In one embodiment, the substrate is heat treated spent coffee grounds.

The substrate of this invention favours the growth of wood-rotting fungi to the exclusion of competing organisms. That is, it exhibits selectivity, thereby facilitating the adoption of non-aseptic conditions for the cultivation of wood-rotting fungi.

The process is particularly applicable to mushrooms, for example, shiitake mushrooms, winter mushrooms, oyster mushrooms, shimeji mushrooms and Coriolus versicolor.

Background of the Invention There are several methods of cultivating mushrooms. For example, it has been traditional to cultivate mushrooms on hardwood logs. However, it may take between about 6 and about 12 months for the first flush of mushrooms to occur and the biological efficiency does not exceed about 15%.

Shiitake mushroom (Lentinus edodes) is an important edible mushroom used for food and medicine (e. g. the extraction and isolation of lentenan from shiitake). For many years it has been mainly grown on natural logs of hardwood trees, in particular the Fagaceae. The fruit bodies usually develop in one year and can be harvested for up to 3 to 6 years. The quality of the mushroom varies greatly depending on the tree species and the fruiting temperatures.

Production of shiitake by the traditional"natural log"method has declined in recent years because of the shortage of suitable tree species.

An alternative technique has been developed using sawdust substrate in what is known as the"synthetic sawdust log"method. In this method, the sawdust is supplemented with other nutrients such as wheat or rice bran, gypsum, and at times fungicides. Both the pH and moisture contents are adjusted before the supplemented sawdust is placed in small plastic bags for sterilisation. While the time required for the first harvest is shortened and the yield is higher than the"natural log"method, the procedures from sterilisation to spawn run demand rather strict aseptic techniques which are costly and not easy to automate.

Furthermore, the quality of the shiitake produced in this manner, even at best, is considered inferior to that produced by the"natural log"method. As with the"natural

log"method, the quality of the mushroom can also vary with the type of sawdust, the consistency of which is not easily controlled by the mushroom growers. Also, in most cases it takes more than two months after spawning for the first harvest.

A compost substrate for shiitake cultivation has previously been developed. For example, Australian Patent No. 582250 is directed to a non-sterile but pasteurised, substrate suitable for growing shiitake mushrooms. The substrate consists of vegetative waste and a mineral fertiliser and is sequentially composted at ambient then mesothermic temperatures. The invention described in Australian Patent 582250 appears not to have been successfully exploited commercially.

US Patent 4,637,163 is directed to a method of producing wood-rotting edible mushrooms on a bed prepared from a carbohydrate, lignin and protein containing compound material. The bed is mainly a cellulose containing material, with minor amounts of proteinaceous and farinaceous substances and water. The process described requires several steps such as pasteurisation, application of temperature and light and addition of material such as peat and pH adjustment.

US patent 4,127,965 is directed to methods and kits for the cultivation of shiitake and similar forest mushrooms. The substrate which is used is cellulosic based and needs to be supplemented with a number of nutrients. The method described in US 4,127,965 also requires an initial phase of aseptic growing conditions.

US patent 4,083,144 is directed to artificial cultivation of shiitake mushrooms by inoculating an artificial solid granular or fibrous culture medium with the spawn of L. edodes. The culture medium must contain nutriments suitable for such growth, such as sawdust, bagasse, rice hulls, rice bran and wheat bran.

US patent 4,083,145 is directed to cultivation of shiitake mushrooms in wide- mouthed bottles or bags. This particular document describes the formation of a plurality of holes in the culture medium along the inner wall of the bottle or bag and inoculating the fungus seed into these holes.

To summarise therefore, prior art techniques suffer from the disadvantages of incurring high production costs and lack of reproducibility in mushroom quality and quantity.

As mentioned above, the shiitake produced by the"natural log"technique is superior to other prior art methods. As also mentioned above, a further disadvantage is the need to sterilise the substrate on which the mushroom is grown. This is costly and does not lend itself to automation.

Disclosure of the Invention Without wishing to be constrained by theory, the present inventors believe that a reason why the alternative substrates used thus far must be sterilised, may be due in part, to the high concentration of soluble nitrogen and carbon sources which renders the substrate more susceptible to contamination.

According to a first aspect of this invention there is provided a substrate for use in the cultivation of wood-rotting fungi said substrate being organic fibrous material substantially depleted of soluble nitrogen and carbon sources, and subjected to heat treatment which increases stability of said substrate and reduces microbial loading, the combined effect of said increased stability and reduced microbial loading facilitating rapid proliferation of said wood-rotting fungi; and wherein said substrate has a moisture content of about 40 to about 75% (dry weight basis), and is substantially free of any nutrient supplement.

According to a second aspect of this invention there is provided a process for preparing a substrate for use in the cultivation of wood-rotting fungi said process comprising the steps of heat treating an organic fibrous material substrate which has been substantially depleted of soluble nitrogen and carbon sources, said heat treating being carried out at a sufficient temperature and for a sufficient period of time in order to increase stability of said substrate and reduce microbial loading, the combined effect of said increased stability and reduced microbial loading facilitating rapid proliferation of said wood-rotting fungi; and wherein said substrate has a moisture content of about 40 to about 75% (dry weight basis).

According to a third aspect of this invention there is provided a process for cultivating wood-rotting fungi, said process comprising adding spawn to the substrate of the first aspect of this invention and leaving for a sufficient time until the resultant wood-rotting fungi are ready for fruiting.

According to a fourth aspect of this invention there is provided wood-rotting fungi when produced by the process of the third aspect of this invention.

According to a fifth aspect of this invention there is provided shiitake mushrooms produced by the process according to the third aspect of this invention.

