CA2070715A1 - Method of cultivating mushrooms - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A culture solution containing a previously cul-tivated mushroom fungus is diluted with a liquid culture medium at dilution rate of 0.05/h or less, and agitated with an agitating power of 0,4 w/L or higher in a culture tank for continuous cultivation of the mushroom fungus at 25°C with a pH of 5. The culture solution is continuously drawn out of the culture tank, and inoculated in a mushroom bed or sawdust, etc. for mushroom cultivation.

Description

'92 06/05 21:19 glue METHOD OF CULTIVATING ~USHROOM5 The present invention royalty Jo a method ox cultivating ~ushrcom fungi continuously in a liquid and growing Wright bodies ox the quilted fungi a spawn.
~escrlptlon ox the Reloan Art:
now methods of culti~tlng mushroom fungi in liquid culture mediums to grow fruit bodily are shown in the invention ~ntltled "Method of cul~ivat~n~ edible mush-lo room" dlscl33ed in Japanese laid-open patent publi~atlon NO. 2-299516 and the ~nvenLi~n entitled mouthed of nlanu~ac-luring liquid spawn ox 8asidiomycete~.
According to the prior art disclosed in the for-men publication, Mazola of aas~diomyce~es are mull pled in a liquid ox primary culture, and then grown into fruit body on Myra beds or true log.
n the method disclosed in the latter public-lion, ~a~idiomycetes are cultivated in a liquid culture m~dlum acc~rdln~ to stationary culture, and then 'broken 20 into small pus, which aye thereafter mixed with qterll-eddy water to Shea there 19 adder a maternal or prevent-in the broken ~a~ldLomycete3 prom being dried, for thereby p~oducln~ liquid spawn ox ~asidiomyce~es.
The disclosed con~ent~on~l educe aye only do-rooted to batch culture, and aye not highly effective or mushroom culture. More specifically, the batch liquid gut-lure causes the fungi to be poulticed without being unit firmly dispersed in the culture medium. Therefore, the rooting ability of the fungi it poor, it wren the fungi art inoculated on a old culture medium, the m~c~lla have a Dow regenerating couplet, Thus, the Mazola spud slowly in the solid culture odium As a result, a large amount ox Mazola is required Jo be supplied, and the fungi have a low Nate of prQd~ctlon. In addition, the batch lit-old culture Ned pow for each lot, and takes long time~fGre it Starr cultivating edible mushrooms.
t is an object of the present invention to pro-I 0~6 21:20 ~005/014 vise a method ox cultivating mushroom ~fflciently.
cording to the present invention, there I
provided a method of calt~vatin~ Shari, comprising the steps of counsel agltat~ng a culture solution Wylie a S m~hroom fungus inoculated, with an agitating power of Owe wow or higher, ~i~ult~neously ~upplylng a llquLd culture dummy continuously or lnterm~ently to the culture oily-toil at dilution rate ox Ooze or lest fox contl~uously cultivating the mushroom fungus in the culture solution, JO continuously drawing out the culture ~olutlon in which the mushroom fungus his been cantlnuously cultivated, Ed thereafter, inoculating the drown owe vulture solu~lon, dip neatly or after concentrator, in a solid culture tedium or cultivation.
The method according to the present invention is capably e~fl~L~ntly producing Myra fungi with bettor rooting blowout for ef~icl~nt cultivation ox Myers.
he above and rather sect details and ad-vantages of toe present inven~ian will become apparent from I the ~ollowlng detailed description ox preferred embodiment Thor, when read in conjunction with the accompanying drawlngY .
The axle figure I schematic diagram of an a putts Pro courting out a method of cultivating edible ushers accardlng to the prevent invention.
method of cultivating edible Miriam accord-in to the present invention will specifically be described below Wyeth reference to the Cole figure.
A culture medium is prep eel ~er~lized~ and cooled in a jar fermenter l, which serves as a culture Yank. Thereafter, a spawn which may have been cultivated in a solid or liquid culture medium it lnocu$a~ed in the lure medium in the jar ~er~entex 1.
Thy spawn is when cultivated while being aerated 35 in the jar fermenter 1. when the fog conc~ntr~tton eke a certain level, e.g., 3.0 mg~mL in wry Tao, a valve 7 in a joint 5 is opened to continuously supply an-. :

