CA1117881A

CA1117881A - Growth promoting method for basidiomycetes

Info

Publication number: CA1117881A
Application number: CA000336570A
Authority: CA
Inventors: Azuma Okubo; Akiyoshi Nakajima; Hitoshi Takita; Toshihiko Wada; Satoru Enomoto; Yutaka Mukaida
Original assignee: Kureha Corp
Current assignee: Kureha Corp
Priority date: 1978-10-03
Filing date: 1979-09-28
Publication date: 1982-02-09
Also published as: PH14315A; DE2939189A1; AU5117579A; FR2437772A1; IT1125434B; AU518829B2; DE2939189C2; GB2032456A; JPS5631112B2; JPS5548388A; GB2032456B; IT7926188A0; FR2437772B1

Abstract

GROWTH PROMOTING METHOD FOR BASIDIOMYCETES

ABSTRACT OF THE DISCLOSURE:
Disclosed is a method for producing the high-quality mycelia of the Basidiomycetes from culture thereof by promoting growth of the fungus as well as differentiation of the cells and attendant organization thereof by adding in the culture medium a straight chain saturated aliphatic alcohol with a carbon number within a specified range.

Description

111781~i~

BACKGROUND OF THE INVENTION:
This invention relates to a method or promoting growth of the fungi of Basidiomycetes by means of cultivation.
Increasing interest is shown recently in use of the fungi of Basidiomycetes as the base material for medicines and health foods, but low rate of growth of such fungi in culti-vation thereof as compared with other microorganisms such as bacteria or yeast is causing a bottleneck for industrial utilization of these fungi.
Addition of nutrients such as inorganic salts, extracts from natural products, etc., in the meidum has been prop~sed as a measure for promoting growth of the basidiomycetes in cultivation thereof. Addition of such nutrients in the culture medium has indeed an effect of promoting cell division of the basidiomycetes, but it can not bring about a similar effect for differentiation of the cells and attendant organi-zation thereof, and hence no desired promotion of growth and propagation is provided. Thus, such measure is unable to realize production of high-quality mycelia of the above-; 20 mentioned fungi at a high rate.
We found that growth of the basidiomycetes is - markedly promoted and also differentiation and organization of the cells are advanced by adding in the culture medium a small quantity of a straight chain saturated aliphatic alcohol with a carbon number within a specified range, thereby allowing obtainment of high-quality mycelia of the fungi.

~788~

The object of this invention, therefore,-is to provide a method capable of promoting growth and propagation of the basidiomycetes and producing the high-quality mycelia of the fungi by means of cultiva-tion thereof.

.~ BRIEF EXPLANATION OF DRAWING:

.
The accompanying dxawing is a graphic representation o the experimental results, showing the relation between the kind of the long-chain alcohol used and the dry culture yield.
In the drawing, numbers 1, 2 and 3 represent the results obtained from one-week culture, two-week culture and three-week culture, respectively.
The invention is described in detail hereinbelow.

. DETAILED DESCRIPTION OF THE INVENTION: .
The straight chain saturated aliphatic alcohol added ln the medium for cultivation of the hasidiomycetes according to this invention is represented by the following general formula:

CnH2n+1H (I) wherein n is an integer of 26 to 36. Examples of such straight chain saturated aliphatic alcohols include the following: ;

