GB2032456A - Growth promoting method for basidiomycetes - Google Patents

Abstract

A method for producing high- quality mycelia of Basidiomycetes by promoting growth of the fungus as well as differentiation of the cells and attendant organization thereof comprises cultivating the fungus in a medium containing at least one straight chain saturated aliphatic alcohol represented by the general formula CnH2n+1OH wherein n is an integer of 26 to 36. o

Description

SPECIFICATION Growth promoting method for basidiomycetes This invention relates to a method for 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 cultivation 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 medium has been proposed 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 organization 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-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.

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 cultivation thereof.

The accompanying drawing is a graphic representation of 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 threeweek culture, respectively.

The invention is described in detail hereinbelow.

The straight chain saturated aliphatic alcohol added in the medium for cultivation of the basidiomycetes according to this invention is represented by the following general formula: CnH2n + ,OH (I) wherein n is an integer of 26 to 36. Examples of such straight chain saturated aliphatic alcohol include the following: n-hexacosanol-l, n-heptacosanol-l. n-octacosanol-l, n-nonacosanol-l, n-triacontanol-l, hentriacontanol-l, n-dotriacontanol-l, n-tritriacontanol-1, n-tetratriacontanol-l, n-pentatriaconfanol-i, 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; noctacosanol-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 these long-chain alcohols, ntriacontanol-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 differentiation of the cells and attendant organization thereof.

The long-chain alcohols represented by the above-shown general formula (I) can produce the above-stated growth promoting effect 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 viewpoints of economy and solubility of long-chain alcohol in water, it is advisable 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, wheat bran, corn steep liquor, peptone, broth, meat extract, soybean flour, powdered cottonseed, 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 employed 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 fungi belonging to the order Agaricales or Aphyllopharales of Homobasidiae, such as for example Armillariella mellea (Fr.) Karst, Trichloma matsutake (S.l. to et Imal) Sing., Lentinus edodes (Berk.) Sing., Coriolus versicolor (Fr.) Fuel., Grifila gigantea (Fr.) Pilot, Favolus Arcularius (Fr.) Ames, etc. The taxological nomenclature of the basidiomycetes 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; it also allows obtainment of the mycelia with small bulk specific gravity and advanced organization in cultivation of for instance Coriolus versicolor (Fr.) Fuel. Further, there are obtained clusters of mycelia in cultivation of Armillaria Mellea (Fr.) Karst, and the mycelia obtained in cultivation of Leatiporus sulphureus (Fr.) Bond. et Sing. present reddish orange which is closely analogous to the color of natural fruit bodies. When Laetiporus sulphureus is cultivated according to the conventional 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 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.

EXAMPLE 1 Each of 20 ml ethanol solutions dissolved therein 6 mg portions of straight chain saturated alcohols with different carbon numbers varying within the range of 26 to 36 was added dropwise into 2 litres of water respectively, and after dissolving, ethanol was evaporated to prepare the aqueous solutions of the alcohols with 3 mg/l concentration each.

The rational formulae and melting points of the straight chain saturated aliphatic alcohols used in this example are shown in Table 1 below.

TABLE 1 Straight chain saturated Rational aliphatic alcohol formula M.p. ("C) n-h exacosanol -1 C26H,30H 78 - 80 n-octacosanol-1 C28H,,70H 81 - 83 n-nonacosanol-1 C2sHssoH 82 - 84 n-tri acontanol-1 C30H610H 85 - 87 n-hentriacontanol-1 C31H630H 86 - 88 n-dotri acontanol -1 C32H6s H 87 - 89 n-tetratriacontanol-1 C34H690H 90 - 91 n-hexatri acontanol-1 C36H730H 91 -92 In the thus prepared long-chain alcohol solutions (2 litres each) were dissolved 120 g of glucose, 1 5 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 1 5 Erlenmeyer flasks (200 ml capacity), and after cotton-plugged, each flask was sterilized at 1 200C for 1 5 minutes. The medium in each of the Erlenmeyer flasks was inoculated with 0.8 mg of the beforehand prepared seed culture of Coriolus Versicolor (Fr.) Fuel. (FERM-P No.2,413) and subjected to stationary culture at 250 C. For the sake of 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 in the respective flasks were separated, washed well with water, ethanol and acetone and then dried at 60--800C.

The relation between the days of cultivation and the obtained dry culture yield is graphically illustrated in the accompanying drawing.

As noted from the drawing, when Coriolus versicolor is cultivated in the media added with straight chain saturated aliphatic alcohols according to this invention, a sharp increase in yield of mycelia is seen after the 14th day from start of the cultivation, indicating a prominent growth and propagation promoting effect of the straight chain alcohols on the basidiomycetes.

EXAMPLE 2 Cultivation carried out by following the same procedure as Example 1 but by using the aqueous solutions of n-triacontanol-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.

