CN109294931A - A kind of solution and its method for preserving for true pleurotus cornucopiae strain suspension preservation - Google Patents
A kind of solution and its method for preserving for true pleurotus cornucopiae strain suspension preservation Download PDFInfo
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Abstract
The invention discloses a kind of solution and its method for preserving for true pleurotus cornucopiae strain suspension preservation, belong to culture presevation field.The solution consists of the following compositions: the solvent of the solution is sterile water, and solute is glucose or polyethylene glycol (PEG), and in the solution, the mass concentration of glucose is 0.5%-1.5%.The method for preserving are as follows: true pleurotus cornucopiae strain block is transferred in cryopreservation tube, backward cryopreservation tube in culture presevation liquid is added, make culture presevation liquid submergence fungus block, sealing, low-temperature preservation.The present invention solves in the prior art; distilled water and physiological saline are as preservative fluid; the problem for causing growth performance heavily suppressed due to lacking nutriment or osmotic pressure etc.; it may be implemented to keep that true pleurotus cornucopiae strain vigor is high, do not degenerate, is easy to operate, is cheap, equipment requirement is low, take up space small significant technical effect; powerful guarantee, application value, economy and social effect with higher are provided for the easy preservation of true pleurotus cornucopiae strain and large-scale planting.
Description
Technical field
The invention belongs to culture presevation fields, and in particular to a kind of solution and its guarantor for true pleurotus cornucopiae strain suspension preservation
Hiding method field.
Background technique
True pleurotus cornucopiae (Hypslzygus marmoreus) have improve the immunity of the human body, it is pre- it is anti-aging, to extend the service life etc. more
Kind of effect, by consumer like and a kind of production kind of great potential.True pleurotus cornucopiae contain needed by human body eight kinds must
Palpus amino acid, especially lysine, arginic content are higher than general mushroom class, facilitate teenager's intelligence development and increase, true pleurotus cornucopiae taste
Road is delicious, and heat is low, enjoys the good reputation of " hear then matsutake, food then beautiful gill fungus ", is a kind of full of nutrition edible and medicinal rare
Edible mushroom, and enter one of the important edible mushroom of the factorial production at present.The strain living resources important as one kind, in order to
It keeps its merit constant, preservation should be carried out to it in time, make the metabolism of true pleurotus cornucopiae in least active or opposing stationary
State could make it not morph within the regular hour and keep viability.In particular with true pleurotus cornucopiae scientific research and
The increasingly increasing of the factorial production scale, be badly in need of it is some be widely used, be easy, true pleurotus cornucopiae strain method for preserving effectively, economic
With preservation medium.
Suspension preserving process is one of the common method of edible fungus species preservation, mainly there is distilled water preservation method and physiology at present
Salt water preserving process.Distilled water preservation method can in room temperature preservation edible fungus species, can preservation 1-20, and bacterial strain pollution is few, but steams
The nutriment contained in distilled water is less, and strain vital movement after long-term preservation is heavily suppressed, slow recovery.
Such as the patent of invention " short term storage methods of large-size fungus species " of publication number CN1952109A, disclose large-scale true
The short term storage method of bacterium strain, comprising the following steps: (1) strain for needing preservation is first inoculated on solid medium and is trained
It supports, if waiting mycelium that solid medium is cut into dry strain block when will cover with plate；(2) sterile steaming is first added in a reservoir
Distilled water, then put strain block into, bottle cap is covered tightly, is saved at 4 DEG C.The short term storage method of large-size fungus species of the invention, letter
Just and economical, strain is not likely to produce variation.
A kind of for another example patent of invention " Boletus aereus parent species low temperature conservation method for Chinese " of Publication No. CN104762208B, it is open
The specific steps of fungi low temperature conservation method for Chinese: by Boletus aereus fructification cultivating mycelia on solid medium, and mycelia is made
Block；Mycelia block is immersed in the sterile chamber containing sterile distilled water, sealing container, under the conditions of 10-15 DEG C, places 10-
30d；It is then placed into 3.5-4.5 DEG C of progress low-temperature preservation.Boletus aereus parent species low temperature conservation method for Chinese provided by the invention, passes through
Gradually cooling mode preservation Boletus aereus parent species, after tested Boletus aereus parent species of this method preservation, mycelia vigor and bacterium
Kind cultivated character is identical as generation parent species, saves Boletus aereus parent species vigor and merit up to 3 years, solves black cattle liver starter
The problem of easy kind of preservation of kind middle or short term, powerful guarantee is provided for large-scale planting.
