CN102511305A - Method for cultivating high-selenium-tolerability strain of edible fungi - Google Patents
Method for cultivating high-selenium-tolerability strain of edible fungi Download PDFInfo
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Abstract
The invention discloses a method for cultivating a high-selenium-tolerability strain of edible fungi. The method is characterized in that the method comprises the the steps of: (1) selecting a culture medium suitable for growth of an edible fungi strain according to the edible fungi strain, inoculating the edible fungi strain into the culture medium to cultivate a tube strain so as to obtain initial bacterium suspension, (2) mixing the culture medium with a selenium source to prepare into selenium-tolerant medium with gradient selenium concentration, inoculating the edible fungi strain on the sterilized selenium-tolerant medium by a bacterium suspension inoculating mode, and gradually carrying out selenium-tolerant cultivation on selenium concentration by a common edible fungi strain cultivation method according to an sequence from low selenium concentration to high selenium concentration, and (3) carrying out continuous transferring cultivation by the bacterium suspension inoculating mode according to the optimal selenium-tolerant concentration of the edible fungi obtained from the step (2), and determining the selenium-tolerant character stability of the strain so as to obtain the high-selenium-tolerability strain of the edible fungi. The tolerability of selenium content in the culture medium is greatly improved a deep cultivation state of liquid after high-selenium tolerability cultivation of the edible fungi strain.
Description
Technical field
The present invention relates to high selenium preparation methods, specifically be meant a kind of method of cultivating the high selenium tolerance of edible mushroom bacterial classification.
Background technology
Selenium is trace element essential in the humans and animals body; Be to constitute the nearly activated centre of 23 kinds of functional proteins such as glutathione peroxidase (GSH-Px), selenoprotein P; Have following effect: body endoperoxide, free radical are removed in (1); Interference-free and the infringement of protection cell structure and function delays senility; (2) metabolism of acceleration carcinogen promotes the dna damage reparation, reduces cancer morbidity; (3) noxious material in the antagonist reduces its toxicity, effectively protects liver; (4) enhancing body cellular immune function improves immunity of organisms.And the famine of selenium can directly cause Keshan disease and Kaschin-Beck disease; Wherein Keshan disease is found the Keshan County, Heilungkiang with the seventies the earliest; Be to be expansion in various degree with heart, spherical in shape when serious, cardiac muscle has and is dispersed in necrosis in different degree kitchen range and the spot district cardiomyopathy for main performance; Kaschin-Beck disease be with the bone joint increase slightly, deformity is a kind of endemic of main performance.
It is the problem that the whole world extensively exists that soil lacks selenium; Although there is world's selenium title all the enshi of China; The Ziyang, Shaanxi also is a rich selenium district, but there is a low selenium band that always extends to southwestern Yunnan Province from the Heilongjiang Province, northeast in China, occupies 72% of China's area.In these low selenium districts; Can't from soil, obtain sufficient selenium in the process of growth of foods such as crops; Thereby form the generally scarce selenium state of food chain; Therefore also be in the state of scarce selenium in these regional human bodies, research shows that the Chinese total amount of day absorption selenium per capita is about 35 μ g, significantly is lower than the selenium daily intaking amount 50-200 μ g that The World Health Organization (WHO) is recommended.Therefore, effective absorption of Selenium Supplement becomes the extremely urgent problem in China low selenium area.
Edible mushroom has traditional health care simultaneously, and selenium is had certain enrichment because of it has short, characteristics such as biomass is big, protein content height of breeding time, it is carried out rich selenium research and development have broad application prospects.But; Present patent mainly concentrates on develops the agricultural product selenium-rich nutritive strengthened, that conduct is directly edible of edible mushroom; Like the industrial planting method (one Chinese patent application 200910067431.9) of the preparation method of selenium enriched oyster mushroom powder (one Chinese patent application 200610135227.2), selenium-enriched agaricus bisporus, do not carry out as yet any with edible mushroom as high selenium carrier, develop as the form of the additive of food, health products.There are two patents in the rich selenium domestication of bacterial classification, to carry out a few thing at present; As: selenium-rich mushroom strain domestication and cultivation method (one Chinese patent application 200810010300.2), selenium-rich phellinus igniarius strain domestication, cultivation, enrichment method (one Chinese patent application 200710011014.3); But the simple two-stage domestication of its process; Its purpose neither be obtained the bacterial classification of maximum anti-selenium concentration, and utilizes the simple two-stage domesticating method in above two kinds of patents can not obtain high selenium tolerance bacterial classification.The present invention therefore.
