CN110402758A - A kind of method of the screening and culturing medium and the lab screening black fungus strain of resistance to heat shock of the black fungus strain of resistance to heat shock - Google Patents
A kind of method of the screening and culturing medium and the lab screening black fungus strain of resistance to heat shock of the black fungus strain of resistance to heat shock Download PDFInfo
- Publication number
- CN110402758A CN110402758A CN201910710755.3A CN201910710755A CN110402758A CN 110402758 A CN110402758 A CN 110402758A CN 201910710755 A CN201910710755 A CN 201910710755A CN 110402758 A CN110402758 A CN 110402758A
- Authority
- CN
- China
- Prior art keywords
- black fungus
- screening
- sawdust
- strain
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/20—Culture media, e.g. compost
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
- A01G18/64—Cultivation containers; Lids therefor
- A01G18/66—Cultivation bags
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/60—Cultivation rooms; Equipment therefor
- A01G18/69—Arrangements for managing the environment, e.g. sprinklers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Environmental Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides the methods of a kind of screening and culturing medium of black fungus strain of resistance to heat shock and the lab screening black fungus strain of resistance to heat shock, belong to black fungus strain screening technique field.Screening and culturing medium of the present invention is suitable for the lab screening black fungus strain of resistance to heat shock, can quick and precisely screen the black fungus bacterial strain of resistance to heat shock using the culture medium；The present invention also provides a kind of methods for screening the black fungus strain of resistance to heat shock indoors using screening and culturing medium described in above scheme, this method is including the use of the culture of primary dcreening operation culture medium and utilizes the culture of secondary screening culture medium, black fungus strain to be screened is passed sequentially through in each step and is protected from light culture, heat shock culture and renewal cultivation, to filter out the black fungus strain of resistance to heat shock.Method of the invention effectively can accurately screen the black fungus bacterial strain of resistance to heat shock.
Description
Technical field
The present invention relates to a kind of screening of black fungus strain screening technique field more particularly to black fungus strain of resistance to heat shock trainings
The method for supporting base and the lab screening black fungus strain of resistance to heat shock.
Background technique
Black fungus (Auricularia heimuer) quality is soft, and sliding crisp and refreshing mouth, is that a kind of food medicine dual-purpose of preciousness is true
Bacterium.Black fungus is full of nutrition, and content is essentially identical in the content and meat of protein, vitamin B2Content be general rice, face
With 10 times of Chinese cabbage, it is 3~5 times higher than pig, beef or mutton；Structure-the Fe of hemochrome in human erythrocyte ball, in agaric
Content is 100 times higher than meat；The content of calcium is 30~70 times of meat；The content of p and s is also higher than meat；Agaric is rich simultaneously
Containing essential amino acid.Black fungus also has certain medical value.In agaric containing polysaccharide, adenosine, melanin, ergosterol,
The chemical components such as phospholipid and multivitamin, and have reducing blood lipid, antithrombotic, it is anti-radiation, it is anti-mutation, it is anti-oxidant, anti-ageing
Always, the bioactivity such as hypoglycemic, raising immunity, anticancer.
The Northeast is due to its unique Cold and cool climate feature, and the black fungus color of production is black, good quality, by consumer's
Consistent favor.Easily occur the bad weathers such as thermal extremes during black fungus produce agaric, high temperature causes black fungus produce agaric difficult, auricle
Yellowish, the harm such as yield reduction, has seriously endangered the sound development of black fungus industry.Black fungus industry rapidly develops,
There is " Bei Ernan expansion ".But since the more northern temperature of southern temperature is high, high temperature etc. is frequently encountered in black fungus production process
Exceedingly odious weather condition.And black fungus fine quality is based on middle low temperature type at this stage, it is therefore desirable to screen high temperature modification
Black fungus kind, for cultivating high temperature black agaric new varieties, to adapt to the exceedingly odious weather such as high temperature and unexpected high temperature day
Situations such as gas.
Summary of the invention
The purpose of the present invention is to provide a kind of screening and culturing medium of black fungus strain of resistance to heat shock and the resistance to heat shocks of lab screening
The method of black fungus strain can quick and precisely screen the black fungus bacterial strain of resistance to heat shock using the culture medium.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of screening and culturing medium of black fungus strain of resistance to heat shock, the screening and culturing medium includes primary dcreening operation training
Support base and secondary screening culture medium；
The primary dcreening operation culture medium is in terms of 1L, the component including following quality: 200~250g of potato, and glucose 20~
25g, thin 8~12g of sawdust, 3~5g of wheat bran, 0.5~1.0g of magnesium sulfate, 1.5~2.0g of potassium dihydrogen phosphate and agar powder 18~
20g；The solvent of the primary dcreening operation culture medium is water；
The thin sawdust includes toothed oak sawdust and birch bits；The diameter of the thin sawdust is 0.1~0.3cm；
The secondary screening culture medium includes the component of following mass parts: 70~80 parts of weed tree sawdust, 10~15 parts of wheat bran, and bean powder 3
~5 parts, 1.0~1.5 parts and 1.0~1.5 parts of gypsum of lime；
The weed tree sawdust includes toothed oak sawdust and birch bits；The weed tree sawdust includes thin sawdust and thick sawdust；The thick sawdust
Diameter be 0.4~0.6cm.
Preferably, the pH value of the primary dcreening operation culture medium is 7.0~7.5.
Preferably, the mass ratio of toothed oak sawdust and birch bits is 0.5~1.5:1~3 in the thin sawdust；The thin sawdust
Moisture content is 8%~10%.
Preferably, the mass ratio of toothed oak sawdust and birch bits is 0.5~1.5:1~3 in the weed tree sawdust；In the weed tree sawdust
The mass ratio of thin sawdust and thick sawdust is 0.5~1.5:0.5~1.5；The moisture content of the thick sawdust is 8%~10%.