In addition, physiological saline preserving process has high osmotic pressure using salt solution, to the sprouting of miscellaneous bacteria spore have compared with
The pollution of strain during preservation can be reduced or avoided in strong inhibition and killing effect；The hyperosmosis of salt solution also can simultaneously
Cell dehydration is caused, apparent inhibiting effect is generated to the vital movement of the mycelia after long-term preservation, recovery is also relatively more slow
Slowly.
Summary of the invention
Technical problem solved by the invention is to overcome defect present in existing culture presevation solution, provides a kind of holding
True pleurotus cornucopiae strain vigor is high, do not degenerate, is easy to operate, is cheap, equipment requirement is low, it is small to take up space, easy to spread true
The solution of pleurotus cornucopiae strain suspension preservation, and its method for preserving in true pleurotus cornucopiae strain suspension preservation, to widen true pleurotus cornucopiae strain
The range of suspension preserving process.
To achieve the goals above, the present invention is as follows using technical solution:
In terms of the contents of the present invention mainly include following two:
A kind of solution for true pleurotus cornucopiae strain suspension preservation, consists of the following compositions:
The solvent of the solution is sterile water, and solute is glucose or polyethylene glycol (PEG).
Preferably, in the solution, the mass concentration of glucose is 0.5%-1.5%.
Preferably, in the solution, the mass concentration of glucose is 0.6-0.8%.
It is highly preferred that the mass concentration of glucose is 0.8% in the solution.
Preferably, in the solution, molecular weight polyethylene glycol 6000-20000, mass concentration 0.05%-0.2%,；
It is highly preferred that in the solution, molecular weight polyethylene glycol 6000, mass concentration 0.05%-0.2%.
It is highly preferred that in the solution, molecular weight polyethylene glycol 6000, mass concentration 0.1%.
It is a further object of the present invention to provide the use true pleurotus cornucopiae strain suspension preservative solutions in the true pleurotus cornucopiae strain of preservation
In method, the specific steps are as follows:
(1) glucose or polyethylene glycol are dissolved with sterile distilled water, is configured to the preservative solution of required concentration and using filter
Film filtering.
(2) will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia covers with plate, with punch with
It is punched on the plate of true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block.
(3) the true pleurotus cornucopiae strain block is transferred in sterilized cryopreservation tube with transfer needle, the backward cryopreservation tube in
Culture presevation liquid is added, makes the culture presevation liquid submergence fungus block, sealing, low-temperature preservation.
Preferably, sterile distilled water described in the step (1) is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121
DEG C, sterilize 20min.The step can effectively ensure that the interference of miscellaneous bacteria in culture presevation.
Preferably, in the step (1), the filter sizes are 0.22 μm.
Preferably, in the step (2), the fungus block diameter of the true pleurotus cornucopiae strain is 6mm-10mm.
It is highly preferred that the fungus block diameter of the true pleurotus cornucopiae strain is 9mm in the step (2).
Preferably, in the step (3), the number that true pleurotus cornucopiae strain block is transferred in the cryopreservation tube is 3-7 block.
It is highly preferred that the number for being transferred to true pleurotus cornucopiae strain block in the cryopreservation tube is 5 pieces in the step (3).
Preferably, the volume of culture presevation liquid, which accounts for, in the step (3), in the cryopreservation tube described freezes pipe volume
65%-95%.
It is highly preferred that in the step (3), in the cryopreservation tube, the volume of culture presevation liquid, which is accounted for, described freezes pipe volume
80%.
Preferably, in the step (3), the temperature of the low-temperature preservation is 2 DEG C -10 DEG C.