Summary of the invention
The purpose of this invention is to provide a kind of method of cultivating the high selenium tolerance of edible mushroom bacterial classification, and then obtain high selenium edible mushroom material, be the material that exploitation is abundanter, more effective benefit selenium product provides high-quality.Technical scheme of the present invention is following:
A kind of method of cultivating the high selenium tolerance of edible mushroom bacterial classification is characterized in that said method comprising the steps of:
(1) select to adapt to the medium of edible fungus species growth according to edible fungus species, edible fungus species is inserted carry out test tube strains in the medium and cultivate and obtain initial bacteria suspension;
(2) medium and selenium source are hybridly prepared into the anti-seleno culture medium of gradient selenium concentration, adopt the bacterial suspension inoculation mode with on the anti-seleno culture medium of edible bacterial vaccine inoculation after the sterilization treatment according to selenium concentration order from low to high according to edible fungus species training method commonly used one by one selenium concentration carry out anti-selenium and cultivate; It is initial bacteria suspension that anti-first selenium is cultivated the bacteria suspension that uses, and anti-selenium is cultivated the bacteria suspension that obtains and cultivated the bacteria suspension that uses for next selenium concentration carries out anti-selenium, and the like; When edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
And the selenium concentration difference of adjacent batch of anti-seleno culture medium stops anti-selenium and cultivates in predetermined threshold the time, and the best of definite edible mushroom tolerance selenium concentration is the selenium concentration of last batch of anti-seleno culture medium;
(3) the best tolerance selenium concentration of the edible mushroom that obtains according to step (2) adopts the bacterial suspension inoculation mode, cultivations of transferring continuously, and the anti-selenium proterties of definite bacterial classification stablizes, and promptly obtains the high selenium tolerance of edible mushroom bacterial classification.
Preferably, in the said method when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold; Adjust the selenium concentration of anti-seleno culture medium; Make the selenium concentration of the selenium concentration of anti-seleno culture medium greater than last batch of anti-seleno culture medium; And the selenium concentration of anti-seleno culture medium is proceeded anti-selenium with the anti-seleno culture medium behind the adjustment selenium concentration according to the method for step (2) then and is cultivated less than the selenium concentration of current batch of anti-seleno culture medium.
Preferably, the selenium concentration of the anti-seleno culture medium of adjustment is to adjust according to the integer multiple of predetermined threshold or predetermined threshold in the said method, when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold, the selenium concentration of adjusting anti-seleno culture medium is the selenium concentration after the selenium concentration of current batch of anti-seleno culture medium deducts the integer multiple of predetermined threshold or predetermined threshold.
Preferably, the selenium concentration of the anti-seleno culture medium of adjustment is to adjust according to the selenium concentration of adjacent batch of anti-seleno culture medium in the said method, when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold, the selenium concentration of adjusting anti-seleno culture medium is the mean value of the selenium concentration of adjacent batch of anti-seleno culture medium.
Preferably, the medium of adaptation edible fungus species growth is the PDA synthetic medium in the said method.
Preferably, in the said method in the every 100ml constant volume of the PDA synthetic medium medium, raw materials usedly be: potato 20g, glucose 2g, agar 1.5g, MgSO
47H
2O 0.15g, KH
2PO
40.3g, VB1 0.001g, pH transfers to 6.0.
Preferably, the compound method of PDA synthetic medium described in the said method comprises the potato and the distilled water mixing that should add earlier, keeps boiling to filter after 30 minutes, adds other raw materials and is settled to volume required step after abundant the mixing.
Preferably, selenium source is selected from sodium selenite, sodium selenate or Se-enriched yeast powder in the said method.
