CN104480158B - A kind of method for improving Chinese caterpillar fungus polysaccharide yield - Google Patents
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Abstract
The invention discloses a kind of method for improving Chinese caterpillar fungus polysaccharide yield, belong to bioengineering field.What the present invention was utilized be one plant separates the Hirsutella sinensis obtained from Cordyceps Sinensis From Tibet fructification（CJMCC No.9046）, utilize molasses（Sugar content 50%）, high F value oligopeptide, yam extract be used as culture medium, by stream plus the microbial technique means such as yam extract and intermittent stirring, mycelium powder is obtained after thalline is expanded into culture, filtering drying step by step, Chinese caterpillar fungus polysaccharide content is up to 13 14% after testing, far above wild cordyceps.Medium nutrient content economical rationality of the present invention, cultural method, without particular/special requirement, and reduces power consumption and reduces production cost to equipment, can be good at improving the yield of Chinese caterpillar fungus polysaccharide, further develops and lay a good foundation for the bacterial strain.
Description
Technical field
The present invention relates to bioengineering field, and in particular to a kind of method of raising Chinese caterpillar fungus polysaccharide yield.
Background technology
Cordyceps sinensis also known as cordyceps sinensis, are that aweto parasitizes Hepialus larva in mesophorbium, coryphile, make larva body
Body ossifys, i.e., the complex that the fructification of aweto is constituted with bombys batryticatus sclerotium, belongs to edible fungi.Cordyceps sinensis is
A kind of traditional rare tonic Chinese herbal medicine material of China, there is the multi-efficiency such as regulation function of immune system, antitumor, antifatigue.Due to
With unique medical value, add that wild resource is constantly reduced, the market price rises violently, cordyceps sinensis is by domestic and international all circles people
The concern of scholar also turns into the focus of scientific research in recent years.
Content highest active material is Chinese caterpillar fungus polysaccharide in cordyceps sinensis, because it can be with anti-oxidant, antitumor and increasing
Favored the effects such as immunity by many people by force.Chinese caterpillar fungus polysaccharide be by mannose,Galactolipin、Arabinose、Glucose、FucoseDeng the polysaccharide of composition.Chinese caterpillar fungus polysaccharide plays the role of reinvigoration, strengthening vital QI to eliminate pathogenic factors, can adjust it is immune, antitumor,
Kidney, anti-aging, antifatigue, regulation a variety of functions such as lipid metaboli and glycometabolism, anti-hepatic fibrosis and radioresistance are protected, are to be worth digging
One mcroorganism treasure-house of pick.
The content of the invention：
It is an object of the invention to provide a kind of method for improving Chinese caterpillar fungus polysaccharide yield.One plant is specifically utilized from west
The Hirsutella sinensis (Hisutella sinensis) separated in cordyceps sporophore is hidden, it is special to pass through using special culture medium
Condition of culture, fermented and cultured obtain mycelium.Find that Chinese caterpillar fungus polysaccharide content is up to 13-14% after testing, far above open country
Raw cordyceps sinensis.
In order to reach above-mentioned purpose, the present invention can be realized by following technical measures：
A kind of method for improving Chinese caterpillar fungus polysaccharide yield, using Hirsutella sinensis strain M-010, in May, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) is preserved within 29th, during the identified bacterial strain is
State's hair spore (Hisutella sinensis), deposit number：CGMCC No.9046, preservation address：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Classification And Nomenclature:Cordyceps sinensis Ophiocordyceps sinensis.
Comprise the following steps：
(1) strain inclined plane culture
Culture medium：Potato culture；
The ring of mycelium one of picking inclined-plane culture, is inoculated in fresh slant medium, is placed in 15-25 DEG C of constant incubator quiet
Put culture 5-15 days；
(2) strain seed culture
Seed culture medium：Molasses 2-6%, high F value oligopeptide 0.2-1.0%, yam extract 0.5-1.0%, remaining is water,
PH is natural.
The mycelium of the fresh inclined-plane growth of the ring of picking one, in liquid seed culture medium, cultivation temperature is 15-25 DEG C, training
It is 100-200rpm, cultivation cycle 5-10 days to support rotating speed；Secondary seed tank is inoculated in 5-15% (V/V) inoculum concentration, cultivated
Temperature is 15-25 DEG C, and culture rotating speed is 100-200rpm, and throughput is 1:0.1-0.5, cultivation cycle 4-7 days；
(3) strain fermentation culture
Fermentation medium：Molasses 4-10%, high F value oligopeptide 0.5-1.5%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 1-3%.