' 92 0B~05 21 21 _ 3 _ 2 I I

other culture medium which ha been sterili~Pd and cooled in a container 3 to the jar fermenter 1 at a constant rate, i.e., a dilution rate D in the range of from Oily to 0~05/h~ At the same tine, the ultra medium in the jar 5 fermenter 1 it agitated by an ~itator 9 to start a Canaan-urea culture step. During the Catalina quill step, a small amount of the culture ~olutlon is taken out ~ontlnu-ouzel or intermittently at certain interval cry checkln~
bacterial contamination and marling fungus concentration.
C when thy Jung concentration is Rowley lncre~sed up to a certain level of about 5 try 7 my n dry stat, the culture solution it continuously drawn out at a constant rate, and concentrated directly or with a centrifuge 11.
Thy concentrated owlet solution 19 thereafter mixed with lo a gas, a liquid, ox a powder, or err mixture, after which the mixture 19 nucleated in a solid culture medium 13 such a A Miriam bad ox tree log Waco have been Toledo.
The inoculated unwise it cultivated into Myra t cult axle tempe~ture. it it possible to spread mushroom 20 rnycella quickly over the solid ultra medium 13 by ionic-cling the fuslgus at many locution or mixing the mushroom bed a f ton the icky fat ion .
It the ions it ' inoculated in a greater amount, then it can spreads Easter, but it has to be cultivated on 25 a larger amount in the culture tank. If the fungus it in-ocula~ed in smaller amount, then it may be Gultivatsd in a Molly mount in the culture ~anXr buy it take a lunger lye to spread in the mushroom bed and is susceptible to bacterial contarnlna~lon ekes: ox its poor antimicrobial 30 activity during the spread in the mushroom bed. In Roy ox the above considerations and also the cleanness of the gut-lure tank, the fungus should be supplies in a weight ran-in from O . l Jo 1 . O g in queue state to 1 kg ox the rnu~hroom bed. The Ritz of thy qupplled gnu may be varied from 35 the above wryness depend rug on the culture envircnmerlt, in-stallatlon, or the like.
The: time await the lure solution is drawn out ' 92 eye 21:21 007/~14 of the culture tan no beware it go inoculated in the solid culture muddle 13 should be a or a possible. If the culture ~olutlon it to by stored aster it has been drown out ox the culture Lctnlc 1, then it. should be stored 5 at log tetnpe~ature ox 5~C or lower for a period ox time of r day or Tartar, In the continuous liquid culture pro-us Decker above, thy spawn it continuously produced.
Louvre, the solid culture Madeline lo more often not contln-uau~ly produced. Doria night, holiday, or other day lo when the spawn lo not inoculated, the spawn that has con-twinkle been manufactured can be stored sod Lyon used when a old culture medium it produced next time. If, however, a large amount of spawn it to be stroll fox a long period ox lime, then a considerable storage installation is 15 required, and more importantly the activity ox the spawn us lowered during storage.
flew of the above Tories problem he lit-vendors have invented a process ox temporarily lowlsrlng the I use medl~m Dillon Nate or reducing it to Nero Uranus 20 the c~n~lnuous llquic~ quoter step when no mushroom bed is produced. it has been found that the cultivated fungus keeps its acelvlty as the spawn even when Thea clutter medium dilution rate I lowered or r~ducecl 'an zero during the continuous liquid culture step The quill medium used in to continuous liquid culture step i-q also waved.
Therefore, the invented process has proven very effective technologl~ally and economically. the extent to which the culture mud dilution Nate it to be lowered should be de-termined bayed on measurements of amounts of fungus and 30 spawn activitl~9 at the lye the dilution rate is varied.
Generally, no reduction in the spawn activity is often ox-perlenced when the ~llutivn raze is reduced to zero if in a very short purl ox time, e.g., halt day. It the dilution rate is to be towered or reduced to zero or a longer pew 35 nod of toe conditions should be determined Case by case.
It is Allah of fact to control the rate attach the culture solution is drawn out ox the ctllture Yank I ~6~05 21:22 go ~008~914 1 and the raze at which the culture medium is upload to the culture tank 1, separately from each other. owe specifically, when mushroom beds are produced, I the rate at which the lure solution is drawn out of the culture tank l it hike than llormal, then the amount of culture solution ion the quill tank 1 lo reduced. At this time, the rat at which the culture medium 13 upload to the culture tank 1 it reduced so chat the culture Moe dllu-lion rate I kowtow at a substantially constant laurel. When lo the amount a culture medium in the vulture tank 1 drops to a certain level, the Rowley of the culture medium prom the culture awoke lo intrepid. Therefore, the amount of gut-lure medium in Lowe culture Yank gradually lncrea~es again back to it original level.