Preferably, transfer continuously in the said method step (3) when cultivating, following situation occurs:
A) bacterial classification can't be grown;
B) the growth cycle differentiation is obvious;
C) strain bio amount difference is obvious;
D) the obvious variable color of bacterial strain;
Then judge and do not satisfy the stable condition of anti-selenium proterties; Otherwise think that anti-selenium proterties is stable.
Preferably; Said method also is included in step (3) and obtains behind the high selenium tolerance of the edible mushroom bacterial classification the high selenium tolerance bacterial classification that is obtained is linked on the solid inclined-plane that contains selenium PDA synthetic medium that contains best anti-selenium concentration and cultivate, and the step of in-80 ℃ of glycerine pipes, preserving as high selenium tolerance bacterial classification.
The value of the described predetermined threshold selenium concentration variation that to be the needs cultivated according to the anti-selenium of edible mushroom confirm with other factors can be constant value, also can be the value of change, the value that reduces like recurrence.
Concrete, the present invention cultivates the method for the high selenium tolerance of edible mushroom bacterial classification, carries out according to following steps:
(1) cultivates carrying out test tube strains in the PDA synthetic medium of edible fungus species access after sterilization treatment;
(2) selenium source is mixed according to certain proportioning with the PDA synthetic medium, be made into and contain seleno culture medium, sterilization then by the series concentration gradient of low (one-level) to high (N level);
(3) adopt the bacterial suspension inoculation mode, the one-level that the test tube strains access step 2 that step 1 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of edible fungus species one-level and cultivates;
(4) adopt the bacterial suspension inoculation mode, the secondary that the one-level bacterial classification of the anti-selenium access step 2 that step 3 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of edible fungus species secondary and cultivates;
(5) the rest may be inferred, grows one of row situation now until edible fungus species: more than a times of (1) strain growth cycle stretch-out to control group; (2) the strain bio amount drops to below 30% of control group, not even growth; (3) the obvious variable color of bacterial strain; Then stopping anti-selenium cultivates;
The medium selenium concentration that (6) inhibition phenomenon in the step 5 will occur is cultivated concentration with the intermediate value selenium concentration that the last selenium concentration of inhibition phenomenon in the step 5 occurs as further optimization;
(7) repeating step 5 and 6 is until the best anti-selenium concentration that finds edible fungus species;
(8) under the anti-selenium concentration of the best, adopt the bacterial suspension inoculation mode, transfer continuously 3 times, guarantee that the anti-selenium proterties of bacterial classification is stable;
(9) the high selenium tolerance bacterial classification that is obtained is linked on the solid inclined-plane that contains selenium PDA synthetic medium that contains best anti-selenium concentration cultivates, and in-80 ℃ of glycerine pipes, preserve as high selenium tolerance bacterial classification.
Said high selenium tolerance bacterial classification is meant that this bacterial classification is through anti-selenium cultivation step by step, up to the bacterial classification that one of following state obtains occurring: more than a times of (1) strain growth cycle stretch-out to control group; (2) the strain bio amount drops to below 30% of control group, not even growth; (3) the obvious variable color of bacterial strain.Said control group adopts the anti-seleno culture medium of last selenium concentration usually.
Said PDA synthetic medium prescription is: in every 100ml constant volume medium, raw materials usedly be: potato 20g, and glucose 2g, agar 1.5g, MgSO47H2O 0.15g, KH2PO4 0.3g, VB1 0.001g, pH transfers to 6.0; Said PDA synthetic medium collocation method is: potato that should add earlier and distilled water mixing, and keep boiling to filter after 30 minutes, add other raw materials and be settled to volume required after abundant the mixing.
Said selenium source is meant sodium selenite, sodium selenate or Se-enriched yeast powder.Said anti-selenium proterties is stable to be meant that following situation do not occur: (1) bacterial classification can't be grown in the process of switching 3 times; (2) growth cycle differentiation is obvious; (3) strain bio amount difference is obvious; (4) the obvious variable color of bacterial strain.
The present invention judges whether the standard of not carrying out anti-selenium cultivation phenomenon occurs obviously suppressing through the edible fungus species growth and carry out.The obvious inhibition of edible fungus species growth appearance phenomenon is meant and one of following state occurs: more than a times of (1) strain growth cycle stretch-out to control group; (2) the strain bio amount drops to below 30% of control group, not even growth; (3) the obvious variable color of bacterial strain.