By the seeding tank bacterium solution of 5-20% (V/V) inoculum concentration inoculation steps (2) into fermentation medium, culture rotating speed is
100-300rpm, throughput is 1:0.2-0.7, after being cultivated 2 days under the conditions of 15-25 DEG C, intermittent stirring is (static to replace with stirring
Carry out), point 3-10 stream plus altogether 0.1-0.3% (V/V) supplemented medium, cultivation cycle is 7-15 days；
(4) zymotic fluid obtained by step (3) fermented and cultured is discarded filtrate by suction filtration through plate-frame filtering, obtains wet mycelium；
(5) wet mycelium that drying obtains step (4) is by obtaining mycelium powder after 50-70 DEG C of dries pulverizing.
The purpose of the present invention can also be realized by following technical measures：
Step (3) described intermittent stirring refers to stirs 2-6h, so circulation every 2-6h；
The extracting method of polysaccharide is in the Chinese caterpillar fungus bacterium powder：Chinese caterpillar fungus bacterium powder is taken, 10 times of quantity bodies of bacterium powder are added
Extracted repeatedly and merging filtrate under long-pending distilled water, boiling water bath, be concentrated into the 1/5 of original volume, added 95% ethanol and low temperature sinks
Shallow lake is stayed overnight, centrifuging and taking precipitation, is then washed respectively with ethanol and acetone, and Thick many candies are obtained after drying.
The Chinese caterpillar fungus polysaccharide content is determined using Phenol sulfuric acid procedure.
Beneficial effects of the present invention：
(1) the special fluid nutrient medium of the present invention, nutritional ingredient economical rationality, cultural method is simple, to equipment without special
It is required that, general Zymolysis Equipment can meet production, further develop and lay a good foundation for the bacterial strain.
(2) by intermittent stirring technique, power consumption is reduced, so as to reduce production cost.
(3) Chinese caterpillar fungus bacterium powder obtained by cultural method of the present invention, Cordyceps sinensis polysaccharide yield is wilder up to 13-14%
Polyoses content improves 62.5-75% in raw cordyceps sinensis.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modifications or substitutions made to the inventive method, step or condition belong to the present invention
Scope.
The percentage of following embodiment medium components is mass percent, is no longer illustrated below.
The Liquid Culture of the Hirsutella sinensis of embodiment 1
1. culture medium
Slant medium：Potato culture；
Seed culture medium：Molasses 2.0%, high F value oligopeptide 0.2%, yam extract 0.5%, remaining is water, and pH is natural；
Fermentation medium：Molasses 4.0%, high F value oligopeptide 0.5%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 1.2%, remaining is water.
2. cultural method
The mycelium of the fresh inclined-plane growth of the ring of picking one, the concussion and cultivate in liquid seed culture medium, rotating speed 100rpm, training
It is 15-25 DEG C to support temperature, is cultivated 5 days, mycelium dry weight reaches the 0.3% of zymotic fluid.
Above-mentioned first order seed is inoculated with secondary seed tank with the inoculum concentration of secondary seed solution volume 5%, cultivation temperature is 15-
25 DEG C, rotating speed 100rpm, throughput is 1：0.1, cultivation cycle 4 days, mycelium dry weight reaches the 0.8% of zymotic fluid.
Secondary seed is inoculated in by fermentation medium with the inoculum concentration of fermentation medium volume 5%, cultivation temperature is 15-25
DEG C, rotating speed 100rpm, throughput：1:0.2, pressure tank is maintained：0.05Mpa, culture 2 days after, with 2 hours it is static, stir within 2 hours
Mix, the intermittent stirring mode circulated successively, respectively in fermentation the 3rd, 4,5 days, each fed-batch cultivation liquid accumulates 0.4% volume
Supplemented medium, flow acceleration 100mL/h, cultivation cycle is 7 days, and now zymotic fluid content of reducing sugar is consumed to 0.1%, fermentation
Mycelium dry weight yield reaches 2.0% after end.
The Liquid Culture of the Hirsutella sinensis of embodiment 2
1. culture medium
Slant medium：Potato culture；
Seed culture medium：Molasses 6.0%, high F value oligopeptide 1.0%, yam extract 1%, remaining is water, and pH is natural；
Fermentation medium：Molasses 10%, high F value oligopeptide 1.5%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 3.0%, remaining is water.
