CN105255736A - Preservation method of hypsizigus marmoreus strain - Google Patents

Preservation method of hypsizigus marmoreus strain Download PDF

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Publication number
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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China
Prior art keywords
wood chip
bacterial classification
preserving
hypsizygus marmoreus
strain
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CN201510807362.6A
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Chinese (zh)
Inventor
陈启永
王旭峰
张红颖
杨华
何乐
班立桐
黄亮
王玉
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Lv Shengpeng Source Tianjin Agricultural Science And Technology Development Corp Ltd
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Lv Shengpeng Source Tianjin Agricultural Science And Technology Development Corp Ltd
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Priority to CN201510807362.6A priority Critical patent/CN105255736A/en
Publication of CN105255736A publication Critical patent/CN105255736A/en
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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

A kind of method for preserving of Hypsizygus marmoreus bacterial classification
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.
CN201510807362.6A 2015-11-19 2015-11-19 Preservation method of hypsizigus marmoreus strain Pending CN105255736A (en)

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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

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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
CN103011963A (en) * 2012-12-18 2013-04-03 成都榕珍菌业有限公司 First-level strain preservation culture medium for Pleurotus eryngii and preparation method of same

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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
CN103011963A (en) * 2012-12-18 2013-04-03 成都榕珍菌业有限公司 First-level strain preservation culture medium for Pleurotus eryngii and preparation method of same

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Cited By (3)

* Cited by examiner, † Cited by third party
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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