TABLE 2

*1 *2 *3 Triacontanol Sugar content Reducing sugar Enzyme Dry culture concentration In filtrate concentration activity yield *4 Days of culture (ppm) (%) in filtrate (%) pH ( /mg) (g/100 ml) Index Comparative Example 0 6.0 5.97 5.00 2.09 0.559 100 0.1 6.0 5.97 5.10 0.86 0.563 101 8 day s 1 6.2 6.05 5.10 0.83 0.492 88 This Invention 2 6.2 5.97 5.10 0.91 0.531 95 3 5.6 5.60 5.15 0.10 0.505 90 Comparative Example 0 4.6 4.57 4.32 9.69 1.451 100 0.1 4.4 4.40 4.40 8.82 1.469 101 14 days 1 4.6 4.65 4.40 7.08 1.670 115 This invention 2 4.6 4.30 4.50 8.00 1.629 112 3 4.6 4.59 4.50 6.81 1.643 113 Comparative Example 0 3.5 3.36 4.40 11.10 1.944 100 0.1 2.8 2.78 4.40 10.48 2.261 116 21 days 1 3.0 2.98 4.40 10.11 2.323 120 This Invention 2 3.0 3.02 4.45 7.78 2.375 122 3 2.7 2.56 4.40 8.52 2.671 137 NOTES: *1: Measured by refraction viscometer.

*2: Phenol oxidase activity in culture filtrate.

*3: Dry culture yield per 100 ml of medium.

*4: The dry culture yield from the triacontanol-non-added medium at each sampling date was given as 100.

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-l in the medium.

It is to be also noted that the mycelila 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).

EXAMPLE 3 There were prepared the aqueous solutions having dissolved therein n-triacontanol-l in concentrations of 1 ppm, 3 ppm and 10 ppm, respectively, according to the method described in Example 1, and in each of these solutions (1 litre) were dissolved 60 g of glucose and 7.5 g of yeast extract to form liquid media. For the sake of comparison, there was similarly prepared a medium by dissolving 60 g of glucose and 7.5 g of yeast extract in 1 litre of water but not containing p- triacontanol-l.

One hundred ml each of the thus prepared media including the comparative medium was pipetted into 500 ml Erlenmeyer flasks, and there were prepared 10 incubators for each medium, totailing 40 incubators in all. After sterilization, 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 250C. 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 Dry culture Phenol oxidase n-tri acontanol-1 yield* 1 activity *2 loading (ppm) (g/100 ml) Index (u/mg) This 1 0.221 119 10.74 invention 3 0.225 121 10.85 10 0.218 117 13.03 Comparative Example 0 0.186 100 9.95 (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.

EXAMPLE 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 solutions containing n%triacontanol-l in concentrations of 1 ppm and 3 ppm, respectively, and in the thus obtained solutions (1 litre each) were dissolved 60 g of glucose, 3 g of yeast extract, 3 g of malt extract and 5 g of peptone to prepare the 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 1 200C for 1 5 minutes.

The media in the respective Erlenmeyer flasks were inoculated with 0.8 mg each of seed cultures of Laetiporus sulphureus (fin) Bond. et Sing. (FERM-P No. 3032)Armillariella mellea Karst. (FERM-P No. 982) and Grifola frondosa (S.R. Gray) (FERM-P No. 3033) and subjected to stationary culture at 25 + 1 OC. For the purpose of comparison, there was conducted a similar cultivation operation by using a medium not containing n-triacontanol-1. After a given period of cultivation, the produced mycelia were separated, washed weii wtth water, ethanol and acetone and dried at 60--800C. The days of cultivation, dry culture yields and indices as determined for the respective mycelia products are shown in Table 4 below.

TABLE 4 n-triacontanol concentration (ppm) Comparative Example This invention 0 1 2 Dry Dry Dry Fungus Days of culture culture culture species cultivation yield* Index** yield Index yield Index Laetiporus 19 0.174 100 0.240 138 0;258 148 sulphureus (Fr.) Bond. et Sing. 29 0.278 100 0.362 130 0.418 150 FERM-P No. 3032 Armillariella 18 1.147 100 1.377 120 1.648 135 mel lea (Fr.) Karst. 28 1.445 100 1.941 134 2.263 157 FERM-P No. 982 Grifola fron- 26 0.887 100 1.034 117 1.044 118 dosa S. F. Gray FERM-P No. 3032 32 1.514 100 1.565 103 1.640 108 (Notes) *Dry culture yield (9/100 ml) per 100 ml of medium.

**The dry culture yield from the tricontanol-non-added medium at each sampling state was given as 100.

The above results indicate that n-tir9acontanol-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 color of the natural fruit bodies, and in the case of Armillariella mellea, there is observed more conspicuous formation of mycelia clusters, while in the case of Grifola frondosa, the mycelial product has a color close to that of the natural products.

Claims (4)

1. A method of cultivating a fungus of the class Basidiomycetes which method comprises cultivating the fungus in a medium containing at least one of straight-chain saturated aliphatic alcohol represented by the general formula: CnH2n + IOH wherein n is an integer of 26 to 36.

2. A method according to claim 1, wherein said medium is a liquid medium containing the at least one alcohol in a concentration of 0.01 to 10 ppm.

3. A method according to claim 1 or 2, wherein the at least one alcohol has from 28 to 32 carbon atoms.

4. A method according to claim 1 substantially as hereinbefore described with reference to any one of the non-comparative runs of Examples 1 to 4.