2. cultural method
The mycelium of the fresh inclined-plane growth of the ring of picking one, the concussion and cultivate in liquid seed culture medium, rotating speed 200rpm, training
It is 15-25 DEG C to support temperature, is cultivated 10 days, mycelium dry weight reaches the 0.25% of zymotic fluid.
Above-mentioned first order seed is inoculated with secondary seed tank with the inoculum concentration of secondary seed solution volume 15%, cultivation temperature is
15-25 DEG C, rotating speed 200rpm, throughput is 1:0.5, cultivation cycle 7 days, mycelium dry weight reaches the 0.7% of zymotic fluid.
Secondary seed is inoculated in by fermentation medium with the inoculum concentration of fermentation medium volume 20%, cultivation temperature is 15-
25 DEG C, rotating speed 300rpm, throughput：1:0.7, pressure tank is maintained：0.05Mpa, after cultivating 2 days, (4 hours quiet for intermittent stirring
Only, stir within 4 hours, circulate successively), respectively in fermentation the 4th, 5,6,7,8,9,10,11,12,13 days, each fed-batch cultivation liquid
The supplemented medium of the volume of volume 0.3%, flow acceleration 80mL/h, cultivation cycle is 15 days, now zymotic fluid content of reducing sugar
It is consumed to mycelium dry weight yield after 0.18%, fermentation ends and reaches 1.8%.
The Liquid Culture of the Hirsutella sinensis of embodiment 3
1. culture medium
Slant medium：Potato culture；
Seed culture medium：Molasses 4.0%, high F value oligopeptide 0.6%, yam extract 0.7%, remaining is water, and pH is natural；
Fermentation medium：Molasses 7%, high F value oligopeptide 1.0%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 2.1%, remaining is water.
2. cultural method
The mycelium of the fresh inclined-plane growth of the ring of picking one, the concussion and cultivate in liquid seed culture medium, cultivation temperature is 20
DEG C, cultivate 7 days, mycelium dry weight reaches the 0.4% of zymotic fluid.
Above-mentioned first order seed is inoculated with secondary seed tank with the inoculum concentration of secondary seed solution volume 10%, cultivation temperature is
15-25 DEG C, rotating speed 150rpm, throughput is 1：0.3, cultivation cycle 5 days, mycelium dry weight reaches the 0.8% of zymotic fluid.
Secondary seed is inoculated in by fermentation medium with the inoculum concentration of fermentation medium volume 15%, cultivation temperature is 15-
25 DEG C, rotating speed 200rpm, throughput：1:0.5, pressure tank is maintained：0.05Mpa, culture 2 days after, with 6 hours it is static, 6 hours
Stirring, the intermittent stirring mode circulated successively, respectively in fermentation the 4th, 5,6,7,8,9,10 days, each fed-batch cultivation liquid product
The supplemented medium of 0.3% volume, flow acceleration 120mL/h, cultivation cycle is 11 days, and now zymotic fluid content of reducing sugar is consumed
To 0.15%, mycelium dry weight yield reaches 1.9% after fermentation ends.
The Liquid Culture of the Hirsutella sinensis of embodiment 4
1. culture medium
Slant medium：Potato culture；
Seed culture medium：Molasses 3.0%, high F value oligopeptide 0.4%, yam extract 0.6%, remaining is water, and pH is natural；
Fermentation medium：Molasses 5%, high F value oligopeptide 0.8%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 1.6%, remaining is water.
2. cultural method
The mycelium of the fresh inclined-plane growth of the ring of picking one, the concussion and cultivate in liquid seed culture medium, cultivation temperature is 15-
25 DEG C, cultivate 6 days, mycelium dry weight reaches the 0.45% of zymotic fluid.
Above-mentioned first order seed is inoculated with secondary seed tank with the inoculum concentration of secondary seed solution volume 8%, cultivation temperature is 15-
25 DEG C, rotating speed 130rpm, throughput is 1：0.2, cultivation cycle 5 days, mycelium dry weight reaches the 0.9% of zymotic fluid.
Secondary seed is inoculated in by fermentation medium with the inoculum concentration of fermentation medium volume 10%, cultivation temperature is 15-
25 DEG C, rotating speed 150rpm, throughput：1:0.4, pressure tank is maintained：0.05Mpa, culture 2 days after, with 6 hours it is static, 6 hours
Stirring, the intermittent stirring mode circulated successively, respectively in fermentation the 4th, 5,6,7 days, each fed-batch cultivation liquid accumulates 0.4% body
Long-pending supplemented medium, flow acceleration 150mL/h, cultivation cycle is 9 days, and now zymotic fluid content of reducing sugar is consumed to
0.14%, mycelium dry weight yield reaches 2.0% after fermentation ends.
