture: below 8%.
Claims (3)
1. a method of cultivating the production Antrodia camphorata mycelium based on solid state surface is characterized in that
1. bacterial classification: the bacterial classification of employing is camphor tree sesame bacterium (Anrodia camphorata), is provided by Fumao Edible Mushroom Co., Ltd., Shanghai of Taiwan-owned enterprise;
2. seed culture
Culture medium prescription g/L: glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, citric acid 0.1～5; Soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L;
The seed culture condition: 20～30 ℃ of cultivation temperature, shaking speed 70～1600r/min, pH 3.0～7.0, and incubation time 2～10d makes seed liquor;
3. solid-state cultivation
Solid medium prescription: adopt food-grade cereal: rice, millet, glutinous rice, one or more fermentations in the wheat bran, container loading amount 60～180g/L, with medium cereal dry weight basis, the weight ratio of cereal and nutrient solution is 6: 1～1: 1; Nutrient solution g/L: glucose 10～30, Na
2HPO
40.1～5, MgSO
40.1～5, soybean hydrolyzate 10～100mL/L, corn steep liquor 10～80mL/L, pH 1.0～7.0; 121 ℃ of sterilization 10～40min;
Solid state surface cultural method: 20～35 ℃ of cultivation temperature, humidity 60%～90%, incubation time 15～120d;
4. quick low temperature drying: cultured material scattered is put in 10～60 ℃ of culturing room oven dry 2～24h, moisture content: below 8%, promptly get Antrodia camphorata mycelium.
2. method of cultivating the production Antrodia camphorata mycelium based on solid state surface according to claim 1 is characterized in that described camphor tree sesame solid state surface cultural method, the solid state surface cultural method in blake bottle:
Solid medium sterilization: 121 ℃ of sterilization 10～40min;
Inoculation: seed liquor and the 0.05～4mL/g nutrient solution of 0.05～4mL/g are connected on the solid medium of gone out bacterium and cooling;
Medium is covered with the measure on blake bottle surface: the certain angle that after the inoculation blake bottle is inclined upwardly, and the rolling blake bottle makes solid medium be covered with the blake bottle wall;
Cultivate: blake bottle is put in 20～35 ℃ of temperature, the culturing room of humidity 60%～90%, fermentation time is 15～120d;
Feed supplement: during cultivating 5～120d, add the solid medium for preparing every 3～5d stream, and make medium be covered with the surface, continue to cultivate, make the Antrodia camphorata mycelium thickening or form big bacterium colony.
3. method of cultivating the production Antrodia camphorata mycelium based on solid state surface according to claim 1 is characterized in that described camphor tree sesame solid state surface cultural method, cultivates on the surface and cultivates in the reactor and the cultural method that sprays solid medium:
Reactor and coupling devices sterilization are cultivated in the surface: reactor and coupling devices are cultivated at 121 ℃ of sterilization 10～120min in the surface;
Medium sterilization: 121 ℃ of sterilization 10～40min;
Inoculation: seed liquor and the 0.05～4mL/g nutrient solution of 0.05～4mL/g are connected on the medium of gone out bacterium and cooling;
Medium is covered with the measure that reactor is cultivated on the surface: will inoculate the back medium is cultivated reactor on the surface by pipeline and nozzles spray surface;
Cultivate: 20～35 ℃ of temperature, 15～120d ferments in the culturing room of humidity 60%～90%;
Feed supplement: during cultivating 15～120d, intermittently spray fresh solid medium every 3～5d, and make solid medium be covered with the surface, continue to cultivate, make the Antrodia camphorata mycelium thickening or form big bacterium colony.
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| CN102047814A (en) * | 2010-10-25 | 2011-05-11 | 青岛农业大学 | Micro ventilation lime wood antrodia camphorate cultivation method |
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| CN1456661A (en) * | 2003-06-03 | 2003-11-19 | 江南大学 | Large scale zhangzhi fungus deep fermenting process |
| CN101151957A (en) * | 2006-09-26 | 2008-04-02 | 国鼎生物科技股份有限公司 | Culture method and uses of antrodia cinnamomea |
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| CN1456661A (en) * | 2003-06-03 | 2003-11-19 | 江南大学 | Large scale zhangzhi fungus deep fermenting process |
| CN101151957A (en) * | 2006-09-26 | 2008-04-02 | 国鼎生物科技股份有限公司 | Culture method and uses of antrodia cinnamomea |
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| CN102047814A (en) * | 2010-10-25 | 2011-05-11 | 青岛农业大学 | Micro ventilation lime wood antrodia camphorate cultivation method |
| CN103202460A (en) * | 2012-01-16 | 2013-07-17 | 张平 | Method for biological fermentation of silybum marianum by using edible fungi |
| CN103444430B (en) * | 2012-06-04 | 2015-07-22 | 慧普生技股份有限公司 | Culture method for Antrodia |
| CN103444430A (en) * | 2012-06-04 | 2013-12-18 | 慧普生技股份有限公司 | Culture method for Antrodia |
| CN102783633A (en) * | 2012-06-28 | 2012-11-21 | 江苏华绿生物科技股份有限公司 | Method for preparing solid fermentation powder of Antrodia camphorata |
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| CN102827895B (en) * | 2012-09-07 | 2014-04-02 | 江南大学 | Double-liquid-phase fermentation method of coupling in-situ extraction antrodia camphorate active product antrondin C |
| CN102872955B (en) * | 2012-09-29 | 2016-01-20 | 佛山市海天(高明)调味食品有限公司 | A kind of cultural method of microbe in solid state culture medium |
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| TWI580779B (en) * | 2012-11-21 | 2017-05-01 | 領先奈米製藥生技股份有限公司 | Solid matrix medium for antrodia cinnamomea and method of culturing antrodia cinnamomea |
| CN103918472A (en) * | 2013-01-15 | 2014-07-16 | 漳州美利德生物工程有限公司 | Antrodia camphorata basswood cultivation method |
| CN103918472B (en) * | 2013-01-15 | 2016-10-26 | 漳州美利德生物工程有限公司 | The cultivation basswood method of Antrodia Camphorata |
| TWI596210B (en) * | 2013-12-02 | 2017-08-21 | Medium for culture of Antrodia camphorata and culture method | |
| CN103907727A (en) * | 2014-03-25 | 2014-07-09 | 李明聪 | Making method of antrodia camphorate tea and antrodia camphorate tea made by adopting making method |
| CN105463038A (en) * | 2016-01-28 | 2016-04-06 | 厦门北化生物产业研究院有限公司 | Method for producing 4-Acetylantroquinonol B through fermentation |
| CN105463038B (en) * | 2016-01-28 | 2018-10-02 | 厦门北化生物产业研究院有限公司 | A kind of method of fermenting and producing 4-Acetylantroquinonol B |
| CN109220537A (en) * | 2017-04-27 | 2019-01-18 | 天明制药股份有限公司 | To improve the cultural method of triterpene compound content in inonotus obliquus |
| CN109220537B (en) * | 2017-04-27 | 2020-11-20 | 天明制药股份有限公司 | Culture method for increasing content of triterpenoids in inonotus obliquus |
| CN112956372A (en) * | 2021-03-11 | 2021-06-15 | 齐齐哈尔大学 | Method for cultivating lucid ganoderma |
| CN114902912A (en) * | 2022-06-01 | 2022-08-16 | 张少先 | Method for cultivating antrodia camphorata through sassafras-tree basswood |
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