~ltern~lvely, the oult~re medium may by up-plied Jo the culture Yank 1 at a constant rate, and when mushroom beds are produced, the culture solution may be drown out of the culture tank 1 at high rat, go 1~3 ox the entire volume at one Tao The culture medium may be supplied or the culture ~olutlon may be drown out inter-mlttently, continuously, or at varying rates.
Thy ~ushraom fungus may be cultivated in the mushroom bed 13 at a room temperature ran~lng prom 20 to 25C and a moisture ranting from 60 to 60 or a period ox US lye ranging from 70 to loo days, and thrower cultl~atQd at a root temperature of 15C and a molester of 90 % or the production of fruit bodies and bench ushers, Mushroom fungi that can be cultivated by the method according to the present invention a not limited to dry tickler species of mushroom, but ghoul prefer ably be wood destroying fungi such us Looniness eddied, Flammulina ~elutipes, laureates outright, ~yophyllu~ us-Myra, Foliate nameko, Ganoderma lucid, CorlQl~ vex color T~melLa fuciform~s, Auricularia ~urlcula, to The liquid culture mec^lLum used by the writhed act cording to the prevent ln~entlon, whether it aye by a pro culture medium prior Jo a continuous culture or a continue '92 06~05 21:23 ~009/01~
- 6 - 2 Do out rid culture m~dlum, is not limited to any particular culture mud us, buy Jay by any of aureole normal liquid culture mediums. For example, the Lowe culture medium may be mad, of soluble starch, Sucrose dextrin, cellulose, glycerin, Joy sauce oily wheat Iran, or the lit he ark of nitrogen used in the method may be petunia, meat extract, yeast tact soybean powder, flea bran, Callahan, polypeptone, gluten, or the like. The organic salt which is added to ho Liquid culture medium may be any of vacua lo phosphates, sulfites, hydrochloride, or the like. TO the llqul~ culture muddle, there may be added vitamin, nucleic acid, etc.
For continuously cultivating a mushroom fungus, it is important that the liquid culture medium ye upload S it dlluti~n Nate ox Owe or lets, and by agitated by the agitator which Lo upload with an agita~lng power at a rate of Owe Walter I or higher. Only when the above requirements are met, toe liquid culture medium can be minuend in pulp form for producing mushroom Fuji I lath strong reptilian ability.
If the agitator were upload lath the agitating power at i Tao smaller than the Nate of 0.4 Wylie, then the Luke culture medium would not be kept in a pump form, and if thy dllutl~n rate were higher than 0 . 05/h, then the con-I cent ration of fungi would be lowered.
Conditions or the pre-culture and continuous culture steps do pharaoh slightly wit unwise storyline and Cal-sure m drum oppositions use, and may be suitably selected depending on the fungus used. Usually, the culture should 30 he carried out it a temperature ranglnr~ f rum 20 to 40C
with a pa ranging from 3 to a at an aeration Nate in the range of from 0.1 to 2.Q v~v~rn (all volume culture tedium volume/mlnute). Toe culture medium may by fully mixed with agitation blades, aeration, or the live. The pricklier step is Cody out for a period of Lowe that may be deter-mined depending on the nature of the fungus sty used but thaw oily normally be in the range of Rome 24 hours '92 06~0~ 21:24 ~910~14 to 20 day. The culture solution thus produced by contlnu-owe cultivating the Messiah unyoke then concentrated directly or a centrifugal portray, after which the unyoke in the concentrated culture solution it nucleated on the S solid culture medium for usher cultl~tion. Since the fungus morphology it a pulp o'er, the Mazola are jot no-queried to be broken up into small pieces, or rather should not be done Jo because the footing ability would be low eyed.
lo In the continuous culture step, a fatty cold or its silt may be added to the culture medium that it sup-piled to the culture tank after the prelature step or it antimicrobial asset fly to suppress hactes~lal contamlna-lion without adversely affecting the multiplication of the lo mushroom Ennui and the production of useful components.
Particularly in the case where a mushroom fungus is to be cultivated in a continuous lulled culture apparatus, fig-old culture muddle stored in a container and a liquid gut-lure medium di9poqed in a joint between the container and 20 thy culture to are liable to gut contaminated and to at-low undeslrablé bacteria to be mul~iplled at a high rate thereon. The addition ox a Patty acid or it salt 'co the culture medium I Thor e~fec~clve to strolls those 1 lulled cut lturc medium .
US the Patty acid or its salt Jo be added should ye of a carbon nabber of 6 or less, or more p~ferahly 4 or lest, Tom thy endpoint of antlmicrubial activity and we-ton sealability.
In order Jo prevent the son~ainer fry being 30 contaminated by undesirable bacteria, to prevent under able bacteria from being multlplie~ in the container, and alto to Lowe the mushroom fungus Jo be normally cultivated in thy continuous culture slap, it it necessary that the the fatty Clyde or its Walt by added to the culture medium such that it has a concentration of Oily to 3.0 (we~ght/volume~ on the culture medium. If the consent-tlon.of toe added fatty acid ox its silt exceeded 3.0 %