Also need judge the steadiness of high selenium tolerance edible fungus species in addition, the applicant confirms that anti-selenium proterties is stable according to following standard: in the process of switching 3 times, following situation do not occur: (1) bacterial classification can't be grown; (2) growth cycle differentiation is obvious; (3) strain bio amount difference is obvious; (4) the obvious variable color of bacterial strain.
Than prior art, the present invention has following beneficial effect:
1, significantly improve the high selenium tolerance of edible mushroom: after cultivating through the high selenium tolerance of edible fungus species, it significantly improves the tolerance of the Se content in the medium under the state that deep liquid is cultivated, generally be the contrast state 3-5 doubly;
2, significantly improve Se content in the edible mushroom: after cultivating through the high selenium tolerance of edible fungus species; It is under the state that deep liquid is cultivated; The ability of its enrichment selenium significantly improves, and the total Se content achievable pair in the body is main with selenomethionine wherein according to more than 10 times of group;
3, significantly shorten the growth cycle that the edible fungus liquid deep layer is cultivated: after cultivating through the high selenium tolerance of edible fungus species, it is under the state that deep liquid is cultivated, and growth cycle foreshortens to the 2/3-1/2 of control group;
4, can produce high selenium material: after cultivating through the high selenium tolerance of edible fungus species; It is under the state that deep liquid is cultivated; Can obtain high selenium material; The high Se content of general bacterial classification can reach more than 1000 mg/kg (dry weight), and the high Se content of glossy ganoderma can reach more than 10000 mg/kg (dry weight).These high selenium materials can be used as the high-quality selenium source additive of food, health products.
Embodiment:
Following examples are used to explain the present invention, but can not be used for limiting scope of the present invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and unaccounted implementation condition is generally the condition in the normal experiment.
Embodiment 1 obtains the step of the high selenium tolerance of glossy ganoderma bacterial classification
(1) preparation of high selenium tolerance lucidum strain:
1) buys lucidum strain (numbering: 5.653) from DSMZ of the Chinese Academy of Sciences.
2) cultivate carrying out test tube strains in the PDA synthetic medium of lucidum strain access after sterilization treatment.
3) with sodium selenite (Na
2SeO
3) mix according to certain proportioning with the PDA synthetic medium, make Na
2SeO
3Content is 2,20,50,100,200,400,600,800, the series concentration gradient of 1000mg/L contain seleno culture medium, it is subsequent use to sterilize then.
4) adopt the bacterial suspension inoculation mode, the 2mg/L that the test tube strains access step 3 that step 2 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of lucidum strain one-level and cultivates.
5) after the anti-selenium cultivation of one-level is accomplished, adopt the bacterial suspension inoculation mode, the 20mg/L that the one-level bacterial classification of the anti-selenium access step 3 that step 4 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of lucidum strain secondary and cultivates.
6) the rest may be inferred, and in the containing in the seleno culture medium of 200mg/L, occur that poor growth, biomass significantly reduce, variable color etc. obviously suppressed phenomenon, stops anti-selenium and cultivates until lucidum strain.
7) the intermediate value selenium concentration 150mg/L with inhibition concentration 200mg/L and last concentration 100mg/L cultivates concentration as further optimizing.
8) the bacterial suspension inoculation mode that adopts the lucidum strain that will grow in the seleno culture medium in containing of 200mg/L insert containing in the seleno culture medium of 150mg/L after sterilization treatment, carry out one-level optimization and cultivate.
9) after one-level optimization is cultivated and accomplished, the one-level that step 8 is cultivated is optimized containing in the seleno culture medium of lucidum strain access 175mg/L (the intermediate value selenium concentration of selenium concentration in the step 8 and inhibition concentration), carry out 2-level optimization and cultivate.
10) in step 9, lucidum strain occurs like the inhibition phenomenon in the step 6, stops to cultivate.
11) the median concentration 160mg/L with selenium concentration in the step 9 and the selenium concentration in the step 7 cultivates concentration as further optimizing.
12) sterilization that the lucidum strain of growing in the step 9 is linked in the step 11 contains in the seleno culture medium, and growth is normal, can be used as the best anti-selenium concentration of this lucidum strain.