Preferably, the diameter of the wheat bran is 0.3~0.5cm；The moisture content of the wheat bran is 7%~9%.
Preferably, the moisture content of the bean powder is 7%~9%.
The present invention also provides a kind of methods of lab screening black fungus strain of resistance to heat shock, comprising the following steps:
1) black fungus strain to be screened is inoculated in primary dcreening operation culture medium described in above scheme, pass sequentially through be protected from light culture,
1.5~3h of heat shock culture and renewal cultivation carry out the first primary dcreening operation under the conditions of 40 DEG C, and mycelia restoration ecosystem speed is selected to be greater than
The bacterial strain of 0.15cm/d is as the second primary dcreening operation bacterial strain；
2) it by step 1) the second primary dcreening operation strain inoculated in primary dcreening operation culture medium, passes sequentially through and is protected from light culture, 44 DEG C of conditions
1.5~3h of lower heat shock culture and renewal cultivation carry out the second primary dcreening operation, and mycelia restoration ecosystem speed is selected to be greater than the bacterium of 0.15cm/d
Strain is used as secondary screening bacterial strain；
3) by step 2) secondary screening strain inoculated secondary screening culture medium described in above scheme, pass sequentially through be protected from light culture,
2~5h of heat shock culture and renewal cultivation carry out the first secondary screening under the conditions of 44 DEG C, and mycelia restoration ecosystem speed is selected to be greater than 0.15cm/
The bacterial strain of d is as the second secondary screening bacterial strain；
4) it by step 3) the second secondary screening strain inoculated in secondary screening culture medium, passes sequentially through and is protected from light culture, 44 DEG C of conditions
4~7h of lower heat shock culture and renewal cultivation carry out the second secondary screening, and mycelia restoration ecosystem speed is selected to be greater than the bacterial strain of 0.15cm/d
As the black fungus strain of resistance to heat shock；
The temperature for being protected from light culture is 22~28 DEG C；The temperature of the renewal cultivation is 25~30 DEG C.
It preferably, further include by the black fungus strain of resistance to heat shock after obtaining the black fungus strain of resistance to heat shock in step 2)
Produce agaric is carried out under the conditions of 30~40 DEG C, selection meets the bacterial strain of high temperature black fungus strain requirement；
The high temperature black fungus strain requires to include: 55g/ bags of yield > of single bag of black fungus；Organoleptic quality evaluations score
> 85 divides, hardness > 4000g, elastic > 0.90, cohesiveness > 0.50, deadlocked degree > 2000g, chewability > 2000g；
Single bag of yield formula of the black fungus is as follows:
The specification of edible black fungus bag for the produce agaric is 16.5~17cm × 36.5~37cm.
Preferably, step 1)~4) described in be protected from light time of culture and stand alone as 6~10d；The time of the renewal cultivation
Stand alone as 3~5d.
Beneficial effects of the present invention: the present invention provides a kind of screening and culturing medium of black fungus strain of resistance to heat shock, the cultures
Base is suitable for the lab screening black fungus strain of resistance to heat shock, can quick and precisely screen the black fungus bacterial strain of resistance to heat shock using the culture medium；
The present invention also provides a kind of method for screening the black fungus strain of resistance to heat shock indoors using screening and culturing medium described in above scheme,
This method is including the use of the culture of primary dcreening operation culture medium and utilizes the culture of secondary screening culture medium, first by black wood to be screened in each step
Ear bacterial strain is protected from light culture under the conditions of being placed in 22~28 DEG C, carry out heat shock under the conditions of 35~45 DEG C, then be placed under the conditions of 25~30 DEG C
Renewal cultivation, to filter out the black fungus strain of resistance to heat shock.Method of the invention effectively can accurately screen the resistance to heat shock of black fungus
Bacterial strain.
Detailed description of the invention:
Fig. 1 shows the flow charts of the method for the lab screening black fungus strain of resistance to heat shock；
Fig. 2 indicates the black fungus strain of no antagonism line；
Fig. 3 indicates that there are the black fungus strains of antagonism line；
Fig. 4 indicates the cluster map of 9 plants of black fungus strains.
Specific embodiment
The present invention provides a kind of screening and culturing medium of black fungus strain of resistance to heat shock, the screening and culturing medium includes primary dcreening operation training
Support base and secondary screening culture medium；
The primary dcreening operation culture medium is in terms of 1L, the component including following quality: 200~250g of potato, and glucose 20~
25g, thin 8~12g of sawdust, 3~5g of wheat bran, 0.5~1.0g of magnesium sulfate, 1.5~2.0g of potassium dihydrogen phosphate and agar powder 18~
20g；The solvent of the primary dcreening operation culture medium is water；
The thin sawdust includes toothed oak sawdust and birch bits；The diameter of the thin sawdust is 0.1~0.3cm；
The secondary screening culture medium includes the component of following mass parts: 70~80 parts of weed tree sawdust, 10~15 parts of wheat bran, and bean powder 3
~5 parts, 1.0~1.5 parts and 1.0~1.5 parts of gypsum of lime；
The weed tree sawdust includes toothed oak sawdust and birch bits；The weed tree sawdust includes thin sawdust and thick sawdust；The thick sawdust
Diameter be 0.4~0.6cm.
In the present invention, the primary dcreening operation culture medium preferably includes the component of following quality in terms of 1L: potato 220~
230g, glucose 22~23g, thin sawdust 10g, wheat bran 4g, magnesium sulfate 0.8g, potassium dihydrogen phosphate 1.8g and agar powder 19g；It is described
The solvent of primary dcreening operation culture medium is preferably distilled water；The pH value of the primary dcreening operation culture medium is preferably 7.0~7.5, more preferably 7.1~
7.3.In the present invention, the effect of the primary dcreening operation culture medium is the black fungus strain that preliminary screening has high temperature resistant character.