~92 ~6/05 21:24 ~011/014 - 8 - 2~7~7~

(WOVE), then the mushroom fungus would nut be normally gut-twitted and useful substance would not be stably produced.
In par~cularly, the con~entratlon of the fatty acid or its salt should by in the range ox prom 0.07 Jo 2.0 S (WOVE). Thy concentration of or higher Jan 0,07 WOVE) Lo effective to prevent undesired bacteria Roy being grown, no to permlc mushroom unwise and useful substance to be produced ef~lc~ently it the continuous liquid culture whereat that c~ncentra~lorl Gould o~trl~ct the production of mushroom fungus and useful substance Lo batch liquid culture process. The. concentration of or let than 2.0 4 WOVE) is alto effective to allow mushroom fungus to be highly normally cultivated and useful substances to be eta-by.
The pi ox the culture medlu~lto which the await aloud or lo lo ha been added, i . e ., the culture odium prior to being supplied to the culture Yank, should be 6 or less ox more preferably be S or lea, for wetter proven-Tony of bacterial contamination.
The fatty acid may, or example, be formic acid, prop ionic acid, berrylike acid, Valerie cold, caprolc cold, or the like, and should preferably be acetic cold in pa-t ocular .
the salt of the fatty cold may, for example, be 25 salts ox alp eel ego., swept of sodium, potas~lum, etch or salts of alkaline earth mote} twig-, salt of p~t~ium~ magnesium, etch The fatty acid or its Walt Jay by ox one material or a combination ox two ore move ma-trials.
In the contlnl~ou~ culture step, lignin may also be added to the culture odium which is supplied Jo the culture tank in which the fungus I being ~ultlvated, for increasing thy ~ungUQ conc~tEation. The llgnln may be added in a small proportion rangln~ Roy Oily to 0.5 WOVE), us should be added in a proportion ran~inq from 0.05 to 0.2 WOW. the elect ox the lignln remain the say Zen it it is added in a proportion higher than the ~92 ~6~5 21:25 ~912/014 20707~L5 above range.
The llgnln may by liqnin ox broad-leaved tree such as beech, oak, or the like, or lignLn ox needle-leaved tree such as pine, cedar, cypress or the like.
Sp~cl~ically, it may ye commercially available deal~alized lig~ln~ uric acid llgnin, or the like, ox vanilla frolic acid, or the like which is a consent monomer of llgnin.
The culture medium which is selected may be sterilized in any way. Fox example, a culture medium to which a fry cold or its Walt ha teen added or not added may be externally strolled with heat or membrane filter-lion either in batch or continuously and then placed in the container. If a fatty Clyde or lo salt has not been added to the culture outside of the container, it may then be add Jo toe stern Ed culture medium in the container in a germ-free environment. Alternatively, culture medium to which a await acid or ltq salt hag been added may be jterlliz~d in a container, or with a con~lnuou~ eerily-ire which Jay be d1spos~d on a suitable position in the~oint between the contalne~ and the culture tank. On the latex swilling arrangement, thy culture medium can easy sly ye ate~iliz~d because bacteria in on unsterilized Cal-turn medium err privet from being m~tlplied by the ad-~ltlon of a Patty acid or its salt.
n the culture tank, there is employed, or ox-ample, a culture tedium which is the same as that in the container except that it contains no Patty Clyde or its salt, and a de trod mushroom fungus is lnocul~ted on thy culture medium or pre-culture. When the fungus is multi-plied to owe exit after a lag phase, the culture medium in the container is continue supplied to the culture tank through the joint.
the old culture medium or cultivating the mushroom fungus by way ox continuous liquid culture may be true logs, or mushier hod compri~lng a grower material such as sedate rice hull or the like to which a nutrient ' 92 06/05 21 26 I 013~014 -10 71~