13) contain employing bacterial suspension inoculation mode under the seleno culture medium at 160mg/L, the bacterial classification of growth in the step 12 is transferred 3 times continuously, guarantee that the anti-selenium proterties of bacterial classification is stablized.
14) the high selenium tolerance lucidum strain that is obtained in the step 13 is linked into 160mg/L and contains on the solid inclined-plane of seleno culture medium and cultivate, and in-80 ℃ of glycerine pipes, preserve as high selenium tolerance lucidum strain.
(2) application of high selenium tolerance lucidum strain:
The high selenium tolerance lucidum strain of above-mentioned acquisition and the same lucidum strain of cultivating without too high selenium tolerance are linked into step by step carry out the rich selenium of liquid deep layer fermenting in the liquid fermentation medium of 200,300,400,500,600,700,800,900,1000,1100,1200,1300,1400 after the sterilization treatment, 1500mg/L sodium selenite and cultivate; Monitor its growth rhythm, and detect the Se content in the ripe ganoderma lucidum mycelium.
(3) implementation result:
The high selenium tolerance of glossy ganoderma improves: after cultivating through the high selenium tolerance of lucidum strain, it can reach 1200mg/L to the tolerance of the Se content in the medium under the state that deep liquid is cultivated, be 3 times of contrast state.
Glossy ganoderma selenium content improves: after cultivating through the high selenium tolerance of lucidum strain; It is under the state that deep liquid is cultivated; The ability of its enrichment selenium significantly improves; Achievable pair is according to more than 10 times of the highest total Se content in the group under the highest degree of anti-selenium (1200mg/L) condition for total Se content in the body, and wherein the form that exists of selenium is main with selenomethionine.
Significantly shorten the growth cycle that the ganoderma lucidum liquid deep layer is cultivated: after cultivating through the high selenium tolerance of lucidum strain, it is under the state that deep liquid is cultivated, and growth cycle foreshortens to 1/2 of control group.
Can produce high selenium material: after cultivating through the high selenium tolerance of lucidum strain; It is under the state that deep liquid is cultivated; Can obtain the high selenium material of glossy ganoderma; The highest Se content can reach more than 18000 mg/kg (dry weight), and exists with organic selenium safely and effectively, can be used as the high-quality selenium source additive of food, health products.
Embodiment 2: the step of obtaining the high selenium tolerance of rainbow conk bacterial classification
(1) preparation of high selenium tolerance rainbow conk bacterial classification:
1) buys rainbow conk bacterial classification (numbering: 5.48) from DSMZ of the Chinese Academy of Sciences.
2) cultivate carrying out test tube strains in the PDA synthetic medium of rainbow conk bacterial classification access after sterilization treatment.
3) with sodium selenate (Na
2SeO
4) mix according to certain proportioning with the PDA synthetic medium, make Na
2SeO
4Content is 2,20,50,100,200,400,600,800, the series concentration gradient of 1000mg/L contain seleno culture medium, it is subsequent use to sterilize then.
4) adopt the bacterial suspension inoculation mode, the 2mg/L that the test tube strains access step 3 that step 2 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of rainbow conk bacterial classification one-level and cultivates.
5) after the anti-selenium cultivation of one-level is accomplished, adopt the bacterial suspension inoculation mode, the 20mg/L that the one-level bacterial classification of the anti-selenium access step 3 that step 4 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of rainbow conk bacterial classification secondary and cultivates.
6) the rest may be inferred, and in the containing in the seleno culture medium of 200mg/L, occur that poor growth, biomass significantly reduce, variable color etc. obviously suppressed phenomenon, stops anti-selenium and cultivates until the rainbow conk bacterial classification.
7) the intermediate value selenium concentration 150mg/L with inhibition concentration 200mg/L and last concentration 100mg/L cultivates concentration as further optimizing.
8) the bacterial suspension inoculation mode that adopts the rainbow conk bacterial classification that will grow in the seleno culture medium in containing of 200mg/L insert containing in the seleno culture medium of 150mg/L after sterilization treatment, carry out one-level optimization and cultivate.