Primary dcreening operation culture medium is usually used in laboratory, for cultivating black fungus strain.By primary dcreening operation culture medium and primary dcreening operation condition,
Preliminary screening provides the black fungus strain of heat-resisting sharp character.
In the present invention, the diameter of the thin sawdust is preferably 0.2cm；The quality of toothed oak sawdust and birch bits in the thin sawdust
Than being preferably 0.5~1.5:1~3, more preferably 1:2；The moisture content of the thin sawdust is preferably 8%~10%, more preferably
8%~9%；The diameter of the wheat bran is preferably 0.3~0.5cm, more preferably 0.4cm；The moisture content of the wheat bran is preferably
7%~9%, more preferably 7%~8%, most preferably 7%~7.5%.
In the present invention, the secondary screening culture medium preferably includes the component of following mass parts: 75 parts of weed tree sawdust, wheat bran 12
Part, 4 parts of bean powder, 1.2 parts and 1.2 parts of gypsum of lime；In the present invention, the effect of the secondary screening culture medium is by similar cultivar
Culture medium in further screening there is the black fungus strain of more high temperature resistant character.
The substances such as wheat bran and bean powder are added in secondary screening culture medium, the substances such as wheat bran and bean powder are cultivation of auricularia auricula kind culture mediums
In common substance, therefore, secondary screening culture medium physicochemical property and nutritive peculiarity have certain phase with cultivation of auricularia auricula kind culture medium
Like property.It, can black fungus strain of the further screening with more high temperature resistant character by secondary screening culture medium and secondary screening condition.
In the present invention, the diameter of the thick sawdust is preferably 0.5cm；The quality of toothed oak sawdust and birch bits in the weed tree sawdust
Than being preferably 0.5~1.5:1~3, more preferably 1:2；The mass ratio of thin sawdust and thick sawdust is preferably 0.5 in the weed tree sawdust
~1.5:0.5~1.5, more preferably 1:1；The moisture content of the thick sawdust is preferably 8%~10%, more preferably 8%~
9%；The weed tree sawdust includes toothed oak sawdust and birch bits；The weed tree sawdust includes thin sawdust and thick sawdust；The thick sawdust it is straight
Diameter is 0.4~0.6cm；The moisture content of the bean powder is preferably 7%~9%, and more preferably 7%~8%.
The present invention also provides a kind of methods of lab screening black fungus strain of resistance to heat shock, comprising the following steps:
1) black fungus strain to be screened is inoculated in primary dcreening operation culture medium described in above scheme, pass sequentially through be protected from light culture,
1.5~3h of heat shock culture and renewal cultivation carry out the first primary dcreening operation under the conditions of 40 DEG C, and mycelia restoration ecosystem speed is selected to be greater than
The bacterial strain of 0.15cm/d is as the second primary dcreening operation bacterial strain；
2) it by step 1) the second primary dcreening operation strain inoculated in primary dcreening operation culture medium, passes sequentially through and is protected from light culture, 44 DEG C of conditions
1.5~3h of lower heat shock culture and renewal cultivation carry out the second primary dcreening operation, and mycelia restoration ecosystem speed is selected to be greater than the bacterium of 0.15cm/d
Strain is used as secondary screening bacterial strain；
3) by step 2) secondary screening strain inoculated secondary screening culture medium described in above scheme, pass sequentially through be protected from light culture,
2~5h of heat shock culture and renewal cultivation carry out the first secondary screening under the conditions of 44 DEG C, and mycelia restoration ecosystem speed is selected to be greater than 0.15cm/
The bacterial strain of d is as the second secondary screening bacterial strain；
4) it by step 3) the second secondary screening strain inoculated in secondary screening culture medium, passes sequentially through and is protected from light culture, 44 DEG C of conditions
4~7h of lower heat shock culture and renewal cultivation carry out the second secondary screening, and mycelia restoration ecosystem speed is selected to be greater than the bacterial strain of 0.15cm/d
As the black fungus strain of resistance to heat shock；
The temperature for being protected from light culture is 22~28 DEG C；The temperature of the renewal cultivation is 25~30 DEG C.
The present invention is preferred to go back before black fungus strain to be screened is inoculated in primary dcreening operation culture medium described in above scheme
The following steps are included: black fungus strain to be screened is inoculated in cPDA culture medium, activation culture, the black wood activated are carried out
It is close to reject affiliation in the black fungus strain of the activation by antagonistic effect and analysis of genetic diversity for ear bacterial strain
Black fungus strain obtains the farther away black fungus strain of affiliation.
In the present invention, the temperature of the activation culture is preferably 25~30 DEG C, and more preferably 28 DEG C；The activation culture
Time is preferably 10~15d, more preferably 12d；The present invention is to the antagonistic effect and analysis of genetic diversity specific embodiment party
Method is not particularly limited, using conventional method in that art.
After the present invention obtains the farther away black fungus strain of affiliation, black fungus strain to be screened is inoculated in above-mentioned side
Primary dcreening operation culture medium described in case passes sequentially through and is protected from light culture, 1.5~3h of heat shock culture and renewal cultivation carry out first under the conditions of 40 DEG C
Primary dcreening operation selects bacterial strain of the mycelia restoration ecosystem speed greater than 0.15cm/d as the second primary dcreening operation bacterial strain；The temperature for being protected from light culture
Preferably 25 DEG C of degree；The time for being protected from light culture is preferably 6~10d, more preferably 8d；The time of the heat shock culture is preferred
For 2h；The temperature of the renewal cultivation is preferably 28 DEG C；The time of the renewal cultivation is preferably 3~5d, more preferably 4d.