n-hexacosanol-l, n-heptacosanol-l, n-octacosanol-l, n-non-acosanol-l, n-triacontanol-l, n-hentriacontanol-l, n-dotri--acontanol-l, n-tritriacontanol-l, n-tetratriacontanol-l, n-pentatriacontanol-l, and n-hexatriacontanol-l. Most preferred ' among these alcohols for the purpose of this invention are those having a carbon number of 28 to 32.
It is very interesting to note the fact that, among these alcohols, those having an odd carbon number exist rarely in the natural products but have a growth and propagation promoting action for the basidiomycetes~ Most of the long-chain aliphatic alcohols existing in plants, such as n-hexacosanol-l, n-octacosanol-l, n-triacontanol-l, etc., are even in their carbon number, and it is reported (Science, Vol.
195, 1339 (1977) and Plant Physiol. Vol~ 61, 855 (1978)) that, among thesa long-chain alcohols, n-triacontanol-l alone has a specific effect of promoting growth of higher plants, but there is available no report on growth activity for the micro-organisms.
Thus, among long-chain alcohols, n-triacontanol-l alone has a specific action for promoting growth of higher plants, while all of the long-chain alcohols with a carbon ~ number of 26 to 36 used in this invention have the activities ;~ for promoting growth of the basidiomycetes as well as differen-tiation of the cells and attendant organization thereof.
The long-chain alcohols represented by the ahove-shown general formula (I) can produce the above-stated growth promoting ef~ect for the basidiomycetes by merely adding a small quantity of said alcohol, or within the range of 0.01 to 10 ppm, in the medium. From the viewpolnts of economy and solubility of long-chain alcohol in water, it is advisable l ~ 7~

to add such alcohol in an amount of 0.05 to 10 ppm in the medium.
It is also possible to use a mixture of two or more of the long-chain alcohols.
Although the long-chain alcohols used in this invention can be chemically synthesized, some of them can be also easily obtained from natural sources through extraction either singly or in the form of a mixture.
¦ As for the culture medium in which the long-chain ¦ alcohol is to be added,there may be used a wide variety of ¦ media which are generally employed for cultivation of the ¦ basidiomycetes~ For instance, there may be used the media ¦ containing carbon sources such as starch, sucrose, maltose, dextrose, wood chips, etc., nitrogen sources such as rice bran, wheak bran, corn steep liquor, peptone, broth, meat extract, soybean flour, powdered conttonseed, yeast extract, malt extract, urea, nitrates, etc., inorganic salts such as calcium salt, magnesium salt, sodium salt, zinc salt, copper salt, iron salt, manganese salt, etc., and other nutrients such as vitamins.
The culture medium may be either solid or liquid, and stationary or submerged culture may be emplo~ed in this invention. Addition of the long-chain alcohol into the medium may be made either before start of the cultivation or in the course of the cultivation after the fungus has grown to a certain degree.
The Basidiomycetes for which this invention is~

appliable include a wide variety of fungi that taxologically belong to the "Basidiomycota", but most preferred for use in this invention are the fuhgi belonging to the order Agaricales or Aphyllopharales of Homobasidiae, such as for example Armil-lariella mellea (Fr~ Karst, Tricholoma matsutake (S. Ito et Imai~ Sing., Lentinus edodes (Berk.) Sing., Coriolus versicolor (Fr.) Quél., Grifola gigantea (Fr.) Pilat, Favolus Arcularius - (Fr.) Ames, etc. The taxological nomenclature of the basidio-mycetes is based on "Coloured Illustrations of Fungi of Japan"
by ROKUYA IMAZEKI and TSUGUO HONGO~
- The outstanding effect of this invention is not limited to promoted growth of the mycelia of the basidiomycetes;
i~ also allows obtainment of the mycelia with small bulk specific gravity and advanced organization in cultivation of for instance ' ~ Coriolus versicolor (Fr.) Quel. Further, there are obtained clusters of mycelia in cultivation of Armillaria Mellea (Fr.) ~ Karst, and the mycelia obtained in cultivation of Laetiporus ;~ sulphureus (Fr.) Bond. et Sing. present reddish orange which is closely analogous to the color of natural fruit bodies. When ~aetiporus sulphureus is cultivated according to the conven-tional method, there are obtained light orange colored mycelia.
The histological change of the mycelia in cultivation of the basidiomycetes according to this invention appears to be based upon with the fact that the long-chain saturated aliphatic alcohol added in the culture medium is greatly concerned with growth and propagation of the fungi, in view of the change of ., q881 phenol oxidase activity, in cultured broth.
The invention is now described in further detail by way of the following examples, but it is to be understood that the scope of this invention is not limited to these examples.

