CN105255736A - Preservation method of hypsizigus marmoreus strain - Google Patents
Preservation method of hypsizigus marmoreus strain Download PDFInfo
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- CN105255736A CN105255736A CN201510807362.6A CN201510807362A CN105255736A CN 105255736 A CN105255736 A CN 105255736A CN 201510807362 A CN201510807362 A CN 201510807362A CN 105255736 A CN105255736 A CN 105255736A
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- wood chip
- bacterial classification
- preserving
- hypsizygus marmoreus
- strain
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000004321 preservation Methods 0.000 title abstract description 13
- 230000001954 sterilising effect Effects 0.000 claims abstract description 36
- 239000001963 growth medium Substances 0.000 claims abstract description 18
- 241000209094 Oryza Species 0.000 claims abstract description 15
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 15
- 235000009566 rice Nutrition 0.000 claims abstract description 15
- 238000011081 inoculation Methods 0.000 claims abstract description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000002023 wood Substances 0.000 claims description 44
- 230000001580 bacterial effect Effects 0.000 claims description 36
- 241001534815 Hypsizygus marmoreus Species 0.000 claims description 21
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims description 10
- 235000011613 Pinus brutia Nutrition 0.000 claims description 10
- 241000018646 Pinus brutia Species 0.000 claims description 10
- 241000219000 Populus Species 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 8
- 239000002994 raw material Substances 0.000 abstract 2
- 238000012546 transfer Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- Mushroom Cultivation (AREA)
Abstract
The invention provides a preservation method of a hypsizigus marmoreus strain. The method comprises a culture medium mixing process, a culture medium sterilization process, a strain inoculation process, a strain culture process and a strain subculture process, wherein the strain subculture process is the process for carrying out primary subculture in the culture medium at every 30-35 days; the strain culture medium is prepared from the following raw materials in percentage by weight: 20%-25% of sawdust, 10%-15% of rice bran and 65% of water, wherein the percentage by weight of the sawdust and the rice bran is the percentage by dry weight. According to the preservation method provided by the invention, the raw material for the culture medium is low in price, easy to purchase, convenient to store, long in strain preservation period and stable in strain character; the strain preserved by the preservation method can be continuously used for 10-20 years.
Description
Technical field
The invention belongs to cell preservation technique field, especially relate to a kind of method for preserving of Hypsizygus marmoreus bacterial classification.
Background technology
Hypsizygus marmoreus contains abundant VITAMIN and multiple amino acids, wherein Methionin, arginic content is higher than general mushroom class, contribute to teenager's intelligence development to increase, and have anticancer, reduce the effect of cholesterol, because the value of Hypsizygus marmoreus is higher, now become one of important kind of national edible fungus industrial production, bacterial classification is as a kind of important Biological resources, in order to keep its good character constant, preservation should be carried out in time to it, the metabolism of microorganism is made to be in state that is least active or geo-stationary, it could be made within the regular hour not morph and keep viability.
Spawn Preservation of Edible Fungi mainly contains liquid nitrogen cryogenics preserving process, the regular transfer methods in inclined-plane, whiteruss method, sand tube preservation method and nature culture precious deposits method etc., liquid nitrogen cryogenics preserving process is a kind of method more advanced at present, but costly, cannot popularization and application in factorial praluction, at present in factorial praluction, generally carry out Tube propagation with PDA substratum, be positioned in 4 DEG C of refrigerators, within every 90 days, carry out a succeeding transfer culture, the preservation term of its bacterial classification is generally 1-2, there is culture medium cost high, cultivation period is long, poor quality, the problems such as pollution rate is high, therefore in the urgent need to the simple and effective culture collection process of one, under the prerequisite effectively keeping bacterial classification proterties, long period can also be used.
Summary of the invention
In view of this, the present invention is intended to the method for preserving of the Hypsizygus marmoreus bacterial classification that proposition is a kind of simply effectively, preservation term is long, cost is low, avoids existing method for preserving high in cost of production shortcoming.