Compared with prior art, the invention has the benefit that
True pleurotus cornucopiae strain preservative fluid and method for preserving provided by the invention, method is easy, and equipment is simple, and preservation effect is better than
Existing physiological saline preserving process and distilled water preservation method, mycelia vigor is identical as generation parent species after preservation, is true pleurotus cornucopiae strain
Easy preservation and large-scale planting provide powerful guarantee, application value, economic significance and social effect with higher.
Preservative fluid of the invention obtains significant technical effect in culture presevation.After measured, wherein in selection grape
Sugar obtains significant skill on mycelial growth rate V, dehydrogenase activity and percent of decolourization DR as in culture presevation liquid
Art effect, and in preservation to June, September, more show glucose solution for the permanent protectiveness of fungi activity, especially
At preservation 9 months, mycelia highest growth rate can reach 0.45, the 0.9%NaCl and distilled water in more same preservation period
It is higher by 0.225 and 0.075 respectively；Wherein, highest dehydrogenase activity can reach 0.7820, the 0.9% of more same preservation period
NaCl and distilled water are higher by 0.4685 and 0.155 respectively.
Equally, in Selection utilization polyethylene glycol as in culture presevation liquid, in mycelial growth rate V, dehydrogenase activity
And significant technical effect is also obtained on percent of decolourization DR.Equally in the long-term preservation of strain, strain can be effectively kept
Activity, at preservation 9 months, mycelia highest growth rate can reach 0.5750, the 0.9%NaCl in more same preservation period with
And distilled water is higher by 0.35 and 0.2 respectively；Wherein, highest dehydrogenase activity can reach 0.7820, more same preservation period
0.9%NaCl and distilled water are higher by 0.4685 and 0.155 respectively.
In addition, the specification for being directed to the strain of preservation in the present invention has carried out specific restriction, the diameter of true pleurotus cornucopiae strain block
For 6mm-10mm, the number that true pleurotus cornucopiae strain block is transferred in cryopreservation tube is 3-7 block, which can effectively keep spawn activity and cultivation
Character is trained, can reach the effect of the effective preservation of strain and the purpose to economize on resources.
It accounts for cryopreservation tube volume ratio simultaneously for the volume of culture presevation liquid in cryopreservation tube and is defined, which can make mycelia
Body keeps more long vigor.
Specific embodiment:
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.Below in conjunction with specific embodiment, invention is further explained.
Embodiment 1: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves glucose with sterile distilled water, is configured to the preservative solution that mass concentration is 0.6%.It passes through in superclean bench
0.22 μm of membrane filtration.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 9mm, fungus block number
It is 5 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 4 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 80% of pipe volume in cryopreservation tube.
Embodiment 2: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves glucose with sterile distilled water, is configured to the glucose solution that mass concentration is 0.8%.It passes through in superclean bench
0.22 μm of membrane filtration.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 10mm, fungus block number
It is 4 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 4 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 65% of pipe volume in cryopreservation tube.
Embodiment 3: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves glucose with sterile distilled water, is configured to the glucose solution that mass concentration is 1.0%.It passes through in superclean bench
0.22 μm of membrane filtration.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 6mm, fungus block number
It is 6 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 10 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 70% of pipe volume in cryopreservation tube.
Embodiment 4: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves glucose with sterile distilled water, is configured to the glucose solution that mass concentration is 1.2%.It passes through in superclean bench
0.22 μm of membrane filtration.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 8mm, fungus block number
It is 4 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 5 DEG C of preservations of low temperature.Wherein culture presevation liquid in cryopreservation tube
Volume freezes the 80% of pipe volume described in accounting for.
Embodiment 5: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 1, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is glucose, mass concentration 0.8%.
Embodiment 6: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 1, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is glucose, mass concentration 1.0%.
Embodiment 7: method for preserving of the glucose solution in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 1, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is glucose, mass concentration 1.2%.
Embodiment 8: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves polyethylene glycol with sterile distilled water, is configured to the polyglycol solution that mass concentration is 0.1%.In superclean bench
It is middle through 0.22 μm of membrane filtration, wherein the molecular weight of polyethylene glycol is 6000.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 7mm, fungus block number
It is 4 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 5 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 80% of pipe volume in cryopreservation tube.