After obtaining the second primary dcreening operation bacterial strain, the present invention in primary dcreening operation culture medium, passes sequentially through the second primary dcreening operation strain inoculated
Be protected from light culture, 1.5~3h of heat shock culture and renewal cultivation carry out the second primary dcreening operation under the conditions of 44 DEG C, select mycelia restoration ecosystem speed
Bacterial strain greater than 0.15cm/d is as secondary screening bacterial strain；The temperature for being protected from light culture is preferably 25 DEG C；It is described be protected from light culture when
Between preferably 6~10d, more preferably 8d；The time of the heat shock culture is preferably 2h；The temperature of the renewal cultivation is preferably
28℃；The time of the renewal cultivation is preferably 3~5d, more preferably 4d.
After obtaining secondary screening bacterial strain, the present invention is by secondary screening strain inoculated secondary screening culture medium described in above scheme, successively
By being protected from light culture, under the conditions of 44 DEG C, 2~5h of heat shock culture and renewal cultivation carry out the first secondary screening, select mycelia restoration ecosystem speed
Bacterial strain of the degree greater than 0.15cm/d is as the second secondary screening bacterial strain；The temperature for being protected from light culture is preferably 25 DEG C；It is described to be protected from light training
The feeding time is preferably 6~10d, more preferably 8d；The time of the heat shock culture is preferably 3~4h；The renewal cultivation
Temperature is preferably 28 DEG C；The time of the renewal cultivation is preferably 3~5d, more preferably 4d.
After obtaining the second secondary screening bacterial strain, the present invention in secondary screening culture medium, passes sequentially through the second secondary screening strain inoculated
Be protected from light culture, 4~7h of heat shock culture and renewal cultivation carry out the second secondary screening under the conditions of 44 DEG C, select mycelia restoration ecosystem speed big
In 0.15cm/d bacterial strain as the black fungus strain of resistance to heat shock；The temperature for being protected from light culture is preferably 25 DEG C；It is described to be protected from light training
The feeding time is preferably 6~10d, more preferably 8d；The time of the heat shock culture is preferably 5~6h；The renewal cultivation
Temperature is preferably 28 DEG C；The time of the renewal cultivation is preferably 3~5d, more preferably 4d.
The present invention preferably further includes by the black fungus strain of resistance to heat shock 30 after obtaining the black fungus strain of resistance to heat shock
Produce agaric is carried out under the conditions of~40 DEG C, selection meets the bacterial strain of high temperature black fungus strain requirement；The temperature of the produce agaric is preferably
30 DEG C, 35 DEG C and 40 DEG C；
The high temperature black fungus strain requires to include: 55g/ bags of yield > of single bag of black fungus；Organoleptic quality evaluations score
> 85 divides, hardness > 4000g, elastic > 0.90, cohesiveness > 0.50, deadlocked degree > 2000g, chewability > 2000g；
Single bag of yield formula of the black fungus is as follows:
The specification of bacterium bag for the produce agaric is preferably 16.5~17cm × 36.5~37cm；The bacterium bag is preferably pasted
The slim cultivating bag of the good polyethylene of wall.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
A kind of method of the lab screening black fungus strain of resistance to heat shock of embodiment 1
1. material
Primary dcreening operation culture medium prescription: potato 200g, glucose 20g, thin sawdust 12g, wheat bran 5g, magnesium sulfate 1.0g, phosphoric acid
Potassium dihydrogen 2.0g, agar powder 18g, 1000 milliliters of distilled water, pH 7.0；Thin sawdust requires to consider to be worth doing for toothed oak sawdust and birch hard miscellaneous
Sawdust, toothed oak sawdust and birch bits mass ratio are 1:2, and thin sawdust diameter is 0.1cm, moisture content 9.1%；Wheat bran diameter is
0.3cm, moisture content 8.5%；
Secondary screening culture medium prescription: weed tree sawdust 80g, wheat bran 15g, bean powder 3g, lime 1g, gypsum 1g；Weed tree sawdust requires to be toothed oak
The hardwood crumbs of sawdust and birch bits, toothed oak sawdust and birch bits mass ratio are 1:2, and weed tree sawdust is thin sawdust and thick sawdust according to 1:
1 (mass ratio) uniformly mixes；Thin sawdust diameter is 0.1cm, moisture content 9.3%；Thick sawdust diameter is 0.4cm, and moisture content is
9.5%；Wheat bran diameter is 0.3cm, moisture content 7.5%；Bean powder moisture content is 7.4%.
2. screening technique
Screening mode flow chart is referring to Fig. 1.
2.1 actication of culture
2017~2019 years respectively in 9 Zhejiang, Fujian and Heilongjiang Province's field acquisition black fungus strains, by strain inoculated
Into cPDA, 28 DEG C of constant temperature are protected from light culture 12d, the black fungus strain activated.
Table 1 is for trying black fungus strain
2.2 antagonistic effects and analysis of genetic diversity, rejecting the closer black fungus strain of affiliation, (affiliation is closer
Black fungus strain, very big may be the same black fungus kind, remove if do not kicked, and there are repetition research, increase workload etc.
Problem) using remaining bacterial strain as strains tested.
2.2.1 antagonistic effect: the antagonism relationship between every group of black fungus obtained by antagonism two-by-two, and three different are supplied
The inoculation block of examination bacterial strain is respectively placed in opposite culture in the plate of 90cm, and any two, which are inoculated between block, is spaced 2~3cm, in 25
DEG C constant temperature is protected from light culture, observes the antagonism between two kinds, every group is repeated 3 times.If two black fungus strains do not have
Antagonism line is generated, but is grown into together, then the two black fungus strain affiliations are closer, and very big may be same strain bacterium
Strain, it is random to reject wherein one plant.Test result is subject to Fig. 2 and Fig. 3, and wherein Fig. 2 indicates the black fungus strain of no antagonism line；
Fig. 3 indicates that there are the black fungus strains of antagonism line.