Each of 20 ml ethanol solutions dissolved therein 6 mg portions of straight chain saturated alcohols with dif-ferent carbon numbers varying within the range of 26 to 36 was added dropwise into 2 litres o~ water respectively, and after dissolving, ethanol was evaporated to prepare the aqueous solutions of the alcohols with 3 mg/Q concentration each.
The rational formulae and melting points of the straight chain saturated aliphatic alcohols used in this example are shown in Table l below.
'': . ' - Table 1 Straight chain saturated Rational aliphatic alcohol formula U.p. (C) n-hexacosanol-l C26H53H 78 - 80 n-octacosanol-l ~28H57H 81 - 83 ~0 n-nonacosanol-l C29H59OH 82 - 84 n-triacontanol-l C30H61H 85 - 87 n-hentriacontanol-l C31H63H 86 - 88 n-dotriacontanol-l C32H65H 87 - 89 n-tetratriacontanol-l C34H69H 90 - 91 n-hexatriacontanol-l C36H73OH 91 - 92 ! ~78~

In the thus prepared long-chain alcohol solutions (2 litres each) were dissolved 120 g of glucose, 15 g of yeast extract and 4 g of malt extract to obtain liquid'culture media.
Seventy-five ml each of the obtained media was pipetted into 15 Erlenmeyer flasks (~00 ml capacity), and after cotton-plugged, each flask was sterilized at 120C for 15 minutes.
I The medium in each of the Erlen~eyer flasks was inoculated ¦ with 0.8 mg of the beforehand prepared seed culture of Coriolus ¦ Versicolor (Fr.) Qu~l. (FERM-P No.21~13) and subjected to ¦ stationary culture at 25C. For the sake o comparison, the ¦ cultivation was carried out in a medium of the same composition ¦ but not added with long-chain alcohol under the same conditions.
¦ After a given period of cultivation, the produced ¦ mycelia ir. the respective flasks were separated, washed well ¦ with water, ethanol and acetone and then dried at 60~- 80C.
¦ ~ The relation between the days of cultivation and the ¦ obtained dry culture yield is graphically illustrated in the accompanying drawing.
¦ As noted from the drawiny, when Coriolus versicolor ¦ is cultivated in the media added with straight chain saturated ¦ aliphatic alcohols according to this invention, a sharp increase in yield o mycelia is seen after the 14th day from start of the cultivation, indicating a prominent growth and propagation ¦ promoting effect of the straiyht chain alcohols on the ¦ basidiomycetes.

~ 7~38~

,:
. EXAMPLE 2 : Cultivation carried out by following the same procedure as Example 1 but by using the aqueous solutions of n-triacon-tanol-l in concentrations of 3 ppm, 2 ppm, 1 ppm and 0.1 ppm, respectively, as straight chain saturated aliphatic alcohol.
As control, there was similarly performed cultivation in a medium same as said above except that no alcohol was added.
The results of measurements on change of culture elements with time are shown in Table 2 below.

~9.78~L

_ _ _ ~ X
* a) o ~ o~ Ln o o ~ u7 ~ ~ O ~D O ~ r~
O O C~ n o o ~ ~ o ~ ~ ~ r~l H ~1 ~1 ~1 ~1 ~I r-l r 1 ~1 ~1 ~1 ~ ~1 I
a:~ rl ~ ~ Lr~ ~1 a) o ~n ~ ~r ~ ~ ~
~1 In ~D ~ ~ O Lll ~ r ~ ~D ~ I` r ~1 0 In Ln ~r L~ ~r ~ ~ ~D ~D a:~ ~ ~ ~
v rd O O O O O ~ ~ i a ~ ~

rl~ ~ ~ 1 0 a~ ~ ~D O ,~ O ~D ~ CO ~ d O ~ OD ~ 'I ~ CO O O ~ ~ ~r .~ I` Lr) ~ ~i OOOO ~ C~ OO1`CO ~
~ .~
O O O O L-) t~l O O O O O O O LO O
:~ o ~ ,~ ~ ~ ~ ~ ~r Ln Ln ~ ~ r P~ Ln Ln ~n Ln Ln ~ ~r ~r ~ ~ ~r ~ r e ~, R ~ .