source such aye rice bran, wheat bran, Or toe like I aided.
For cultivation efficiency, the mushroom bed is referred to the tree logs.
Experiment examples will be described below 5 err numerical acuminate ox vantages of the present in-venison .
PIPER ZIP 1:
Conditions for turning a liquid culture medium into a pulp form, and effects of llgnin lull be given be-1 0 low .
1. Experimental condition:
Angus strain: fungus of hentlnus edodeQ Al No.
46S) I Culture medium com~o~itlon:
[l] Without liS~nin:
Polypeptone 0 4 eta extract 0 . 4 Glucose S . 0 Monob~sic put slum phosphate 0 . l Magnealum sulfate o . l Calcium hydrochloride 0 . l 12] With llgnin Polypep~one 0, 4 Yeas extract 0 . 4 %
2 5 Glucose S . 0 Monoba~ic potassium phosphate Owl %
Magnesium sulfate 0.1 Calcium hydrochloride 0.1 De~lkali~ed Lenin 0 . l %
* Unit: W/V
I Culture medium phi So I Cultivating temporary: 25C
I Aeration rate: 0,5 v/v/ln 6) ~ilutlon rate: 0 . 02/h 2. E;~perlmenta~ equipment:
200 jar ~ermerlter. Agitator with two blade.
In pre-culture step, 200 my of spawn which had been 92 06~05 21 26 ~014~019 cultivated in a Sakaguchi's Luke for 30 days was ionic-late and cultivated while being agitate with an agitating power of O . 4 watt Lotte Wylie) fox 10 days . Thereafter, the cul~lvat~d fungus wee continuously cultivated, and her-S vetted on thy Thea day.
* Method ox mooring the agitating potter:
lo With no water in the 20~ L jar ferments, toe joy-toting Lydia are rotated, and consumed power PA) it de-termlned at ho rotating speed of the a~ltatlng blades.
lo I when, 150 liters ox water ore poured into the 200 L jar Serment~r, and the agi~atlng blades are rotated. A
Cowan power I) is deter~lned at the retooling speed of the agltn~lng blades.