2.2.2ISSR analysis of genetic diversity: ISSR amplification is carried out using 10 ISSR primer pairs in table 2.
2 ISSR primer sequence of table
Reaction system: 2 μ 10 × Ex of L TaqBuffer (Takara), 2 μ L dNTP (0.2mmol/L), primer (0.5 μ
Mol/L), 0.5U archaeal dna polymerase (Takara), 1 μ L template DNA (50ng/ μ L), ddH2O is mended to 20 μ L.
Response procedures: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 45~55 DEG C of renaturation 30s, 72 DEG C of extension 2min, 35
Circulation；72 DEG C of extension 7min.
It filters out that polymorphism is high, amplification is stablized, reproducible primer repeat amplification protcol 3 times, is analyzed for data.
The electrophoresis result for comparing analysis ISSR amplified production has band to be denoted as 1, and no band is denoted as 0, constructs 0/1 initial number
According to matrix.Genetic similarity between sample is calculated using NTsys_2.02 software, it is poly- to carry out genetic similarity with UPGMA method
Class.
Analysis of genetic diversity and UPGMA clustering are carried out with NTsys_2.02 software.The hereditary phase of 9 plants of fungus strains
As shown in table 3 like coefficient, genetic similarity shows 9 plants of fungus strain affinities farther out, wherein Zhejiang between 0.4~0.9
Black No. 3 minimum with black No. 7 similarity factors in Zhejiang, is 0.4, and black No. 3 and black No. 6 similarity factor highests are 0.9.9 plants of agaric fungus
The UPGMA cluster map of strain is as shown in figure 4, be that will originally be divided into two major classes group for sample at 0.67 in similarity factor, wherein Zhejiang black 1
Number, Zhejiang is No. 3 black, good fortune it is black No. 3 and black No. 8 similitudes of good fortune it is higher, gather for one kind；Zhejiang is No. 7 black, good fortune is No. 2 black, it is No. 2 black, black 3
Number and black No. 6 similitudes it is higher, gather for one kind.Therefore, 9 plants of fungus strain affinities farther out, can preliminary judgement be difference
Black fungus strain, can be used for the screening of subsequent black fungus high-temperature resistant strain.
Similarity factor between 3 black fungus strain of table
2.3 the black fungus strain of resistance to heat shock primary dcreening operation
1) it is punched at identical cell age using 1cm punch, in the primary dcreening operation culture medium that the black fungus strain of activation is inoculated with,
25 DEG C of constant temperature are protected from light culture 8d, are transferred to heat shock 2h in 40 DEG C of constant incubators, are transferred in 28 DEG C of constant incubators and restore training
It supports, renewal cultivation 4d, observes mycelia recovery situation, measure mycelia resume speed；
2) it filters out mycelia and restores good, bacterial strain of the mycelia restoration ecosystem speed greater than 0.15cm/d is adopted as new strains
It is punched at identical cell age with 1cm punch, by the primary dcreening operation culture medium of new strains inoculation, 25 DEG C of constant temperature are protected from light culture 8d, turn
Heat shock 2h in 44 DEG C of constant incubators is moved on to, renewal cultivation in 28 DEG C of constant incubators is transferred to, renewal cultivation 4d observes mycelia
Recovery situation measures mycelia resume speed；
2.4 black fungus strain of resistance to heat shock secondary screenings
1) by secondary screening strain inoculated in secondary screening culture medium, 25 DEG C of constant temperature be protected from light culture to mycelia it is long to 10cm when, transfer
The heat shock 2h into 44 DEG C of constant incubators, is transferred to renewal cultivation in 28 DEG C of constant incubators, and it is extensive to observe mycelia by renewal cultivation 4d
Multiple situation, measures mycelia resume speed；
2) new strains are inoculated in secondary screening training as new strains by the bacterial strain using mycelia restoration ecosystem speed greater than 0.15cm/d
Support in base, 25 DEG C of constant temperature be protected from light culture to mycelia it is long to 10cm when, be transferred to heat shock 4h in 44 DEG C of constant incubators, be transferred to 28
Renewal cultivation in DEG C constant incubator, renewal cultivation 4d observe mycelia recovery situation, measure mycelia resume speed, filter out bacterium
Silk restores good, and bacterial strain of the mycelia restoration ecosystem speed greater than 0.15cm/d is as bacterial strain to be verified.
The verifying of 2.5 black fungus strains of resistance to heat shock
Cultivation produce agaric verification experimental verification is carried out, respectively by edible black fungus bag (the slim cultivating bag of 16.5cm × 36.5cm polyethylene)
Carry out produce agaric under the conditions of 30 DEG C, 35 DEG C, 40 DEG C, the bacterial strain of resistance to heat shock can under the conditions of 30 DEG C, 35 DEG C, 40 DEG C produce agaric, resistance to height
Warm bacterial strain screening standard: produce agaric success, and there was no significant difference for yield compared with control group (30 DEG C) for black fungus yield, black fungus auricle
There was no significant difference with control group for sensory evaluation score, and there was no significant difference with control group for black fungus auricle edible quality.
1) single bag of yield: 1 plant of high temperature black fungus strain produce agaric filtered out is chosen, as a result, it has been found that 30 DEG C, 35 DEG C and 40
Single bag of yield of black fungus is respectively 58.72 ± 0.24g, 58.15 ± 0.25g and 56.22 ± 0.19g, single bag of yield under the conditions of DEG C
It is all larger than 55g.
2) organoleptic quality: 10 professional peoples that please receive organoleptic examination training form evaluation group, referring to 4 black fungus of table
Sensory evaluation index takes two digits random number to sample and sampfe order and carries out blind mark, is averaged and comments for sense organ
Valence score.