The Liquid Culture of the Hirsutella sinensis of embodiment 5
1. culture medium
Slant medium：Potato culture；
Seed culture medium：Molasses 5.0%, high F value oligopeptide 0.8%, yam extract 0.8%, remaining is water, and pH is natural；
Fermentation medium：Molasses 9%, high F value oligopeptide 1.2%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 2.7%, remaining is water.
2. cultural method
The mycelium of the fresh inclined-plane growth of the ring of picking one, the concussion and cultivate in liquid seed culture medium, cultivation temperature is 15-
25 DEG C, cultivate 6 days, mycelium dry weight reaches the 0.42% of zymotic fluid.
Above-mentioned first order seed is inoculated with secondary seed tank with the inoculum concentration of secondary seed solution volume 12%, cultivation temperature is
15-25 DEG C, rotating speed 180rpm, throughput is 1：0.4, cultivation cycle 6 days, mycelium dry weight reaches the 0.85% of zymotic fluid.
Secondary seed is inoculated in by fermentation medium with the inoculum concentration of fermentation medium volume 18%, cultivation temperature is 15-
25 DEG C, rotating speed 260rpm, throughput：1:0.6, pressure tank is maintained：0.05Mpa, culture 2 days after, with 6 hours it is static, 6 hours
Stirring, the intermittent stirring mode circulated successively, respectively in fermentation the 3rd, 4,5,6,7,8,9,10,11 days, each fed-batch cultivation
Liquid accumulates the supplemented medium of 0.3% volume, flow acceleration 100mL/h, and cultivation cycle is 12 days, and now zymotic fluid reduced sugar contains
Amount is consumed to mycelium dry weight yield after 0.13%, fermentation ends and reaches 2.1%.
The measure of the Chinese caterpillar fungus polysaccharide content of embodiment 6
The content to fermentation mycelium powder and the Cordyceps sinensis polysaccharide of wild cordyceps is detected respectively.
Take 10g above-described embodiments to treat test sample, plus 10 times of volumes distilled water, extract 3h under boiling water bath, 3 times extracted repeatedly simultaneously
Filtrate is merged, the 1/5 of original volume is concentrated into, adding 95% ethanol, simultaneously low-temperature precipitation is stayed overnight to 4 times of volumes, centrifuging and taking precipitation,
Then with respectively washing 2 times of ethanol and acetone, dry, obtain Thick many candies.
Accurate weigh is dried to each 50mg of Cordyceps sinensis polysaccharide of constant weight, is placed in 100mL volumetric flasks, plus distilled water is settled to quarter
Degree, shakes up.Sample liquid 1.0mL is taken, distilled water is supplemented to 2.0mL, adds 50g/L phenol 1.0mL, concentrated sulfuric acid 5.0mL, rapidly
Vibration shakes up, and develop the color 20min at room temperature, determines OD488, 3 of parallel determination and according to the regression equation of standard curve calculate
The Chinese caterpillar fungus polysaccharide content for going out the mycelium powder of Liquid Culture is 13.6%, and Chinese caterpillar fungus polysaccharide contains in wild cordyceps
Measure as 7.8%, mycelium powder improves 74.4% than Chinese caterpillar fungus polysaccharide content in wild cordyceps.
The measure of other nutritional ingredients of embodiment 7
The content to fermentation mycelium powder and other nutritional ingredients of wild cordyceps is detected respectively.
1. cordyceps sinensis acidity test
The measure of cordycepic acid is carried out using liquid chromatography, chromatographic column is Inertsil ODS-3 (4.6mm × 250mm, 5 μ
M), 30 DEG C of column temperature.Detector is differential refraction detector.Mobile phase is acetonitrile:Water=80:20, gradient elution, flow velocity 0.8mL/
min。
The content for determining the cordycepic acid of the mycelium powder of Liquid Culture is 8.2%, and cordycepic acid contains in wild cordyceps
Measure as 7.5%, mycelium powder improves 9.3% than cordycepic acid content in wild cordyceps.