3) The agitating power it determined according Jo (I V where 18 the volume of the jar fermenter.
3. Experimental results:
eye experimental results are Lyon in the lot-towing tale 1:
.
~gitatlng Culture medium with no Gullet medium with power w/L lignin lignin __ CultureFungu~ Culture Fungus my 0.2 I A 6.5 6.5 0.3 5.5 a _ so .4 5 lo 0 1 0 c 13.0 C 13.0 2 5 It 0 ___!~______ 3 0 C 12,0 _ _ C L_ A: Pi tired form B: Pi tired pulp form C: pulp Norm It can be seen from the above expel men that the morphology of the culture medium is turner into a pup I form when the agitating power is a least 0.4 Wylie. When '92 ~6,~05 21 31 ~001~011 the agitatln~ power ranges from 0.4 to 3.0 Wylie, the rotate in speed of the agitator range prom loo to 250 rum It can also by teen that the amount of fungus produced 1 larger with the culture medium containing ll~nln, S ~EXP~RIMEN~Al. EXAMPLE 2:
Amounts and tooting abilities of Eying podded according Jo ln~entlve and conventional method in the ox-psri~ental example 2 will be de3crlbed below.
A. Inventive Method:
lo l. ~xperl~ental conditions:
The same as those of the experimental example 1:
2. Experimental in~tallatlon:
The tame a those ox the experimental exam~lu 1:
* Roy pr~-culture step was alto the same as what ox the experiment example 1. The continuously cult lad unwise way harvested on the Thea day.
B. Conventional method thatch method):
(1) Fungus strain: fungus of Lentinus erodes oriole Jo.
455~
Culture eddy oompositlon:
The tame as that of the ~xperlmen~al example 1.
13) Cultlv~ting process:
Spawn was aerobically cultivated in a Sakaguchl'~ flask having a volume of 500 my or JO days. 200 my Or the gut-US tlvated solution was cultivated a spawn in a 150 L of fig-rid culture medium in a 200 L jar fermenter or lo day on-dew the following conditions:
Cul~lvating temperature: 25C
Culture tedium pi: 5,0 Ae~atlon rate: 0.5 vim Agitating power: lo we 2. ~xperi~antal inst~llstlon:
200 L or fermenter.
* Method ox measuring the footing ability:
Owl L of rich bran was mixed with l ox sawdust, and the molter of the mixture was adjusted to SO I.
Thereafter, A wide-mouthed flask ox 200 L way parked with ' 92 013~05 I 32 UP [~1002~011 - 13 - ~17~5 50 g of the mixture, and then twirled with saturated steam at 11~ jar US minutes. Tory the st~rilixed mlx~ur~
we cooled Jo room temperature, 1 AL of culture solution was aided to the cooled mixture and mixed therewith. The mixture wee the pelt in a petrol dish having a dlam~ter of 9 I and a thlckn~s ox 3 cm. The mlx~ure in thy petrol dish wee then cultivated at 30~C or I hours. The number of colonies of L~n~ln~ eddied fungi produced on the surface of the culture medium was counted as a footing ability.
3. paramountly results:
The experimental results are given it the Sol-towing table 2:
__ Fungus Reptilian alto mammal ~number/~etri dish) __ _ Inventive method 13 . O 92 _ _ Conventlanal method 5.2 I
_ _ _ _ It will b understood that the inventive method is capable of producing more gnu with hlghe~ rooting ability than the conventional method.
INVENTIVE; fax A culture modicum A way composed of 25 g ox grape sugar, 4 ox eighteen, 4 g of yeast extract, 2 of ~onoba-sly potassium pop hate 2 g ox h~pt~hydrate of mine I'm slate and 1000 my of waxer, 100 my ox the culture medium A was put in a Sakaguchi~s flask having a volume of 500 my, st~silized wick steam at }~0C under 1 atmospheric pressure for 15 Nanette, and then cooled down to zoom Tom 2$ putter. The, a fugue of Looniness erodes (Morn Jo. 465) was inoculated in the culture medium A, and cultl~ated at 25C or Lo days. 800 L of the culture medium A and ~00 my of ~lllcone manu~actuxe~ by Tray industries, Ltd. were placed in jar fermenter having a volume of 1000 L, suer-lucid with swam at 120C under 1 atmospheric pi sure or 20 Nanette and cooled down to 25~C. on piece ox the cultivated fungus were thin inoculated as spawn in the '92 ~6~5 21:33 ~003~
- 14 - 2 I I.