9) after one-level optimization is cultivated and accomplished, the one-level that step 8 is cultivated is optimized containing in the seleno culture medium of rainbow conk bacterial classification access 175mg/L (the intermediate value selenium concentration of selenium concentration in the step 8 and inhibition concentration), carry out 2-level optimization and cultivate.
10) in step 9, the rainbow conk bacterial classification occurs like the inhibition phenomenon in the step 6, stops to cultivate.
11) the median concentration 160mg/L with selenium concentration in the step 9 and the selenium concentration in the step 7 cultivates concentration as further optimizing.
12) sterilization that the lucidum strain of growing in the step 9 is linked in the step 11 contains in the seleno culture medium, and the rainbow conk bacterial classification occurs like the inhibition phenomenon in the step 6, stops to cultivate.
13) the median concentration 130mg/L with selenium concentration in the step 11 and the selenium concentration in the step 7 cultivates concentration as further optimizing.
14) sterilization that the rainbow conk bacterial classification of growing in the step 12 is linked in the step 13 contains in the seleno culture medium, and growth is normal, can be used as the best anti-selenium concentration of this rainbow conk bacterial classification.
15) contain employing bacterial suspension inoculation mode under the seleno culture medium at 130mg/L, the bacterial classification of growth in the step 14 is transferred 3 times continuously, guarantee that the anti-selenium proterties of bacterial classification is stablized.
16) the high selenium tolerance rainbow conk bacterial classification that is obtained in the step 15 is linked into 130mg/L and contains on the solid inclined-plane of seleno culture medium and cultivate, and in-80 ℃ of glycerine pipes, preserve as high selenium tolerance rainbow conk bacterial classification.
(2) application of high selenium tolerance rainbow conk bacterial classification:
The high selenium tolerance rainbow conk bacterial classification of above-mentioned acquisition and the same rainbow conk bacterial classification of cultivating without too high selenium tolerance are linked into step by step carry out the rich selenium of liquid deep layer fermenting in the liquid fermentation medium of 200,300,400,500,600,700,800,900,1000,1100,1200,1300,1400 after the sterilization treatment, 1500mg/L sodium selenate and cultivate; Monitor its growth rhythm, and detect the Se content in the ripe prepared from coriolus versicolor mycelium.
(3) implementation result:
The high selenium tolerance of rainbow conk improves: after cultivating through the high selenium tolerance of rainbow conk bacterial classification, it can reach 1000mg/L to the tolerance of the Se content in the medium under the state that deep liquid is cultivated, be the contrast state 3-4 doubly.
The rainbow conk Se content improves: after cultivating through the high selenium tolerance of rainbow conk bacterial classification; It is under the state that deep liquid is cultivated; The ability of its enrichment selenium significantly improves; Total Se content in the body under the highest degree of anti-selenium (1000mg/L) condition achievable pair according in the group the 8-12 of high total Se content doubly more than, wherein the form that exists of selenium is main with selenomethionine.
Significantly shorten the growth cycle that the rainbow conk deep liquid is cultivated: after cultivating through the high selenium tolerance of rainbow conk bacterial classification, it is under the state that deep liquid is cultivated, and growth cycle foreshortens to 2/3 of control group.
Can produce high selenium material: after cultivating through the high selenium tolerance of rainbow conk bacterial classification; It is under the state that deep liquid is cultivated; Can obtain the high selenium material of rainbow conk; The highest Se content can reach more than the 7000-9000 mg/kg (dry weight), and exists with organic selenium safely and effectively, can be used as the high-quality selenium source additive of food, health products.
Embodiment 3: the step of obtaining the high selenium tolerance of Xingbao mushroom bacterial classification
(1) preparation of high selenium tolerance Xingbao mushroom bacterial classification:
1) buys Xingbao mushroom bacterial classification (numbering: 5.775) from DSMZ of the Chinese Academy of Sciences.
2) cultivate carrying out test tube strains in the PDA synthetic medium of Xingbao mushroom bacterial classification access after sterilization treatment.
3) with sodium selenite (Na
2SeO
3) mix according to certain proportioning with the PDA synthetic medium, make Na
2SeO
3Content is 2,20,50,100,150,200,300, the series concentration gradient of 400mg/L contain seleno culture medium, it is subsequent use to sterilize then.