4 black fungus sensory evaluation index of table
Note: mouthfeel is evaluated as black fungus room temperature and is soaked 6h, is evaluated after then draining surface moisture.
Organoleptic quality evaluations scores are as shown in table 5, as a result, it has been found that black fungus sense organ under the conditions of 30 DEG C, 35 DEG C and 40 DEG C
Quality evaluation score is respectively 88.30 ± 1.42,86.60 ± 1.26 and 86.40 ± 1.17, and sensory evaluation score is all larger than 85
Point.
5 black fungus sensory evaluation score of table
3) edible quality: edible quality measurement is carried out using TA.XT.plus TextureAnalyser food texture measurement.
Edible quality index includes hardness, elasticity, cohesiveness, deadlocked degree, chewability.
High-temperature resistant strain requirement: single bag of yield is greater than every bag of 55g, it is desirable that organoleptic quality evaluations score is greater than 85 points, it is desirable that
Hardness is greater than 4000g in edible quality, and elasticity is greater than 0.90, and cohesiveness is greater than 0.50, and deadlocked degree is greater than 2000g, and chewability is big
In 2000g.
The results are shown in Table 6 for black fungus fructification edible quality, as a result, it has been found that the black fungus strain is in 35 DEG C of high temperature and 40
It can normal produce agaric under the conditions of DEG C.(30 DEG C) of normal produce agaric are control, and black fungus fructification edible quality, discovery are measured after produce agaric
Black fungus hardness is maximum under the conditions of 30 DEG C, is 6058.9 ± 552.9g, and black fungus hardness is minimum under the conditions of 40 DEG C, be 4122.5 ±
838.1g, black fungus hardness is all larger than 4000g under the conditions of three kinds of temperature；Black fungus elasticity is best under the conditions of 30 DEG C, and 35 DEG C and 40
Black fungus elasticity is consistent under the conditions of DEG C, is 0.93, is all larger than 0.90；Black fungus cohesiveness difference is unknown under condition of different temperatures
It is aobvious, wherein black fungus cohesiveness is minimum under the conditions of 35 DEG C, it is 0.50 ± 0.08, black fungus cohesiveness is big under the conditions of three kinds of temperature
In 0.50；The deadlocked degree of black fungus is maximum under the conditions of 30 DEG C, and deadlocked degree is minimum under the conditions of 40 DEG C, for 2115.6 ± 420.9g, three kinds
The deadlocked degree of black fungus is all larger than 2000g under the conditions of temperature；Black fungus chewability is maximum under the conditions of 30 DEG C, is 3058.1 ± 325.5,
Deadlocked degree is minimum under the conditions of 35 DEG C, is 2058.1 ± 308.7, black fungus chewability is all larger than 2000g under the conditions of three kinds of temperature.Food
Meet high-temperature resistant strain requirement with each index of quality.
Therefore, the high temperature black fungus strain filtered out indoors by this patent, have high-temperature stability, can meet compared with
Produce agaric requirement under the conditions of high-temperature.
6 black fungus Taste quality evaluation of table
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Institute of Microbiology, Heilongjiang Academy of Sciences
<120>method of a kind of screening and culturing medium of black fungus strain of resistance to heat shock and the lab screening black fungus strain of resistance to heat shock
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gagagagaga gagagayg 18
<210> 2
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtgacgactc tctctctct 19
<210> 3
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agagagagag agagagg 17
<210> 4
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acacacacac acacacc 17
<210> 5
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tgcacacaca cacac 15
<210> 6
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
agcagcagca gcagcagcg 19
<210> 7
<211> 17
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
acacacacac acacacc 17
<210> 8
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tgtgtgtgtg tgtgtgrc 18
<210> 9
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atgatgatga tgatgatg 18
<210> 10
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
agagagagag agagagyc 18
Claims (9)
1. a kind of screening and culturing medium for the black fungus strain of resistance to heat shock, which is characterized in that the screening and culturing medium includes primary dcreening operation culture
Base and secondary screening culture medium；
The primary dcreening operation culture medium is in terms of 1L, the component including following quality: 200~250g of potato, 20~25g of glucose, carefully
18~20g of 8~12g of sawdust, 3~5g of wheat bran, 0.5~1.0g of magnesium sulfate, 1.5~2.0g of potassium dihydrogen phosphate and agar powder；It is described
The solvent of primary dcreening operation culture medium is water；
The thin sawdust includes toothed oak sawdust and birch bits；The diameter of the thin sawdust is 0.1~0.3cm；
The secondary screening culture medium includes the component of following mass parts: 70~80 parts of weed tree sawdust, 10~15 parts of wheat bran, and bean powder 3~5
Part, 1.0~1.5 parts and 1.0~1.5 parts of gypsum of lime；
The weed tree sawdust includes toothed oak sawdust and birch bits；The weed tree sawdust includes thin sawdust and thick sawdust；The thick sawdust it is straight
Diameter is 0.4~0.6cm.
2. screening and culturing medium according to claim 1, which is characterized in that the pH value of the primary dcreening operation culture medium be 7.0~
7.5。
3. screening and culturing medium according to claim 1 or 2, which is characterized in that toothed oak sawdust and birch bits in the thin sawdust
Mass ratio be 0.5~1.5:1~3；The moisture content of the thin sawdust is 8%~10%.
4. screening and culturing medium according to claim 3, which is characterized in that the matter of toothed oak sawdust and birch bits in the weed tree sawdust
Amount is than being 0.5~1.5:1~3；The mass ratio of thin sawdust and thick sawdust is 0.5~1.5:0.5~1.5 in the weed tree sawdust；Institute
The moisture content for stating thick sawdust is 8%~10%.