u~ ~ ~ l~ r~ Ln r- o l-- o ~n o ~n ~D ~D co ~ ~D ~0 C ~ ~ ~ ~ o ~ ~D Ln ~ ~D ~ Ln ~ ~ ~ O Ln ~i ~
U a) rl Ln Ln ~D Ln Ln ~r ~ r ~ ~ t`~ ~ ~ N ~1 3 ~ ~ ~ .~
~ 8.~ ~ ~ o ,, ~
~ c ~ ~ e s~
r~l ~ ~ ~ O~oO
~ O ~ O O ~ ~ ~ ~0 ~ U~ 0 0 0 r~ U e R ~ L4 ~D ~ D Ln ~ ~ r ~ ~ ~ ~ r~7 N h r1 0 0 n~
E~ ~ ~ a) o ~
~C~P .
. _ _ _. _ 0 ~1)3 C: ~ ~I ~1 ~I
O C O O ~ O O ~ O O ~
'E~ ~'0 ~ ~
_ ~ ~ O g ~ ',1 rl .~ rl ~ ~c ~ ~ ~ ~ ~ ~ a .~ ~ .~ ~ .~ ~

a ~ c ~ aJ c ~ ~ c ........
h ~ " h ~1 ,~ h ~1 ~1 ~I N ~ ~
o x 3 ~ 3 o x 3 # ~ * *
o ~ E~ ~ ~ E~ ~ ~ E~
~ ~ _ _ o ~o ~, ~
~1 a .~

l ~

It is seen from the above table that n-triacontanol-l rather controls growth of mycelia of Coriolus versicolor in the early phase of cultivation, but it comes to show its activity for promoting growth of mycelia visibly from around 14th day of cultivation. It is also noted that the sugar content and reducing sugar concentration in the used medium and filtrate pH are not much different from those of the control, but the phenol oxidase enzyme activity associated with the intracellular factors changes very specifically by presence of n-triacontanol-1 in the medium.

It is to be also noted that the mycelia obtained from cultivation according to this invention were smaller in bulk specific gravity and more solid in tissue than those of the Comparative Examples (control).

There were prepared the aqUeQus solutions having dissolved therein n-triacontanol-l in concentrations of l ppm, 3 ppm and 10 ppm, respectively, according to the method described in Example l, and in each o~ these solutions (1 litre) ~lere dissolved 60 g of glucose and 7.5 g of yeast extract to form liquid media. For the sake o comparison, there was similarly prepared a medium by dissolving 60 g of glucose and 7.5 g of yeast extract in 1 litre of ~ater but not containing n-triacon-tanol-l.
One hundred ml each of the thus prepared media including the comparative medium was pippet.ted into 500 ml 1117BBl Erlenmeyer flasks, and there were prepared 10 incubators for each medium, totalling 40 incubators in all. After steriliza-tion, the medium in each the Erlenmeyer flask was inoculated with 120.8 mg of seed culture of Coriolus versicolor (Fr.) Quél.
(FERM-P No.2413) and then subjected to shaking culture at 25C.
After 5-day shaking culture, the produced mycelia from the respective cultures were separated, washed, dried and measured in weight~ Also, the phenol oxidase activity of each culture filtrate was determined. The results are shown in Table 3 below.

Table 3 n-triacon- Dry culture Phenol oxidase tanol-l yield *1 Index activity 2 present (ppm) (g/100 ml) (u/mg~

This 1 0.221 11910.74 invention 3 0.225 12110.85 0.218 11713.03 _ _ , Comparative ~ 0.186100 9.95 Example (Notes) *1: Dry culture yield per 100 ml of medium.