Embodiment 9: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves polyethylene glycol with sterile distilled water, is configured to the polyglycol solution that mass concentration is 0.05%.In superclean bench
It is middle through 0.22 μm of membrane filtration, wherein the molecular weight of polyethylene glycol is 6000.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 6mm, fungus block number
It is 5 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 5 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 70% of pipe volume in cryopreservation tube.
Embodiment 10: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves polyethylene glycol with sterile distilled water, is configured to the polyglycol solution that mass concentration is 0.2%.In superclean bench
It is middle through 0.22 μm of membrane filtration, wherein the molecular weight of polyethylene glycol is 6000.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 7mm, fungus block number
It is 4 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 5 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 85% of pipe volume in cryopreservation tube.
Embodiment 11: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
(1) distilled water is cooling stand-by through high-temperature sterilization.Sterilising conditions are as follows: 121 DEG C, sterilizing 20min obtains sterile distillation
Water dissolves polyethylene glycol with sterile distilled water, is configured to the polyglycol solution that mass concentration is 0.15%.In superclean bench
It is middle through 0.22 μm of membrane filtration, wherein the molecular weight of polyethylene glycol is 6000.
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia soon covers with plate, be existed with punch
It is punched on plate with true pleurotus cornucopiae mycelia, obtains true pleurotus cornucopiae strain block, the diameter of true pleurotus cornucopiae strain block is 8mm, fungus block number
It is 4 pieces.
(3) the true pleurotus cornucopiae strain block is aseptically transferred to sterilized cryopreservation tube with transfer needle, moved into later
Culture presevation liquid makes the culture presevation liquid submergence fungus block, sealing, 5 DEG C of preservations of low temperature.
Wherein the volume of culture presevation liquid accounts for and described freezes the 80% of pipe volume in cryopreservation tube.
Embodiment 12: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 8, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is polyethylene glycol, mass concentration 0.1%, molecular weight 10000.
Embodiment 13: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 8, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is polyethylene glycol, mass concentration 0.05%, molecular weight 20000.
Embodiment 14: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 8, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is polyethylene glycol, mass concentration 0.1%, molecular weight 20000.
Embodiment 15: method for preserving of the Aqueous Solutions of Polyethylene Glycol in true pleurotus cornucopiae strain suspension preservation
Preservative fluid is prepared according to method prepared by embodiment 8, the true pleurotus cornucopiae strain of preservation is unique the difference is that culture presevation liquid
Ingredient is polyethylene glycol, mass concentration 0.2%, molecular weight 20000.
The measurement of 17 culture presevation performance of embodiment
For the true pleurotus cornucopiae strain preservation sample that different embodiments prepare, it is measured respectively and was saved through 3,6,9 months
Culture presevation performance test afterwards, i.e. mycelial growth rate, mycelia dehydrogenase activity, the test of LBL culture medium percent of decolourization.
Wherein:
Control group 1: culture presevation solution is the NaCl solution that mass concentration is 0.9%, the same embodiment of other preparation methods
1；
Control group 2: culture presevation solution is distilled water, and the preparation method is the same as that of Example 1 for other；
Experimental group 1,2,3,4 respectively corresponds the embodiment of the present invention 1,5,6,7；
Experimental group 5,6,7,8,9 respectively corresponds the embodiment of the present invention 8,12,13,14,15.
Measuring method is as follows:
Culture presevation effect detection 1: mycelial growth rate V
Take one had the cryopreservation tube of strain recover at 25 DEG C to cultivate 3 days by 3 months, 6 months and preservation in 9 months,
Then one piece of strain block is taken to be inoculated on PDA plate, 48h measures colony diameter after mycelium germination, it is calculated by the following formula:
Mycelial growth rate V (cm/d)=(colony diameter 2- colony diameter 1)/time (d)
Culture presevation effect detection 2: mycelia dehydrogenase activity
From taking mycelium 0.1g to be fitted into 15mL centrifuge tube in plate, 2mL distilled water is added, adds 0.2mL, 5mg/mL
MTT solution, be protected from light 35 DEG C of water-baths, stand 3 hours.Supernatant is removed, 2mL DMSO is added, is protected from light 35 DEG C of water-baths, stands 4 hours.