For achieving the above object, technical scheme of the present invention is achieved in that a kind of method for preserving of Hypsizygus marmoreus bacterial classification, comprise the mixing of substratum, medium sterilization, strain inoculation, spawn culture, bacterial classification subculture process, the weight percent of described culture presevation culture medium prescription is: wood chip 20-25%, rice bran 10-15%, water 65%, described wood chip, rice bran is the per-cent of dry weight weight, said components is mixed in proportion and carries out preparation substratum, the substratum prepared is put into sterilising plant and carries out sterilizing, Hypsizygus marmoreus strain inoculation is cultivated to the substratum of sterilizing, within every 30-35 days, carry out a subculture, namely bacterial classification inoculated in every 30-35 days on the above-mentioned bacterium culture medium prepared and carry out succeeding transfer culture, the Subculture of recirculation bacterial classification.
As preferably, described wood chip comprises poplar wood chip and pine wood chip, and wherein poplar wood chip accounts for the 30%-50% of wood chip gross dry weight ratio, and pine wood chip accounts for the 50%-70% of wood chip gross dry weight ratio.
As preferred, the weight percent of described culture medium prescription is: wood chip 22%, rice bran 13%, water 65%, and wherein said wood chip, rice bran weight percent are the per-cent of dry weight weight.
As preferably, the weight percent of described culture medium prescription is: wood chip 20-25%, rice bran 10-15%, water 65%, and wherein said wood chip, rice bran weight percent are the per-cent of dry weight weight.
As preferred, described wood chip comprises poplar wood chip and pine wood chip, and described poplar wood chip accounts for 48% of gross dry weight ratio, and described pine wood chip accounts for 52% of wood chip gross dry weight ratio.
As preferably, described medium sterilization process is: first 95 DEG C of sterilizing 55-65 minute, then 105 DEG C of sterilizing 25-35 minute, afterwards 121 DEG C of sterilizing 40-60 minute, finally stewingly puts 80-100 minute.
As preferred, described medium sterilization process is: first 95 DEG C of sterilizings 60 minutes, then 105 DEG C of sterilizings 30 minutes, 121 DEG C of sterilizings afterwards 50 minutes, finally stewingly puts 90 minutes.
As preferably, the inoculation condition of described strain inoculation process is that the substratum after sterilizing is cooled to 18-22 DEG C, then inoculates in an aseptic environment.
As more there being choosing, the growing environment of described Hypsizygus marmoreus bacterial classification is: temperature is 20-22 DEG C, and humidity is 60-70%, and gas concentration lwevel is 1500-2500ppm, in actual production, gas concentration lwevel is 2000ppm is preferred embodiment.
Succeeding transfer culture is to coming from culture (comprising cell, tissue or its segment) that explant breeds by changing fresh culture and constantly cutting or be separated, carrying out the cultivation of continuous multi-generation.
The advantage that the present invention has and positively effect are: compared with prior art, and the prices of raw and semifnished materials of method for preserving of the present invention are cheap, are easy to buy, it is convenient to store, and the culture presevation phase is long, utilizes the bacterial classification of this method for preserving preservation can use 10-20 continuously, and bacterial classification proterties is stablized, the Hypsizygus marmoreus bacterial classification adopting this method to cultivate is with short production cycle, and mycelium growth vigor is vigorous, energetic, after being inoculated into planting material, field planting is fast, and pollution rate is low, not easily aging.
Embodiment
Embodiment one
Substratum is prepared: the weight percent of culture medium prescription is: wood chip 22%, rice bran 13%, water 65%, wherein the weight percent of wood chip, rice bran is dry weight weight percent, poplar wood chip accounts for 40% of wood chip gross dry weight ratio, pine wood chip accounts for 60% of wood chip gross dry weight ratio, said components is carried out preparation substratum in proportion, first poplar wood chip, pine wood chip, rice bran is added in spice pot; Stir and mix for 30-40 minute, then add water according to formula moisture ratio, continue to be stirred to Compost moisture content in spice pot consistent.