4) adopt the bacterial suspension inoculation mode, the 2mg/L that the test tube strains access step 3 that step 2 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of Xingbao mushroom bacterial classification one-level and cultivates.
5) after the anti-selenium cultivation of one-level is accomplished, adopt the bacterial suspension inoculation mode, the 20mg/L that the one-level bacterial classification of the anti-selenium access step 3 that step 4 is cultivated prepares contains in the seleno culture medium, carries out the anti-selenium of Xingbao mushroom bacterial classification secondary and cultivates.
6) the rest may be inferred, and in the containing in the seleno culture medium of 150mg/L, occur that poor growth, biomass significantly reduce, variable color etc. obviously suppressed phenomenon, stops anti-selenium and cultivates until the Xingbao mushroom bacterial classification.
7) the intermediate value selenium concentration 120mg/L with inhibition concentration 150mg/L and last concentration 100mg/L cultivates concentration as further optimizing.
8) the bacterial suspension inoculation mode that adopts the Xingbao mushroom bacterial classification that will grow in the seleno culture medium in containing of 100mg/L insert containing in the seleno culture medium of 120mg/L after sterilization treatment, be optimized cultivation, and growth is normal, can be used as the best anti-selenium concentration of this Xingbao mushroom bacterial classification.
9) contain employing bacterial suspension inoculation mode under the seleno culture medium at 120mg/L, the bacterial classification of growth in the step 9 is transferred 3 times continuously, guarantee that the anti-selenium proterties of bacterial classification is stablized.
10) the high selenium tolerance Xingbao mushroom bacterial classification that is obtained in the step 9 is linked into 90mg/L and contains on the solid inclined-plane of seleno culture medium and cultivate, and in-80 ℃ of glycerine pipes, preserve as high selenium tolerance Xingbao mushroom bacterial classification.
(2) application of high selenium tolerance Xingbao mushroom bacterial classification:
The high selenium tolerance Xingbao mushroom bacterial classification of above-mentioned acquisition and the same Xingbao mushroom bacterial classification of cultivating without too high selenium tolerance are linked into step by step carry out the rich selenium of liquid deep layer fermenting in the liquid fermentation medium of 100,150,200,300,400,500,600,700,800,900 after the sterilization treatment, 1000mg/L sodium selenate and cultivate; Monitor its growth rhythm, and detect the Se content in the ripe Xingbao mushroom mycelium.
(3) implementation result:
The high selenium tolerance of Xingbao mushroom improves: after cultivating through the high selenium tolerance of Xingbao mushroom bacterial classification, it can reach 800mg/L to the tolerance of the Se content in the medium under the state that deep liquid is cultivated, be the contrast state 4-5 doubly.
The Xingbao mushroom Se content improves: after cultivating through the high selenium tolerance of Xingbao mushroom bacterial classification; It is under the state that deep liquid is cultivated; The ability of its enrichment selenium significantly improves; Achievable pair is according to more than 10 times of the highest total Se content in the group under the highest degree of anti-selenium (800mg/L) condition for total Se content in the body, and wherein the form that exists of selenium is main with selenomethionine.
Significantly shorten the growth cycle that the Xingbao mushroom deep liquid is cultivated: after cultivating through the high selenium tolerance of Xingbao mushroom bacterial classification, it is under the state that deep liquid is cultivated, and growth cycle foreshortens to 1/2 of control group.
Above-mentioned instance only is explanation technical conceive of the present invention and characteristics, and its purpose is to let the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.