cooled culture odium I. The fungus way cultlva~ed while by aerated at 25C for 7 day. The concentration ox the fungus was gradually increased to a level of 3 Smalley it dry state. A culture m~diu~. ox the same compost lion as the S culture m~dl~m A was added to toe tar errantry a a flu lion rote D ox O.OlJh, and a contln~ou~ queller step aye twitted. The concentration ox the fungus was further in-created, end reached lo gym in dry state in days.
The Cannes vulture was Charlie out at 25C
lo with a pi of 5 at an aeration rate of Owe TV lo an ago stating power of 3.0 Wylie.
Then, cultivated mushrooms were produced using the quilt solution ~btalned thus far aye spawn. More ~p~c~lcally, 1 L of sawdust and Cot I raw rice bran wore mixed, and the ~olsture ox the mixture jag adjusted to 60 I. 1 kg of the mixture was then packed in a jag of polypropylene having a porous filter a a vent hole, and ~terlllzed at 120C owe 1 hour and thin cooled down to room temperature. To the cooled mixture, there way added 30 my of the continuously cultiY~t~3d fungi ox culture sultan desc~bed above Aster thy were mixed, the fungus way cultivated it ~2~C with a moisture ranS~lng foe 60 to MU
for 80 days. Thereaf~ex, the fungus was cultivated at l5~C
with a moisture ox 90 % for the psoduc~lon of royalty bodies.
US Aster 7 days, 160 go ox Lentinus dyes was arrested per unit area ox culture medium. Then 70 g/kg was havened after JO days, and 90 gig way harvested after 40 days.
TIP EXAMPLE 2:
A culture medium was composed ox 30 g of grape sugar, 6 g of depone 4 9 ox yeast extract, g ox monoba-sly pota~aium phosphate, 0.5 of heptahydrate ox magne3lum sulfate, and l L of water. loo of the culture medium B
was put in a Sakaguchl~ ala k having a volume so SO my.
A guying Or Flyweight nam~ko (Hokuken Ann N217) was Cal-tlvated in thy culture medium 8 it 25C for jays Wylie ltwas bluing shaken. 800 of thy culture medium B and 400 my of silicone manufactured by Tray Industries, Ltd. were I ~6~05 ~1:33 isle ~Q7~
- lo -played in a jar fermenter having a volume ox loo L, stern ilized with Sue at 120C under 1 atmospheric prowar for 15 minutes, and cooler down to ~5C. Ten pus ox the cultivate fungus were then inoculated as spawn in the cooler culture Dow B. The fungus was cultivated white balmy neratcd at 25~C fox 5 day. The concentration of to fungus wee gradually lncLea~e~ to Level a 3 mg~mL in dry state. A culture Moe ox the same composition as the culture medium B way placed in another or having a volume lo of loo I, and acetic acid was added to the culture odium so that toe acetic acid ha a concentration ox owe I. The culture medium was then serialized and cooled. Thereafter, the vulture odium was added to the above culture tedium B
at a d~lutlon raze D ox O . 02~h, and contlnu~us culture I step was started. Thy coneentratlo~ of the fungus was fur-then increased, and reached lo mum in dry state in 5 days .
The continuous culture way carried out at 25~C
with à pi of 5 it on aeration Nate of 0.5 vJv~m with an ago itatlng power ox 3.0 w/L.
The number of bacteria and the amount of fungus~ln dry state) were determined as whey varied wit time.
The results are given in the Sallowing table 3:
Table 3 . _ _ _ _ Days of cult- 5 10 lo I
ration _ _ _ Number of bee- O O O O
I= _ , __ Amount ox fun- 7.5 Lowe 10 9 11.2 I___ __ Days of cult- 25 30 40 US
__ ____ Number of bee- O O O O
I__ _ __ _ Amount of fun- 11.7 11.3 10.4 __ __ ' 92 06~05 21:34 UP
1~1 005~01 1 2~71~

The number ox Australia in the culture so-Lucia wow measured us follows: The culture solution was filtered by gauze in a swarmer conditl~ to remove the fungus of Foliate nameko. Thy culture solution was then S dll~tcd with physiologic alone at a ratio selected in TV ow of the number bizarrely 1 my a the diluted culture 90-Litton we Ted iota 7 my ox a culture elm composed of l ~WJV) ox teat extract, 1 wove of palypeptone, 0.5 of yeast extract, 1 & of glucose, and 1.5 of ajar, the culture medium having a pi ox 7Ø The mlxtu~e way then cultl~ated at 37C for 24 hours, and the number ox colonies produced was measured a the number of viable bacteria.
Thy, cultivated mushroom were px~duced using the culture solution stained thus far as spawn Gore lo specifically, 1 L of audit and 0.2 L of raw floe Iran were mixed, end the oilier ox the mixture was adjusted to 60 %. 1 kg ox the mixture was then packed in jag of polypropyl~nc having a porous liter as a vent hole, end sterilized at 12~C for l hour and then cooled down to room temperature. To the cooled mixture, there was added, prom byway, 30 AL of thy continuously cultivated fungus or gut-lure solution de~crlbed above, two added culture solution being not mixed. The ~unguq was cultl~ated at 22C with a moisture ranging from 60 Jo I for 60 jays. Thereafter, the fungus was cultivated at 15C with a moisture I 90 %
for the production ox fruit bodies. Aster 20 days, 180 gig ox Pullout nameko was harvested per unit area ox gut-tore medium. Then, 70 gfk~ was harvested aster US day, and 50 gig was rutted aster 45 day.
3 0 NATIVE: EN E 3:
100 my ox the culture medium A (see the liven-live example l above) was put in a 5akaguchi's ask having a volume of 500 my, strolled Thea strum at 120DC under 1 atmospheric pressure or 15 ~inutl~s, and when cooled down 35 to room temperature. when, I ~unguq ox Lentlnu~ educe Myra No . 4 651 was inoculated in ho culture medium A, and cultivated it 25DC for lo days while it was busing shaken.