Medium sterilization: the substratum prepared is loaded in culturing bottle, chooses 200 bottles and test, substratum is put into sterilising plant and carry out sterilizing, first 95 DEG C of sterilizings 60 minutes, then 105 DEG C of sterilizings 30 minutes, 121 DEG C of sterilizings afterwards 50 minutes, finally stewingly put 90 minutes.
Strain inoculation: the substratum after sterilizing is cooled to 18-22 DEG C, is positioned on Bechtop by cooled substratum, by Hypsizygus marmoreus strain inoculation on the substratum of sterilizing.
Spawn culture environment: postvaccinal Hypsizygus marmoreus bacterial classification is positioned over culturing room, culturing room's temperature is 20-22 DEG C, and humidity is 60-70%, and gas concentration lwevel is 2000ppm, and incubation time is 30 days.
Bacterial classification subculture: inoculate in the substratum after sterilizing in an aseptic environment by the bacterial classification after cultivating, carry out a succeeding transfer culture, recirculation in every 30 days, bacterial classification proterties can keep not degenerating for 10 years.
Carry out factorial praluction with this bacterial classification, directly can carry out field planting, field planting after stain rate is 0.3%.
Comparative example
The preparation of substratum: PDA culture medium prescription per-cent is as follows: potato 17%, glucose 1.6%, agar 1.4%, water 80%, take one by one in culture medium prescription ratio, potato is cut into small pieces and puts into vessel and then add water, be heated to boiling on the heaters, maintain 20-30 minute, filter on measuring cup with gauze, keep the skin wet again to aequum after filtration, NaOH or HCl with 10% carries out adjust ph to 6-7, add glucose, agar heats, and constantly stir with glass rod, after agar dissolves completely, keep the skin wet again to aequum, the substratum of preparation is distributed in vitro, dispensed loading amount is about 1/5 of test tube height, packing quantity is 200.
Medium sterilization: at 104kPa sterilized under pressure 20-30 minute.
Strain inoculation: the substratum after subject to sterilization is cooled to 18-22 DEG C, is positioned on Bechtop by cooled test-tube culture medium, by Hypsizygus marmoreus strain inoculation on the test-tube culture medium of sterilizing.
Spawn culture environment: cultivated in 4 DEG C of refrigerators by postvaccinal test tube slant setting, incubation time is 90 days.
Bacterial classification succeeding transfer culture: the bacterial classification after cultivating is inoculated in PDA substratum in an aseptic environment.Within every 90 days, carry out a succeeding transfer culture, recirculation.
Carry out factorial praluction with the bacterial classification of this method for preserving preservation, field planting after stain rate is 1%.
Effect test
Choose 200 bottles of bacterial classifications in embodiment one and comparative example respectively as effect test, can be found out by table 1, embodiment one is 89% at the cultivating rate of the 2nd year, maintains higher cultivating rate, embodiment one is 46.8% at the cultivating rate of the 10th year, and from the 10th year, degradation phenomena was serious.
Comparative example is 35% at the cultivating rate of the 2nd year, and serious degradation phenomena has appearred in bacterial classification, needs again to buy bacterial classification and carries out subsequent production.
By relatively finding out, the bacterium culture medium prepared by the method for embodiment one and method for preserving can maintain the use of more than 10 years, for large-scale factorial praluction has saved cost.
Table 1 embodiment one compares with the cultivating rate of comparative example
Above embodiments of the invention have been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the present patent application scope change and improve, and all should still belong within patent covering scope of the present invention.
Claims (9)
1. the method for preserving of a Hypsizygus marmoreus bacterial classification, comprise the mixing of substratum, medium sterilization, strain inoculation, spawn culture, bacterial classification subculture process, it is characterized in that: described bacterial classification subculture process is described bacterial classification every process of carrying out a subculture for 30-35 days in described substratum.
2. the method for preserving of Hypsizygus marmoreus bacterial classification according to claim 1, it is characterized in that: the weight percent of described culture medium prescription is: wood chip 20-25%, rice bran 10-15%, water 65%, wherein said wood chip, rice bran weight percent are the per-cent of dry weight weight.