Claims (10)
1. method of cultivating the high selenium tolerance of edible mushroom bacterial classification is characterized in that said method comprising the steps of:
(1) select to adapt to the medium of edible fungus species growth according to edible fungus species, edible fungus species is inserted carry out test tube strains in the medium and cultivate and obtain initial bacteria suspension;
(2) medium and selenium source are hybridly prepared into the anti-seleno culture medium of gradient selenium concentration, adopt the bacterial suspension inoculation mode with on the anti-seleno culture medium of edible bacterial vaccine inoculation after the sterilization treatment according to selenium concentration order from low to high according to edible fungus species training method commonly used one by one selenium concentration carry out anti-selenium and cultivate; It is initial bacteria suspension that anti-first selenium is cultivated the bacteria suspension that uses, and anti-selenium is cultivated the bacteria suspension that obtains and cultivated the bacteria suspension that uses for next selenium concentration carries out anti-selenium, and the like; When edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
And the selenium concentration difference of adjacent batch of anti-seleno culture medium stops anti-selenium and cultivates in predetermined threshold the time, and the best of definite edible mushroom tolerance selenium concentration is the selenium concentration of last batch of anti-seleno culture medium;
(3) the best tolerance selenium concentration of the edible mushroom that obtains according to step (2) adopts the bacterial suspension inoculation mode, cultivations of transferring continuously, and the anti-selenium proterties of definite bacterial classification stablizes, and promptly obtains the high selenium tolerance of edible mushroom bacterial classification.
2. method according to claim 1 is characterized in that in the said method when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold; Adjust the selenium concentration of anti-seleno culture medium; Make the selenium concentration of the selenium concentration of anti-seleno culture medium greater than last batch of anti-seleno culture medium; And the selenium concentration of anti-seleno culture medium is proceeded anti-selenium with the anti-seleno culture medium behind the adjustment selenium concentration according to the method for step (2) then and is cultivated less than the selenium concentration of current batch of anti-seleno culture medium.
3. method according to claim 1 is characterized in that in the said method that the selenium concentration of the anti-seleno culture medium of adjustment is to adjust according to the integer multiple of predetermined threshold or predetermined threshold, when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold, the selenium concentration of adjusting anti-seleno culture medium is the selenium concentration after the selenium concentration of current batch of anti-seleno culture medium deducts the integer multiple of predetermined threshold or predetermined threshold.
4. method according to claim 1 is characterized in that in the said method that the selenium concentration of the anti-seleno culture medium of adjustment is to adjust according to the selenium concentration of adjacent batch of anti-seleno culture medium, when edible fungus species grows now one of row situation:
When i) the anti-seleno culture medium of strain growth cycle stretch-out to last selenium concentration is cultivated more than one times;
When ii) the strain bio amount anti-seleno culture medium that drops to last selenium concentration is cultivated below 30%, even growth;
The iii) obvious variable color of bacterial strain;
When the selenium concentration difference of adjacent batch of anti-seleno culture medium during greater than predetermined threshold, the selenium concentration of adjusting anti-seleno culture medium is the mean value of the selenium concentration of adjacent batch of anti-seleno culture medium.
5. method according to claim 1 is characterized in that the medium of adaptation edible fungus species growth in the said method is the PDA synthetic medium.
6. method according to claim 5 is characterized in that in the said method in the every 100ml constant volume of the PDA synthetic medium medium, raw materials usedly is: potato 20g, glucose 2g, agar 1.5g, MgSO
47H
2O 0.15g, KH
2PO
40.3g, VB1 0.001g, pH transfers to 6.0.
7. method according to claim 6; The compound method that it is characterized in that PDA synthetic medium described in the said method comprises the potato and the distilled water mixing that should add earlier; Keep boiling to filter after 30 minutes, add other raw materials and be settled to volume required step after abundant the mixing.
8. method according to claim 1 is characterized in that selenium source is selected from sodium selenite, sodium selenate or Se-enriched yeast powder in the said method.
9. method according to claim 1 is characterized in that transferring continuously in the said method step (3) when cultivating, and following situation occurs:
A) bacterial classification can't be grown;
B) the growth cycle differentiation is obvious;
C) strain bio amount difference is obvious;
D) the obvious variable color of bacterial strain;
Then judge and do not satisfy the stable condition of anti-selenium proterties; Otherwise think that anti-selenium proterties is stable.
10. method according to claim 1; It is characterized in that said method also is included in step (3) and obtains behind the high selenium tolerance of the edible mushroom bacterial classification the high selenium tolerance bacterial classification that is obtained is linked on the solid inclined-plane that contains selenium PDA synthetic medium that contains best anti-selenium concentration and cultivate, and the step of in-80 ℃ of glycerine pipes, preserving as high selenium tolerance bacterial classification.
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