5. screening and culturing medium according to claim 1 or 2, which is characterized in that the diameter of the wheat bran is 0.3~0.5cm；
The moisture content of the wheat bran is 7%~9%.
6. screening and culturing medium according to claim 1 or 2, which is characterized in that the moisture content of the bean powder is 7%~9%.
7. a kind of method of the lab screening black fungus strain of resistance to heat shock, comprising the following steps:
1) black fungus strain to be screened is inoculated in primary dcreening operation culture medium described in claim 1~6 any one, passes sequentially through and keeps away
Optical culture, 1.5~3h of heat shock culture and renewal cultivation carry out the first primary dcreening operation under the conditions of 40 DEG C, select mycelia restoration ecosystem speed big
In 0.15cm/d bacterial strain as the second primary dcreening operation bacterial strain；
2) it by step 1) the second primary dcreening operation strain inoculated in primary dcreening operation culture medium, passes sequentially through and is protected from light culture, heat under the conditions of 44 DEG C
Swash 1.5~3h of culture and renewal cultivation carries out the second primary dcreening operation, bacterial strain of the mycelia restoration ecosystem speed greater than 0.15cm/d is selected to make
For secondary screening bacterial strain；
3) it by step 2) secondary screening strain inoculated secondary screening culture medium described in claim 1~6 any one, passes sequentially through and keeps away
Optical culture, 2~5h of heat shock culture and renewal cultivation carry out the first secondary screening under the conditions of 44 DEG C, and mycelia restoration ecosystem speed is selected to be greater than
The bacterial strain of 0.15cm/d is as the second secondary screening bacterial strain；
4) it by step 3) the second secondary screening strain inoculated in secondary screening culture medium, passes sequentially through and is protected from light culture, heat under the conditions of 44 DEG C
Swash culture 4~7h and renewal cultivation carry out the second secondary screening, select mycelia restoration ecosystem speed be greater than 0.15cm/d bacterial strain as
The black fungus strain of resistance to heat shock；
The temperature for being protected from light culture is 22~28 DEG C；The temperature of the renewal cultivation is 25~30 DEG C.
8. preparation method according to claim 7, which is characterized in that obtain the black fungus strain of resistance to heat shock in step 2)
It afterwards, further include that the black fungus strain of resistance to heat shock is carried out to produce agaric under the conditions of 30~40 DEG C, selection meets high temperature black agaric
The bacterial strain that bacterial strain requires；
The high temperature black fungus strain requires to include: 55g/ bags of yield > of single bag of black fungus；Organoleptic quality evaluations score > 85
Point, hardness > 4000g, elastic > 0.90, cohesiveness > 0.50, deadlocked degree > 2000g, chewability > 2000g；
Single bag of yield formula of the black fungus is as follows:
The specification of edible black fungus bag for the produce agaric is 16.5~17cm × 36.5~37cm.
9. preparation method according to claim 7, which is characterized in that step 1)~4) described in be protected from light culture time it is only
It stands as 6~10d；The time of the renewal cultivation stands alone as 3~5d.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910710755.3A CN110402758B (en) | 2019-08-02 | 2019-08-02 | Screening culture medium of heat-resistant auricularia auricula-judae strains and method for screening heat-resistant auricularia auricula-judae strains indoors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910710755.3A CN110402758B (en) | 2019-08-02 | 2019-08-02 | Screening culture medium of heat-resistant auricularia auricula-judae strains and method for screening heat-resistant auricularia auricula-judae strains indoors |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110402758A true CN110402758A (en) | 2019-11-05 |
| CN110402758B CN110402758B (en) | 2021-09-07 |
Family
ID=68365461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910710755.3A Active CN110402758B (en) | 2019-08-02 | 2019-08-02 | Screening culture medium of heat-resistant auricularia auricula-judae strains and method for screening heat-resistant auricularia auricula-judae strains indoors |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110402758B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112243798A (en) * | 2020-10-22 | 2021-01-22 | 山东同康农业开发有限公司 | Energy-saving and efficient tremella strain breeding and planting method |
| CN114317284A (en) * | 2021-12-28 | 2022-04-12 | 黑龙江华腾生物科技有限公司 | Black fungus autumn cultivation strain, domestication method and cultivation method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1194089A (en) * | 1998-04-07 | 1998-09-30 | 杭州常青保健食品有限公司 | High temperature resistant mushroom and its breeding and cultivating method |
| CN105145114A (en) * | 2015-08-19 | 2015-12-16 | 黑龙江省科学院微生物研究所 | Method evaluating black fungus mycelium high temperature resistance feature |
| CN107557307A (en) * | 2017-10-24 | 2018-01-09 | 广西壮族自治区农业科学院微生物研究所 | A kind of lab screening method of high temperature resistant cloud ear bacterial strain |
| CN107657141A (en) * | 2016-07-23 | 2018-02-02 | 东北林业大学 | A kind of construction method of black fungus quality monitoring system |
| CN108753676A (en) * | 2018-07-20 | 2018-11-06 | 山东农业大学 | A kind of selection of Stropharia rugoso-annulata high-temperature resistant strain |