'92 ~6~05 21:35 00~0 1 1 800 L of the culture medium A and 400 AL of silicone menu-lectured by Tray Indust~1es, kid. were place in a jar fermenter having a vilely of Lowe L, twirled with steam at 1~0C under l at~ospherLc pressure or JO mln~ltes, and 5 cooled down Jo 25C. Ten posses of the ctllti-rated Angus were then inoculated a spawn in the cooled culture medium . The fungus way cultivated while Boeing aerated at 25C
fox 7 clays. The concentration of the forgoes way gradually increased to a level of 3 mg,'mL in dry Sue. A culture lo medium of the same composition as the culture medium A way placed in another tar having a volume ox loo L, and deal-allied lignin was added to toe culture medium so that the dea1kal1zed llgrlin had a concen~ratlon of Owl 4. The Cal-turn metlum way then sty Ed end cooled. Thereafter, the culture tedium way added to the above culture medium B
at a dilution aye of 0.01~h, sod a continuous culture step way started. ye concentration of the fungus was our-then increased, and reached 15 mg~mL in duly state in 7 yo-yo .
Tic continuous culture was called it it 25C
with a pi of S at an aeration rate ox OHS vim with an ago ltatlng power ox 3.0 wife.
when, cultivated usher were produced using toe culture solution tanned thus far as spawn. More I specifically, l L ox sawdust and a Al L of raw flee bran were mixed, and the moisture of the mixture was adjusted to 60 I. l I of the mixture way then packed in a bag of pol~propy1ene having a porous filter us a Kent hole, and strolled at 120DC or hour and then cooled down to zoom 30 temperature To the cooled mixture, the way added I my of thy continuously cultivated f~ngu3 or vulture ~olutlo~
described above After whey were McCoy, the fungus way culSlvated at 22~C with a Miss ranging from 60 to I
or 80 days. thereafter, the ~un~u3 was cultivated at 15~C
with outwore of I for toe production ox fruit bodiless.
After 7 day, 160 gfkg of enhance erodes way harvested pox unit aroma ox culture medium. Then, 70 gJkg was harvested 92 06~05 21:35 by after 20 days, arid 8û q/kg way harvested after 40 Dow.

Claims (12)

1. A method of cultivating mushroom, comprising the steps of:
continuously agitating a culture solution with a mushroom fungus inoculated with an agitating power of 0 . 4 w/L or higher, simultaneously supplying a liquid culture medium continuously or intermittently to said culture solution at a dilution rate of 0.05/h or less for continuously culti-vating the mushroom fungus in the culture solution;
continuously drawing out said culture solution in which the mushroom fungus has been continuously culti-vated; and thereafter, inoculating the drawn-out culture solution, directly or after concentrated, in solid cul-ture medium for cultivation.

2. A method according to claim 1, further in-cluding the steps of starting to agitate and dilute said culture solution when the concentration of the fungus in the culture solution reaches a first concentration level, and drawing out said culture solution when the concentra-tion of the fungus in the culture solution reaches a second concentration level which is higher than said first concen-tration level.

3. A method according to claim 2, wherein said first concentration on level is 3.0 mg/mL, and said second concentration level ranges from 5.0 to 7.0 mg/mL.

4. A method according to claim 1, further in-cluding the step of temporarily lowering said dilution rate to zero or closely to zero when said liquid culture medium is supplied to said culture solution.

5. A method according to claim 1, further in-cluding the step of temporarily preventing the culture so-lution from being drawn out as long at the culture solution is of a predetermined amount or less.

6. A method according to claim 2, further in-cluding the step of previously cultivating the mushroom fungus before the mushroom fungus is continuously culti-vated in said culture solution, until the concentration of the fungus in said culture solution reaches said first con-centration level.

7. A method according to claim 2, further in-cluding the step of measuring the concentration of the fun-gus in said culture solution continuously while the mush-room fungus is being cultivated in said culture solution.

8. A method according to claim 1, further in-cluding the step of adding fatty acid or its salt to said liquid cultured medium when the mushroom fungus is culti-vated in said culture solution

9. A method according to claim 8, wherein said fatty acid has a carbon number of 6 or less.

10. A method according to claim 8, wherein said fatty acid or its salt is added in a ratio ranging from 0.01 to 3.0 wt./vol.

11. A method according to claim 1, further in-cluding the step of adding lignin to said liquid culture medium when the mushroom fungus is cultivated in said cul-ture solution.

12. A method according to claim 11, wherein said lignin is added in a ratio of 0.001 wt./vol. or higher.