3. Hypsizygus marmoreus culture collection process according to claim 2, is characterized in that: the weight percent of described culture medium prescription is: wood chip 22%, rice bran 13%, water 65%, and wherein said wood chip, rice bran weight percent are the per-cent of dry weight weight.
4. the method for preserving of Hypsizygus marmoreus bacterial classification according to claim 1 and 2, it is characterized in that: described wood chip comprises poplar wood chip and pine wood chip, described poplar wood chip accounts for the 30%-50% of wood chip gross dry weight ratio, and described pine wood chip accounts for the 50%-70% of wood chip gross dry weight ratio.
5. the method for preserving of Hypsizygus marmoreus bacterial classification according to claim 4, is characterized in that: described wood chip comprises poplar wood chip and pine wood chip, and described poplar wood chip accounts for 48% of gross dry weight ratio, and described pine wood chip accounts for 52% of wood chip gross dry weight ratio.
6. the method for preserving of the Hypsizygus marmoreus bacterial classification according to claim 1 or 2 or 3, it is characterized in that: described medium sterilization process is: first 95 DEG C of sterilizing 55-65 minute, then 105 DEG C of sterilizing 25-35 minute, afterwards 121 DEG C of sterilizing 40-60 minute, finally boil in a covered pot over a slow fire and put 80-100 minute.
7. the method for preserving of Hypsizygus marmoreus bacterial classification according to claim 6, is characterized in that: described medium sterilization process is: first 95 DEG C of sterilizings 60 minutes, then 105 DEG C of sterilizings 30 minutes, 121 DEG C of sterilizings afterwards 50 minutes, finally stewingly puts 90 minutes.
8. the method for preserving of the Hypsizygus marmoreus bacterial classification according to claim 1 or 2 or 3 or 5, is characterized in that: the inoculation condition of described strain inoculation is that the substratum after sterilizing is cooled to 18-22 DEG C, then inoculates in an aseptic environment.
9. the method for preserving of Hypsizygus marmoreus bacterial classification according to claim 8, is characterized in that: the culture environment in described spawn culture stage is: temperature is 20-22 DEG C, and humidity is 60-70%, and gas concentration lwevel is 1500-2500ppm.
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| CN201510807362.6A CN105255736A (en) | 2015-11-19 | 2015-11-19 | Preservation method of hypsizigus marmoreus strain |
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| CN201510807362.6A CN105255736A (en) | 2015-11-19 | 2015-11-19 | Preservation method of hypsizigus marmoreus strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108315261A (en) * | 2018-04-13 | 2018-07-24 | 四川省农业科学院土壤肥料研究所 | A kind of edible mushroom storage medium and preparation method thereof |
| CN109735454A (en) * | 2019-02-22 | 2019-05-10 | 天津农学院 | A kind of sawdust preservation medium for shimeji mushroom strain and preservation method thereof |
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| CN102835253A (en) * | 2012-09-21 | 2012-12-26 | 山东正汉生物科技集团有限公司 | Optimization process for factory production of hypsizygus marmoreus by adopting liquid strains |
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2015
- 2015-11-19 CN CN201510807362.6A patent/CN105255736A/en active Pending
Patent Citations (3)
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| CN102816012A (en) * | 2012-09-21 | 2012-12-12 | 山东正汉生物科技集团有限公司 | Preservation culture medium for hypsizygus marmoreus stock culture and method for preparing preservation culture medium |
| CN102835253A (en) * | 2012-09-21 | 2012-12-26 | 山东正汉生物科技集团有限公司 | Optimization process for factory production of hypsizygus marmoreus by adopting liquid strains |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315261A (en) * | 2018-04-13 | 2018-07-24 | 四川省农业科学院土壤肥料研究所 | A kind of edible mushroom storage medium and preparation method thereof |
| CN109735454A (en) * | 2019-02-22 | 2019-05-10 | 天津农学院 | A kind of sawdust preservation medium for shimeji mushroom strain and preservation method thereof |
| CN109735454B (en) * | 2019-02-22 | 2022-03-25 | 天津农学院 | A kind of sawdust preservation medium for shimeji mushroom strain and preservation method thereof |
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