| CN109837217A (en) * | 2018-12-26 | 2019-06-04 | 澳门荣世联商贸一人有限公司 | High temperature resistant Produced from Pleurotus ostreatus and its screening technique and application |
| KR20190072238A (en) * | 2017-12-15 | 2019-06-25 | 동의대학교 산학협력단 | Producing method of Auricularia auricular fruit body |
-
2019
- 2019-08-02 CN CN201910710755.3A patent/CN110402758B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1194089A (en) * | 1998-04-07 | 1998-09-30 | 杭州常青保健食品有限公司 | High temperature resistant mushroom and its breeding and cultivating method |
| CN105145114A (en) * | 2015-08-19 | 2015-12-16 | 黑龙江省科学院微生物研究所 | Method evaluating black fungus mycelium high temperature resistance feature |
| CN107657141A (en) * | 2016-07-23 | 2018-02-02 | 东北林业大学 | A kind of construction method of black fungus quality monitoring system |
| CN107557307A (en) * | 2017-10-24 | 2018-01-09 | 广西壮族自治区农业科学院微生物研究所 | A kind of lab screening method of high temperature resistant cloud ear bacterial strain |
| KR20190072238A (en) * | 2017-12-15 | 2019-06-25 | 동의대학교 산학협력단 | Producing method of Auricularia auricular fruit body |
| CN108753676A (en) * | 2018-07-20 | 2018-11-06 | 山东农业大学 | A kind of selection of Stropharia rugoso-annulata high-temperature resistant strain |
| CN109837217A (en) * | 2018-12-26 | 2019-06-04 | 澳门荣世联商贸一人有限公司 | High temperature resistant Produced from Pleurotus ostreatus and its screening technique and application |
Non-Patent Citations (6)
| Title |
|---|
| 朱兰宝等: "《专家释疑解难农业技术丛书 食用菌优质高产栽培技术问答》", 31 March 2013, 金盾出版社 * |
| 王进等: "紫外诱变选育秦巴山区黑木耳耐高温菌株的研究", 《陕西理工学院学报（自然科学版）》 * |
| 陈恒雷: "低能N+束诱变选育耐高温阿魏菇研究", 《中国优秀博硕士学位论文全文数据库 (硕士) 农业科技辑》 * |
| 马瑞霞等: "《普通高等教育"十三五"规划教材 食用菌栽培学》", 31 August 2017, 中国轻工业出版社 * |
| 高英: "《食用菌栽培与制种技术》", 31 March 2003, 延边人民出版社 * |
| 黄瑞贤等: "《黑木耳袋栽小口出耳新技术》", 29 February 2016, 吉林人民出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112243798A (en) * | 2020-10-22 | 2021-01-22 | 山东同康农业开发有限公司 | Energy-saving and efficient tremella strain breeding and planting method |
| CN114317284A (en) * | 2021-12-28 | 2022-04-12 | 黑龙江华腾生物科技有限公司 | Black fungus autumn cultivation strain, domestication method and cultivation method |
| CN114317284B (en) * | 2021-12-28 | 2023-09-05 | 黑龙江华腾生物科技有限公司 | Autumn strain planting and domestication method and cultivation method for black fungus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110402758B (en) | 2021-09-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zamani et al. | Genetic relationships among pomegranate genotypes studied by fruit characteristics and RAPD markers | |
| Jaunet et al. | Variation in genotype and aggressiveness of Ralstonia solanacearum race 1 isolated from tomato in Taiwan | |
| Peng et al. | Unique microbial diversity and metabolic pathway features of fermented vegetables from Hainan, China | |
| CN110402758A (en) | A kind of method of the screening and culturing medium and the lab screening black fungus strain of resistance to heat shock of the black fungus strain of resistance to heat shock | |
| Begum et al. | Morphological and microsatellite analysis of intravarietal heterogeneity in ‘Beneshan’mango (Mangifera indica L.) | |
| Liu et al. | Morphological and molecular diversity in a germplasm collection of seed pumpkin | |
| Liao et al. | Genetic diversity of inner quality and SSR association analysis of wild kiwifruit (Actinidia eriantha) | |
| CN107653194B (en) | It is a kind of for cultivating the culture medium and cultural method of preeminent hickory chick | |
| Sankar et al. | Genetic diversity analysis of sweet orange (Citrus sinensis Osbeck) varieties/clones through RAPD markers | |
| WO2021047032A1 (en) | Primers for molecularly labeling new strain of flammulina fennae and molecular labeling method | |
| CN106119350B (en) | Early stage identification ' Hort 16A ' Kiwi berry opening pollination offspring's property method for distinguishing | |
| CN110257264A (en) | A method of screening agaricus bisporus high-temperature resistant strain | |
| CN108330194A (en) | A kind of method and its kit of determining build | |
| Banwo et al. | Sourdough Bread from the Blend of Cassava, Sweet Potato, and Soybean Flours Using Lactobacillus Plantarum and Pichia Kudriavzevii | |
| CN106916837A (en) | Hyperosmosis glycerine protein kinase gene RkHog1 and its recombinant expression carrier | |
| CN106350591B (en) | Construct SSR primer, constructed finger-print and its application of coloured silk Ye Luanshu Variety fingerprinting | |
| CN111850155A (en) | Application of specific target primer in simultaneous and rapid identification of two pathogenic bacteria of strawberry infection | |
| KR20210038410A (en) | Specific molecular markers for discrimination of one set of autonomy and red terrestrial species, and differentiation methods and applications | |
| CN106893769A (en) | Recombinant nucleic acid fragment RecCR012602 and its detection method | |
| Katoch et al. | Application of PCR based diagnostics in the exploration of Parastagonospora nodorum prevalence in wheat growing regions of Himachal Pradesh | |
| CN105420354B (en) | Conventional rice variety Huaihe River rice No. 5 and Huaihe River rice No. 18 identification methods based on InDel label | |
| Azam et al. | Microsatellite marker based characterization of mango cultivars | |
| Erlinawati et al. | Genetic diversity analysis of daluga (Cyrtosperma merkusii) using sequence-related amplified polymorphism in Siau, Sangihe and Talaud Islands, North Sulawesi, Indonesia | |
| CN110885758A (en) | New Flammulina finna strain and molecular marker primer and molecular marker method thereof | |
| KR102395587B1 (en) | Biomarker for discrimination between Ligularia fischeri and Caltha palstris |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |