CN103571788A - Collection and storage method of cordyceps militaris ascospores - Google Patents
Collection and storage method of cordyceps militaris ascospores Download PDFInfo
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Abstract
The invention provides a collection and storage method of cordyceps militaris ascospores. The collection method comprises the following steps: adding sand into a multi-spore separating and collecting container which can be sealed; sterilizing and drying; putting the selected matured sporocarp of cordyceps militaris ascocarps into the multi-spore separating and collecting container containing the sand; vibrating the multi-spore separating and collecting container so as to collect the sand containing matured ascospores which are ejected out or not; sealing the collected sand in a dry and sterile container; and preserving the container at room temperature by keeping out of the sun or in a refrigerator in a cold storage manner. According to the collection method, the operation is simple and the ascospore collecting rate is increased; the preserved ascospores can retain the inheritable characters of the original parents of cordyceps militaris after being activated and novel breeds for stress resistance can be screened.
Description
Technical field
The invention belongs to cordyceps cultivation technology field, particularly, relate to a kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link..
Background technology
Cordyceps militaris (L.) Link. Cordyceps militaris(L.ex Fr) Link, has another name called Cordyceps militaris (L.) Link., Cordyceps militaris, colonizes on the insect pupal cells such as lepidopteran, Coleoptera, Diptera.The region, Plain that is grown in low height above sea level, is mainly distributed in the ground such as northeast, North China, northwest.Modern study shows, Cordyceps militaris (L.) Link. contains the chemical compositions such as cordycepin (3'-Deoxyadenosine), cordycepic acid (PEARLITOL 25C), adenosine, Cordyceps polysaccharide, ergosterol, superoxide-dismutase (SOD), selenium (Se).Have and improve immune function of human body, anticancer, antibiotic, antifatigue, anti-ageing, anticonvulsion, hypoxia tolerance, calmness, the establishing-Yang effect such as reinforce the kidney.Also there is the effects such as fat-reducing, weight reducing, beauty treatment simultaneously.
In recent years, a lot of research all shows, tame Cordyceps militaris (L.) Link. chemical composition and pharmacological action are similar to Cordyceps sinensis, but price is well below Cordyceps sinensis, and therefore, the Application and Development of Cordyceps militaris (L.) Link. has great potential market.Tissue Culture of Cordyceps militaris will obtain sporophore, and key is that this cordyceps militaris link bacterial strain will have the ability that forms sporophore, and the genetic background that different strains possesses is different, so that they possess the ability of sporophore of formation is variant.Yet in actual production, even advantage cordyceps militaris link bacterial strain, along with bacterial classification go down to posterity, transfer increase or the prolongation of preservation time of number of times, microorganism resource is very easily degenerated.
Spore separation method, is to sprout into mycelia with sexual spore or the asexual spore of edible mushrooms, cultivates into the method for bacterial classification.This bacterial classification vitality is stronger, but variant between spore individuality, and natural differentiation phenomenon is more serious, and variation is large, need could on producing, apply through fruiting experiment.Current spore separation method mainly contains: (one) monospore partition method: be each or every test tube is only got a sporidium, allow it sprout into the method that mycelium obtains pure strain.Less employing in monospore separation of produced, and technical sophistication, generally adopt many spores partition method.(2) many spores partition method be many spore inoculatings on same substratum, allow a kind of method that their are sprouted, pangamy obtains edible mushrooms pure strain.Concrete operation method, has following several: 1. kind of mushroom spore launches method: the kind mushroom that strong eight or nine minutes of selection adaptation is ripe, stem with aseptic water washing number all over after with sterilized gauze or absorbent cotton, filter paper, blot surface-moisture again.In inoculation tank or sterilisable chamber, the lamella of planting mushroom is hung upside down below glass funnel with iron wire down, funnel covers on culture dish; Upper end aperture clogs with cotton.Culture dish is placed on an enamel tray that is covered with gauze, and standing 12～20 hours, the spore on lamella will be scattered in culture dish.Form the Powdered spore print of one deck.With inoculating needle, pick streak inoculation on the agar of a small amount of spore in test tube outside or culture dish.Treat spore germination, while generating bacterium colony, select spore germination early, bacterium colony that growing way is good carries out test tube cultivation.Also available spore collector is collected spore.Press said procedure, raise gently bell glass, kind of a mushroom handle is inserted on the wire frame of spore collector down, be placed on culture dish centre.Build immediately lens, with gauze, the clock palm is around filled in.And on gauze, fall a little mercuric chloride or sterilized water.Moving into 20 ℃ of left and right thermostat containers cultivates.2. streak method on pleat: select ripe kind mushroom, with inoculating needle, directly insert between pleat sheet, smear gently and get the spore that pleat sheet surface sporophore is not yet launched, then streak inoculation on substratum.3. the outstanding method of hook: several lamella or a fritter auricle (black fungus, Auricularia polytricha (Mout) Sacc., Tremella) of getting ripe cap, the top that hangs on the substratum in triangular flask with aseptic Stainless Steel Wire (or other suspension materials such as iron wire, cotton thread), does not make to touch substratum or surrounding bottle wall.Put under optimal temperature and cultivate, transfer.4. attaching method: ripe lamella or auricle are got to a fritter, and the nutrient agar that use is dissolved or gum arabic, paste etc. are attached on the test tube wall directly over pipe arrangement slant medium.Cultivation through 6～12 hours, treats that spore drops on inclined-plane, immediately spore is transplanted in new test tube and is cultivated together with part nutrient agar.Mother's kind that spore separation obtains, further purification and rejuvenation, when mother plants field planting about one week, when mycelia is covered with inclined-plane, select mycelia healthy and strong, grow vigorous without aging, without the mother who infects the mattress of mixing, plant test tube, and then tube expansion.Mother's kind that spore separation obtains, must be accredited as after good quality strain by fruiting experiment, just can supply production and application.There are two deficiencies in aforesaid method: the first, and complicated operation.The second, can only collect the ripe spore being launched out, and the ripe ripe spore not launched out but can not be collected.
Summary of the invention
The object of this invention is to provide a kind of thecasporous Collection and preservation method of Cordyceps militaris (L.) Link., to overcome above-mentioned deficiency.
In order to realize the object of the invention, the thecasporous collection method of a kind of Cordyceps militaris (L.) Link. of the present invention, it comprises the steps:
1) sand is put into many spores separated and collected container that can seal, sterilizing, dry;
2) Cordyceps militaris (L.) Link. ascoma fully-developed sporophore is put into many spores separated and collected container of step 1);
3) many spores separated and collected container that vibrates, collects the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out.
Wherein, described step 1) sand is neutral dry sand, and it obtains by the following method:
A) river sand is crossed to 60 mesh sieves, discarded macrobead and impurity;
B) with 80 orders, sieve again, discard fine sand, collect unsifted sand;
C) remove the irony in sand;
D), with after the salt acid soak sand 24h of 10%-20%, the acid of desalting, washes bubble with water to neutral, dries sand.
Further, in step c), can use magnet to remove the irony in sand.
Many spores separated and collected container that the present invention uses, sealable Glass Containers or the metal vessel that can have any shape.
Wherein, the laying method that the sand described in step 1) is put into many spores separated and collected container that can seal be by etc. after the sand of quality mixes with distilled water, 1:3-10 is placed in many spores separated and collected container by volume.
Wherein, the sterilizing described in step 1) is 121 ℃, moist heat sterilization 30min.
Wherein, dry described in step 1) is 80 ℃ of dry 24-48h.
In above-mentioned sterilizing and dry step, many spores separated and collected container all strictly sealing carry out sterilizing and dry.
Wherein, step 2) the abundant sporophore head of the preferred 1-2cm ascoma of Cordyceps militaris (L.) Link. ascoma fully-developed sporophore, puts into many spores separated and collected container of step 1), and room temperature is placed 10-30 days, to sporophore complete drying.
Wherein, step 2) described sporophore is put into many spores separated and collected container, and the sporophore number of putting into and the number mass ratio of container sand are 1: 2.5-5g.
Step 2 in Cordyceps militaris (L.) Link. thecaspore collection method provided by the invention), in, room temperature is placed 10-30 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore to fine sand by nature, have the ascoma of part maturation not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
Wherein, the vibration described in step 3) is with 80-200r/min speed oscillation 24-48h by many spores separated and collected container.
The thecasporous collection method of a kind of Cordyceps militaris (L.) Link. provided by the invention also comprises takes out dry sporophore.
The present invention also provides a kind of Cordyceps militaris (L.) Link. thecasporous store method, and the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out that aforesaid method is obtained is placed in dry sterile chamber sealing preservation.
Further, to preserve the thecasporous storage conditions of Cordyceps militaris (L.) Link. be room temperature lucifuge or 0-6 ℃ of refrigeration in the present invention.
The invention provides the application of Cordyceps militaris (L.) Link. thecaspore collection method in Cordyceps militaris (L.) Link. breeding.
The present invention utilizes the rubbing effect of fine sand, thereby the rubbing effect that is subject to fine sand when dry ripe ascoma and immature ascoma is broken and discharge thecaspore wherein, falls into sand carrier.By collecting the visual sand of naked eyes, thereby collect the sightless Cordyceps militaris (L.) Link. thecaspore of naked eyes, so present method not only can collect the thecaspore of ejection, can also collect the thecaspore not ejecting, thecasporous collection rate reaches 100%, has improved thecasporous collection effciency.And the thecaspore that available technology adopting catapult technique is collected is the parton cystospore launching out after ascoma maturation, thecaspore quantity is less than 1% of all thecaspore total amounts in sporophore ascoma.The present invention further takes out dry sporophore, and thecaspore is activated or saved backup, and has finally realized the object of the thecasporous separation of Cordyceps militaris (L.) Link., collection and preservation.Cordyceps militaris (L.) Link. thecaspore collection method provided by the invention is simple to operate, use manpower and material resources sparingly, improved thecasporous collection rate simultaneously, the Cordyceps militaris (L.) Link. thecaspore store method providing can effectively keep thecasporous biological activity in 2-4, the inherited character that has kept original parent, for seed selection novel bacterial provides effective ways, be with a wide range of applications.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The cultivation of embodiment 1 Cordyccps-militaris-(L.)-link. Sporophore
1, bacterial classification: the strain number that adopts autonomous screening to obtain is HX-64 cordyceps militaris link bacterial strain (purchased from Beijing City Agriculture and Forestry Institute Bio-Centers).
2, substratum (1L) for liquid shaking bottle: peeled potatoes 300g(liquor), glucose 10g, peptone 2g, potassium primary phosphate 0.5g, sal epsom 1g, water 1000ml, pH7.2.
3, sporophore is produced nutritive medium: the ratio of soya bean 2%, glucose 1%, milk powder 5%, potassium primary phosphate 0.5%, sal epsom 0.05%, VB11 0mg/1000ml is made into nutritive medium, adjusts pH 5.6～7.2.
4, the cultivation box (diameter 8cm goes to the bottom for high 10cm, upper base diameter 12cm) that adopts PP material to make.Every box rice 40g and sporophore are produced nutritive medium 56g packing mixing, disinfection inoculation.
5, mycelium culture management
Culturing room's optimal temperature is 18～25 ℃, and humidity is 70～85%, and lucifuge is cultivated 8 days.Mycelia starts aerlbic culture after being covered with surface.
6, sporophore is cultivated
After by kind, treat that mycelia is covered with whole culture vessel the dark container bottom of pricking, and starts to see light, but avoids sunlight direct projection.If culturing room's light is too dark, can supplements fluorescent lamp and shine.Mycelia sees after light, on culture material surface or surrounding see there is orange chromogenesis, and there is the orange button of grain of rice shape, button becomes sporophore after extending.Sporophore breeding phase, temperature should be 20～23 ℃, surpasses 25 ℃ of poor growths, and humidity should bring up to 80～85%.The mycelia atrophy over 27 ℃ can not go out bacterium bundle.In sporophore late stage of culture, strengthen gradually circulation and the exchange of room air, if finding to pollute, should remove immediately culturing room's cultivation stage.
7, gather: adopt rear mean fresh and reach 28.7 grams/cultivation box.
The embodiment thecasporous Collection and preservation of 2 Cordyceps militaris (L.) Link. (1)
(1) preparation of many spores carrier adsorption sand:
River sand is sieved with 60 orders, discard macrobead and impurity, then sieve with 80 orders, remove fine sand.With magnet, suck irony, put into 10% salt acid soak for container, the acid of desalting after 24 hours, washes bubble with water for several times to neutral, and sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the husky about 25g of many spores carrier adsorption of handling well mixes with equivalent distilled water and put into 250mL triangular flask, then whole 121 ℃ of moist heat sterilization 30min.After sterilizing, put into 80 ℃, loft drier and be dried 24 hours until complete drying.
(3) selection of the separated parent's sporophore of many spores:
Choose not polluted of later stage of just growing in culture bottle in embodiment 1, the sporophore growth cycle is short, bar shaped is neat, and even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the abundant head of ascoma is cut to 1cm with scissors, put into many spores separated and collected bottle that sterilizing-drying is crossed, collect 5 for every bottle.
Room temperature is placed 10 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore to fine sand by nature, have the ascoma of part maturation not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static dry end, many spores separated and collected bottle is put into shaking table, with the rotating speed of 80r/min, shake 24 hours.
Due to the rubbing effect of fine sand, thereby dry ripe ascoma and immature ascoma are received that the rubbing effect of fine sand is broken and then are discharged thecaspore wherein.Then dry sporophore is partly taken out, collect the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out.Finally realized the object of Cordyceps militaris (L.) Link. thecasporous separation, collection.The present embodiment can be collected the whole thecaspores in ascoma in sporophore, and collection rate is 100%.
(5) preservation of many spores
Small test tube, with 160 ℃ of 2 hours dry sterilizations, is sub-packed in the ripe thecasporous sand that launches out ripe thecaspore and do not launch out that contains of collecting in small test tube, then, with solid paraffin sealing, prevents that moisture from entering, and room temperature keeps in Dark Place 2 years.
The embodiment thecasporous Collection and preservation of 3 Cordyceps militaris (L.) Link. (2)
(1) preparation of many spores carrier adsorption sand:
River sand is sieved with 60 orders, discard macrobead and impurity, then sieve with 80 orders, remove fine sand.With magnet, suck irony, put into 15% salt acid soak for container, the acid of desalting after 24 hours, washes bubble with water for several times to neutral, and sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the husky about 50g of many spores carrier adsorption of handling well mixes with equivalent distilled water and put into 250mL triangular flask, then whole 121 ℃ of moist heat sterilization 30min.After sterilizing, put into 80 ℃, loft drier and be dried 48 hours until complete drying.
(3) selection of the separated parent's sporophore of many spores:
Choose not polluted of the later stage of just growing in culture bottle in embodiment 1, the sporophore growth cycle is short, bar shaped is neat, and even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the abundant head of ascoma is cut to 2cm with scissors, put into many spores separated and collected bottle that sterilizing-drying is crossed, approximately collect 10 for every bottle.
Room temperature is placed 30 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore to fine sand by nature, have the ascoma of part maturation not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static dry end, many spores separated and collected bottle is put into shaking table, with the rotating speed of 200r/min, shake 48 hours.