2. cordycepin is determined
The measure of cordycepin is carried out using liquid chromatography, chromatographic column is Inertsil ODS-3 (4.6mm × 250mm, 5 μ
M), 30 DEG C of column temperature.Detector is diode array UV-detector, Detection wavelength 260nm.Mobile phase acetonitrile:Water=80:20,
Flow velocity 0.8mL/min.
The content of cordycepin is that the content of cordycepin in 0.60%, wild cordyceps is in the mycelium powder of Liquid Culture
0.48%, mycelium powder improves 25.0% than cordycepin content in wild cordyceps.
In addition, other every nutritional ingredients to the mycelium powder are compared with wild cordyceps, it is summarized as follows
Table.
Claims (1)
1. it is a kind of improve Chinese caterpillar fungus polysaccharide yield method, it is characterised in that use Hirsutella sinensis strain M-010, in
On June 20th, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number：CGMCC
No. 9046, carry out as follows：
（1）Solid slope culture:Slant medium selects potato culture；Hirsutella sinensis strain M-010 is inoculated with Ma Ling
Potato medium slant, is cultivated 5-15 days under the conditions of 15-25 DEG C；
（2）Seed culture:Inoculation step（1）Mycelia is into one-level shake-flask seed culture medium, with 100-200rpm rotating speed cultures 5-
10 days, then by 5-15%（V/V）Inoculum concentration be inoculated with shake-flask seed liquid to secondary seed tank culture, throughput：1:0.1-0.5,
Culture 4-7 days；The seed culture temperature is 15-25 DEG C；
Seed culture medium：Molasses 2-6%, high F value oligopeptide 0.2-1.0%, yam extract 0.5-1.0%, remaining is water, and pH is natural；
（3）Fermented and cultured:By 5-20%（V/V）Inoculum concentration inoculation step（2）Seeding tank bacterium solution into fermentation medium, culture
Rotating speed is 100-300rpm, and throughput is 1:0.2-0.7, after being cultivated 2 days under the conditions of 15-25 DEG C, using under intermittent stirring
Divide 3-10 stream plus altogether 0.1-0.3%（V/V）Supplemented medium, cultivation cycle be 7-15 days；
Fermentation medium：Molasses 4-10%, high F value oligopeptide 0.5-1.5%, remaining is water, and pH is natural；
Supplemented medium：Yam extract 1-3%；
（4）Suction filtration:By step（3）Zymotic fluid discards filtrate through plate-frame filtering obtained by fermented and cultured, obtains wet mycelium；
（5）Drying:By step（4）The wet mycelium of acquisition is by obtaining mycelium powder after 50-70 DEG C of dries pulverizing；
Step（3）The intermittent stirring refers to stirs 2-6h, so circulation every 2-6h；
The extracting method of polysaccharide is in the Chinese caterpillar fungus bacterium powder：Chinese caterpillar fungus bacterium powder is taken, 10 times of quantity volumes of bacterium powder are added
Extracted repeatedly and merging filtrate under distilled water, boiling water bath, be concentrated into the 1/5 of original volume, add 95% ethanol and low-temperature precipitation mistake
Then night, centrifuging and taking precipitation is washed respectively with ethanol and acetone, and Thick many candies are obtained after drying.
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| CN110129207B (en) * | 2019-04-22 | 2024-04-05 | 江苏农林职业技术学院 | Liquid fermentation medium for high-yield antioxidant cordyceps sobolifera mycelium and production method of antioxidant cordyceps sobolifera mycelium electuary |
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| CN1796539A (en) * | 2004-12-24 | 2006-07-05 | 青海月王青藏药业有限责任公司 | Ferment for producing aweto in large scale and technique for processing power of fungus |
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| CN1796539A (en) * | 2004-12-24 | 2006-07-05 | 青海月王青藏药业有限责任公司 | Ferment for producing aweto in large scale and technique for processing power of fungus |
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| 中国被毛孢液体发酵工艺优化及其多糖制备;张曰辉;《中国优秀硕士学位论文全文数据库农业科技辑》;20140415;第2014卷(第4期);第D047-185页 * |
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Correction item: Patentee|Address Correct: Jinan Guoli Biotechnology Co., Ltd|Second floor, xiaohonglou, No.41, Jiefang Road, Jinan, Shandong Province 250013 False: Shandong Guoli Biotechnology Co., Ltd|1101, building 2, Xinsheng building, 1299 Xinluo street, hi tech Zone, Jinan City, Shandong Province 250100 Number: 24-02 Volume: 36 |
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