; *2: Phenol oxidase activity of the culture filtrate.
,, As apparent from the above results, the prominent effect of this invention was confirmed in shaking culture, too.
.

~ ~... ... ~ 8~

EX~MPLE 4 Twenty-five ml of an ethanol solution containing 7.5 mg of n-triacontanol-l was added dropwise into 2.5 litres of water and n-tiracontanol-l was perfectly dissolved in water.
Then ethanol was evaporated away to obtain the aqueous solution co~taining n-triacontanol-l in concentr~tion of 3 ppm. A portion of this solution was diluted with water to contain 1 ppm of n-triancontanol-l. In the thus obtained solutions (1 litre each) were dissolved 60 g of glucose, 3 g of yeast extract, 3 g malt extrac~ and 5 g of peptone to prepare the - 10 liquid culture media. Fifty ml each of the thus prepared media was pipetted into ten 200 ml capacity Erlenmeyer flasks, and after cotton-plugged, each of the flasks was sterilized at 120C for 15 minutes.
The media in the respective Erlenrneyer flasks were inoculated with 0.8 mg each of seed cultures of Laetiporus sulphureus (Fr.) Bond. et Sing. tFERM-P No.3032), Armillariella mellea (Fr.) Karst. (FERM-P No.982) and Grifola frondosa (S.R.
Gray) (FERM-P No.30333 and subjected to stationary culture at 25 +1C. For the purpose of comparison, there was conducted a similar cultivation operation by using a medium not contain-ing n-triacontanol-l. After a given period of cultivation, the produced rnycelia were separated, washed well with water, ethano and acetone and dried at 60 - 80C. The days of cultivation, dry culture yields and indices as determined for the respective mycelial products are shown in Table 4 below.

, .~lt l~i7889L

Table 4 n-triacontanol concentration (ppm) Comparative Exa~ple This invention Fungus Days of species cultivation 0 1 3 Dry Dry Dry culture Index** culture Index culture Index yield* yield yield Laetiporus 190.174 100 0.240 138 0.258 148 sulphureus (Fr.) FERM P No.3032 29 0.278 100 0.362 130 0.418 150 Armillariella 18 1.147 100 1.377 120 1.648 135 mellea (Fr.) FERM-P No.982 28 1.445 100 1.941 134 2.263 157 ,., Grifola fron- 26 0.887 100 1.034 117 1.044 118 dosa S.F.Gray FE~I-P No 3032 321.514 100 1.565 103 1.640 108 :'.
(Notes) * Dry culture yield (g/100 ml) per 100 ml of medium.
** The dry culture yield from the tricontanol-non-: added medium at each sampling date was given as 100 ,, : The above results indicate that n-tiracontanol-l .. takes an excellent growth and propagation promoting effect on the above-shown basidiomycetes.
It is to be also noted that the mycelia obtained from the process of this invention are distinguishable in color from those of the comparative examples, that is, in the case of Laetiporus sulphureus, the produced mycelia present reddish orange which is closely analogous to the ,, ~788~ .

color of the natural fruit bodies, and in the cas~ of Armil-lariella mellea, there is observed more conspicuous formation of mycelial clusters, while in the case of Grifola frondosa, the mycelial product has a color close to that of the natural products.

.,

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of cultivating fungus of Basidiomycetes characterized in that said fungus is cultivated in a medium containing at least one of straight-chain saturated aliphatic alcohols represented by the general formula:

CnH2n+1OH
wherein n is an integer of 26 to 36.

2. A method according to Claim 1, wherein said medium is a liquid medium containing at least one of said straight-chain saturated aliphatic alcohols in a concentration of 0.01 to 10 ppm.

3. A method according to Claim 1 or 2, wherein said straight chain saturated aliphatic alcohols are those having a carbon number of 28 to 32.

4. A method according to Claim 1, wherein said alcohol is n-triacontanol-l