Due to the rubbing effect of fine sand, thereby dry ripe ascoma and immature ascoma are received that the rubbing effect of fine sand is broken and then are discharged thecaspore wherein.Then dry sporophore is partly taken out, collect the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out.Finally realized the object of Cordyceps militaris (L.) Link. thecasporous separation, collection.The present embodiment can be collected the whole thecaspores in ascoma in sporophore, and collection rate is 100%.
(5) preservation of many spores
Small test tube is with 160 ℃ of 2 hours dry sterilizations, the ripe thecasporous sand that launches out ripe thecaspore and do not launch out that contains of collecting is sub-packed in small test tube, then with solid paraffin sealing, prevent that moisture from entering, storage conditions is 0 ℃ of refrigeration 4 years.
The embodiment thecasporous collection of 4 Cordyceps militaris (L.) Link. (3)
(1) preparation of many spores carrier adsorption sand:
River sand is sieved with 60 orders, discard macrobead and impurity, then sieve with 80 orders, remove fine sand.With magnet, suck irony, put into 20% salt acid soak for container, the acid of desalting after 24 hours, washes bubble with water for several times to neutral, and sand is dried.
(2) sterilizing of multi spore collection container and drying treatment:
Get after the husky about 50g of many spores carrier adsorption of handling well mixes with equivalent distilled water and put into 500mL triangular flask, then whole 121 ℃ of moist heat sterilization 30min.After sterilizing, put into 80 ℃, loft drier and be dried 38 hours until complete drying.
(3) selection of the separated parent's sporophore of many spores:
Choose not polluted of the later stage of just growing in culture bottle in embodiment 1, the sporophore growth cycle is short, bar shaped is neat, and even thickness, color Exocarpium Citri Rubrum, ascoma fully-developed culture bottle put into Bechtop after bottle surface sterilization.
(4) collection of many spores:
Choose ascoma fully-developed sporophore, through aseptic technique, the abundant head of ascoma is cut to 2cm with scissors, put into many spores separated and collected bottle that sterilizing-drying is crossed, approximately collect 8 for every bottle.
Room temperature is placed 25 days, until sporophore complete drying.In the meantime, the ascoma that a part is ripe can launch thecaspore to fine sand by nature, have the ascoma of part maturation not yet to launch thecaspore just dry, some not full ripe ascoma does not enter the period of launching, and thecaspore is still retained in dry ascoma.
After static dry end, many spores separated and collected bottle is put into shaking table, with the rotating speed of 150r/min, shake 40 hours.
Due to the rubbing effect of fine sand, thereby dry ripe ascoma and immature ascoma are received that the rubbing effect of fine sand is broken and then are discharged thecaspore wherein.Then dry sporophore is partly taken out, collect the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out.Finally realized the object of Cordyceps militaris (L.) Link. thecasporous separation, collection.The present embodiment can be collected the whole thecaspores in ascoma in sporophore, and collection rate reaches 100%.
(5) preservation of many spores
Small test tube is with 160 ℃ of 2 hours dry sterilizations, the ripe thecasporous sand that launches out ripe thecaspore and do not launch out that contains of collecting is sub-packed in small test tube, then with solid paraffin sealing, prevent that moisture from entering, storage conditions is 6 ℃ of refrigerations 3 years.
The thecasporous activation of Cordyceps militaris (L.) Link. and the cultivation of embodiment 5 Collection and conservations
Get respectively the Cordyceps militaris (L.) Link. thecaspore of embodiment 2-4 Collection and preservation, under aseptic condition, open many spores preservation pipe, get the part grains of sand on PDA slant medium, grow after bacterium colony switching more once.Or get the grains of sand in suitable liquid nutrient medium (formula of 1L liquid culture medium is peeled potatoes 300g(liquor), glucose 10g, peptone 2g, potassium primary phosphate 0.5g, sal epsom 1g, water 1000ml) in, fruiting experiment or the inclined-plane of transferring again after multiplication culture, directly done.