It takes 100 μ l of supernatant liquid to measure light absorption value OD, DMSO at 490nm wavelength and makees blank.
Culture presevation effect detection 3:LBL culture medium percent of decolourization
LBL culture medium is made according to the proportion of table 1.Ligand approach is identical as PDA culture medium.
1 LBL culture medium prescription of table
It is transferred in a 5ml cryopreservation tube from the mycelia block for picking up 2 diameter 9mm in plate, access contains 35ml after being homogenized 30s
In the 50ml centrifuge tube of LBL culture medium, stationary culture is carried out under the conditions of 25 DEG C.500 μ l of culture solution is taken after Liquid Culture 16d,
5000r/min is centrifuged 15min at 4 DEG C, pipettes 100 μ l supernatants and ELISA Plate is added, and uses extinction at microplate reader measurement 615nm
Angle value imports percent of decolourization formula and calculates percent of decolourization:
DR=[1- (A615nm of strain/A615nm of blank)] × 100%
(DR is expressed as a percentage, A615nm of strainFor the light absorption value after inoculating strain culture, A615nm of blankFor blank
The light absorption value of culture solution).
Preservation effect is as shown in table 2, the results showed that several glucose solutions for participating in test protect true pleurotus cornucopiae strain suspension
The result of hiding is effectively, wherein 0.8% glucose solution preservation effect is preferable.The preservation effect of preservation initial stage glucose solution
It is weaker than physiological saline and distilled water, but with the growth of preservation time, the preservation effect of glucose solution, which shows, to be gradually better than
The trend of physiological saline and distilled water illustrates that glucose preservative fluid is suitable for medium-term and long-term preservation.
Preservation effect of 2 glucose solution of table to true pleurotus cornucopiae strain
From Table 2, it can be seen that selecting glucose as in culture presevation liquid, in mycelial growth rate V, dehydrogenase
Significant technical effect is obtained on activity and percent of decolourization DR, especially in preservation to June, September, more shows glucose
Solution is significantly better than physiological saline and distilled water for the permanent protectiveness of fungi activity, especially at preservation 9 months, bacterium
Silk highest growth rate can reach 0.45, the 0.9%NaCl and distilled water in more same preservation period be higher by respectively 0.225 and
0.075；Wherein, highest dehydrogenase activity can reach 0.7820,0.9%NaCl and the distilled water difference in more same preservation period
It is higher by 0.4685 and 0.155.
In addition, it should be noted that, in the continuous mode of percent of decolourization, association of the percent of decolourization as measurement mycelia growth performance
The percent of decolourization of same sex reference index, strain is bigger, shows that the PH of culture medium in Liquid Culture is changed significantly, this is exhaled with mycelia
Suction effect is related, and under normal circumstances, with the raising of mycelial growth rate, mycelia growth is more vigorous, then mycelia respiration
Stronger, percent of decolourization is high, but percent of decolourization it is high mycelia growth it is not necessarily high because percent of decolourization is also permeability-related with film, and film
Permeability again it is related with the osmotic pressure of solution.It, can and from the application strain in glucose solution during preservation 9 months
To find out, growth rate is significantly higher than distilled water group and 0.9%NaCl, and its percent of decolourization is same compared to 0.9%NaCl
Also there is different degrees of raising trend.
Equally, in Selection utilization polyethylene glycol as in culture presevation liquid, in mycelial growth rate V, dehydrogenase activity
And significant technical effect is also obtained on percent of decolourization DR.Equally in the long-term preservation of strain, strain can be effectively kept
Activity, at preservation 9 months, mycelia highest growth rate can reach 0.5750, the 0.9%NaCl in more same preservation period with
And distilled water is higher by 0.35 and 0.2 respectively；Wherein, highest dehydrogenase activity can reach 0.7820, more same preservation period
0.9%NaCl and distilled water are higher by 0.4685 and 0.155 respectively.