After the Cordyceps militaris (L.) Link. thecaspore activation that embodiment 2-4 preserves, the method for recording according to embodiment 1 is carried out the cultivation of Cordyccps-militaris-(L.)-link. Sporophore.Cultivation results is found, the Cordyceps militaris (L.) Link. thecaspore that embodiment 2-4 preserves all can be cultivated and obtain sporophore, adopt rear mean fresh and reach respectively 28.7,28.9,28.8 grams/cultivation box, the sporophore that the output of sporophore and biological character and embodiment 1 cultivation obtain, all without significant difference, illustrates that the thecasporous method of Collection and preservation Cordyceps militaris (L.) Link. of the present invention can retain parent's inherited character.The present embodiment presentation of results: the Cordyceps militaris (L.) Link. thecaspore of the collection in above-described embodiment 2-4 bacterial classification under room temperature or general refrigerator condition is preserved 2-4 and still had biological activity, and fruiting body yield and biological transformation ratio, without significant difference, have retained the inherited character of original parent substantially.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the thecasporous collection method of Cordyceps militaris (L.) Link., is characterized in that, comprises the steps:
1) sand is put into many spores separated and collected container that can seal, sterilizing, dry;
2) Cordyceps militaris (L.) Link. ascoma fully-developed sporophore is put into many spores separated and collected container of step 1);
3) many spores separated and collected container that vibrates, collects the ripe thecasporous sand that contains the ripe thecaspore launching out and do not launch out.
2. collection method as claimed in claim 1, is characterized in that, described step 1) sand is neutral dry sand, and it obtains by the following method:
A) river sand is crossed to 60 mesh sieves, discarded macrobead and impurity;
B) with 80 orders, sieve again, discard fine sand, collect unsifted sand;
C) remove the irony in sand;
D), with after the salt acid soak sand 24h of 10%-20%, the acid of desalting, washes bubble with water to neutral, dries sand.
3. collection method as claimed in claim 1, it is characterized in that, the laying method that sand described in step 1) is put into many spores separated and collected container that can seal be by etc. after the sand of quality mixes with distilled water, 1:3-10 is placed in many spores separated and collected container by volume.
4. collection method as claimed in claim 1, is characterized in that, the sterilizing described in step 1) is 121 ℃, moist heat sterilization 30min.
5. collection method as claimed in claim 1, is characterized in that, dry described in step 1) is 80 ℃ of dry 24-48h.
6. collection method as claimed in claim 1, is characterized in that step 2) described sporophore puts into many spores separated and collected container, and the sporophore number of putting into and the mass ratio of container sand are 1: 2.5-5g.
7. collection method as claimed in claim 1, is characterized in that, the vibration described in step 3) is with 80-200r/min speed oscillation 24-48h by many spores separated and collected container.
8. the collection method as described in as arbitrary in claim 1-7, is characterized in that, also comprises dry sporophore is taken out in step 3).
9. the thecasporous store method of Cordyceps militaris (L.) Link., it is characterized in that, the ripe thecasporous sand that launches out ripe thecaspore and do not launch out that contains that the arbitrary described method of claim 1-6 is collected is placed in dry sterile chamber sealing preservation, and storage conditions is room temperature lucifuge or 0-6 ℃ of refrigeration.
10. the application of the arbitrary described thecaspore collection method of claim 1-7 in Cordyceps militaris (L.) Link. breeding.
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| CN109122049A (en) * | 2018-08-03 | 2019-01-04 | 内蒙古农业大学 | A kind of more spore separators of Cordyceps militaris and method |
| CN117063780A (en) * | 2023-10-17 | 2023-11-17 | 四川朕源生物科技有限公司 | Cordyceps sinensis ascospore harvesting culture medium and harvesting method thereof |
| CN117063780B (en) * | 2023-10-17 | 2023-12-26 | 四川朕源生物科技有限公司 | Cordyceps sinensis ascospore harvesting culture medium and harvesting method thereof |
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