In the continuous mode of percent of decolourization, and from the application strain in polyglycol solution during preservation 9 months,
As can be seen that its growth rate is significantly higher than distilled water group and 0.9%NaCl, and its percent of decolourization is same compared to 0.9%NaCl
Sample also has different degrees of raising trend.
It should be noted that the glucose of the technical effect of 2-4 of embodiment of the present invention same concentrations corresponding with embodiment 5-7
Solution has similar technical effect.
The strain that the embodiment of the present invention 9,10,11 is prepared is in mycelial growth rate V, dehydrogenase activity and percent of decolourization
Technical effect on DR have with significant technical effect similar in embodiment 8 (experimental group 5), wherein strain grows speed in mycelia
In rate, preservation 3 monthly to reach 0.9 or more, and preservation 6 monthly to reach 0.82 or more, preservation 9 monthly reach 0.5 with
On.And in dehydrogenase activity, preservation 3 monthly to reach 1.2 or more, and preservation 6 monthly to reach 1.1 or more, preservation 9
It is monthly to reach 0.7 or more.
Claims (10)
1. a kind of solution for true pleurotus cornucopiae strain suspension preservation, it is characterised in that: consist of the following compositions:
The solvent of the solution is sterile water, and solute is glucose or polyethylene glycol；In the solution, the quality of glucose is dense
Degree is 0.5%-1.5%.
2. a kind of solution that can be used for true pleurotus cornucopiae strain suspension preservation as described in claim 1, it is characterised in that: the solution
In, the mass concentration of glucose is 0.6-0.8%.
3. a kind of solution that can be used for true pleurotus cornucopiae strain suspension preservation as described in claim 1, it is characterised in that: the solution
In, the molecular weight of polyethylene glycol is 6000-20000, mass concentration 0.05%-0.2%.
4. a kind of solution that can be used for true pleurotus cornucopiae strain suspension preservation as claimed in claim 3, it is characterised in that: the solution
In, the molecular weight of polyethylene glycol is 6000, mass concentration 0.05%-0.2%.
5. being used for side of the true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as described in claim 1-4 is any
Method, it is characterised in that: specific step is as follows:
(1) glucose or polyethylene glycol are dissolved with sterile water, be configured to the preservative solution of required concentration and use membrane filtration；
(2) it will be inoculated on PDA plate to the true pleurotus cornucopiae strain of preservation, when mycelia covers with plate, with true pleurotus cornucopiae mycelia
It is punched on plate, obtains true pleurotus cornucopiae strain block；
(3) the true pleurotus cornucopiae strain block is transferred in sterilized cryopreservation tube with transfer needle, the backward cryopreservation tube in be added
Culture presevation liquid, and fungus block is submerged, it seals, low-temperature preservation.
6. a kind of method for true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as claimed in claim 5,
It is characterized by: sterile water described in the step (1) is through high-temperature sterilization, cooling stand-by, the high-temperature sterilization condition are as follows: 121
DEG C, sterilize 20min.
7. a kind of method for true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as claimed in claim 5,
It is characterized by: the diameter of the true pleurotus cornucopiae strain block is 6mm-10mm in the step (2).
8. a kind of method for true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as claimed in claim 5,
It is characterized by: the number for being transferred to true pleurotus cornucopiae strain block in the cryopreservation tube is 3-7 block in the step (3).
9. a kind of method for true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as claimed in claim 5,
It is characterized by: the volume of culture presevation liquid accounts for the 65%- for freezing pipe volume in the cryopreservation tube in the step (3)
95%.
10. a kind of method for true pleurotus cornucopiae strain suspension preservative solution in the true pleurotus cornucopiae strain of preservation as claimed in claim 5,
It is characterized by: the temperature of the low-temperature preservation is 2 DEG C -10 DEG C in the step (3).
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| CN109735454A (en) * | 2019-02-22 | 2019-05-10 | 天津农学院 | A kind of sawdust storage medium and its method for preserving for true pleurotus cornucopiae strain |
| CN109735454B (en) * | 2019-02-22 | 2022-03-25 | 天津农学院 | Wood chip preservation medium for hypsizigus marmoreus strains and preservation method thereof |
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