The starting material which provides the organic fibrous material from which the substrate is derived is suitably coffee beans, coffee pulp and coffee grounds, tea leaves, bagasse, sawdust, wheat straw or other agricultural material containing fibrous residue which have been substantially depleted of soluble nitrogen and carbon sources.

For example, in the case of coffee beans, this step may be carried out by continuous extraction of ground coffee beans with super-heated water or super-heated water plus steam for about six hours. This process of extraction leads to"spent coffee grounds". Similar extraction leads to"spent tea leaves","spent bagasse"or"spent agricultural waste material"where tea leaves; bagasse; or sawdust, wheat straw or other agricultural waste material respectively, is the starting material.

The preferred spent starting material for use in this invention is spent coffee grounds. In general, all varieties of coffee may be suitably used as spent coffee grounds in the present invention.

Wood-rotting fungi include wood-rotting mushrooms, which include shiitake mushrooms, winter mushrooms, oyster mushrooms, shimeji mushrooms and Coriolus versicolor. The method of cultivation is especially suitable for shiitake mushrooms.

In general, while the moisture content of the substrate is from about 40 to about 75 % (dry weight basis), it is preferred that this range is from about 50 to about 60% (dry weight basis). When dry starting material is used, the moisture content is suitably adjusted by addition of water.

One of the advantages of the processes of the present invention is that there is no requirement to adjust the pH during the process of preparing the substrate of this invention.

In the event that the chosen spent starting material is nutritionally poor for the cultivation of mushrooms, it may be necessary to supplement it with approximately 5-10% of spent coffee grounds for reasons to be explained below.

Any method which does not adversely affect the mushroom growing ability of spent starting material and which removes the soluble nitrogen and carbon sources would theoretically be suitable. In practice, the higher costs of such extraction (for example, by the use of a solvent or solvents) compared to hot water extraction, would exclude other methods on the basis of commercial viability.

The soluble nitrogen and carbon sources may be suitably removed from the starting material by, for example repeated extraction with hot water, super-heated water, steam and/or combinations thereof.

The spent starting material is then subjected to heat treatment. Preferably the heat treatment is carried out using steam with forced aeration. The ratio and delivery of steam to air mixture may be controlled by a timing mechanism and temperature probe placed in the material being heat treated, in order to achieve the desired temperature and percentage moisture. The placement of such a probe (or probes) is effected in

accordance with methods known in the art. A range of heating temperatures from around about 45 to about 80°C is suitable. This range may be about 40 to about 65°C, preferably about 50 to about 65°C and more preferably from about 50 to about 60°C.

A range of duration of heat treatment is also suitable. This range is discussed presently.

When spent coffee grounds are used, heat treatment depends on the initial microbial loading of the grounds and the type of wood-rotting fungi to be cultivated.

For example, in the case of shiitake mushrooms, which are slow growing, heat treatment is suitably approximately 1 day at about 60°C, or according to one protocols (a) to (e) set out below. In the case of oyster mushrooms, which are slower growing than shiitake mushrooms, heat treating may be reduced below 1 day.

Therefore, in general, heat treatment may be carried out for approximately 1 day at about 60°C, or according to one of the following protocols: (a) Heating for about four days beginning at about 50°C on day one and progressing to about 65°C by day four, increasing by about 5°C per day.

(b) Heating for about three days beginning at about 55°C on day one and progressing to about 65°C by day three, increasing by about 5°C per day.

(c) Heating for about two days beginning by about 60°C on day one and progressing to about 65°C by day two.

(d) Heating for about two days at about 60°C for both days.

(e) Heating for about two days at about 65°C continuously.

In the case of heat treatment for one day or less, coffee for example should preferably have been freshly prepared or stored under hygienic conditions. Suitably, heating may also be carried out over about four days, starting at about 50°C, with increments of about 5°C daily, reaching a final temperature of about 65°C on the fourth day.

It has been found for example, that spent coffee grounds are a rich source of lipids, containing approximately 20-25 % on a dry weight basis. It has been found that this level of lipids is beneficial in both the vegetative and reproductive stages of shiitake growth for example. It has also been found that the presence of lipids in the substrate increases the uptake of glucose by the mushroom mycelia.

It has been demonstrated that heat treatment and spawning can be carried out in bulk and in small quantities. For example, a container of approximately lm high with a cross section of 2m2 is suitable for bulk processing.

In the cultivation process, spawn used is suitably grain spawn. Preferably, the grain spawn is fully grown millet spawn. The millet spawn has several advantages over the traditional rye or wheat grain spawn. For example, for a given weight of spawn there are many more points of inoculum. The smaller size of fully grown millet spawn means that it does not rupture easily to release nutrients which are prone to cause contamination.

It has been demonstrated that the lipid profile and degree of lipid unsaturation can be influenced significantly by temperature. It is also known that immersion of the substrate colonised by the mycelium in chilled water, at a temperature of about 5-about 10°C, aids in the initiation of sporophores. This is known as"cold shock"in the industry and is used commercially. Thus, this procedure of using cold shock may be advantageous for the use of the process of this invention. Cold shock treatment can be repeated until after approximately 3 to 4 months when the yield decreases to the extent that it is no longer of any commercial value.

It has been reported that the addition of a fungicide may stimulate mycelial growth and early fruiting. Thus, although it is not essential for carrying out this invention, the addition of a fungicide may be advantageous. The same stimulating effect of mycelial growth has also been observed in the preparation of grain spawn.

Reference is made to the second and third aspects of this invention. Specifically, the substrate which is prepared according to the second aspect and which is used according to the third aspect is subjected to three phases, namely depleting said substrate of soluble nitrogen and carbon; heat treating the resultant substrate; and using that substrate for cultivation of the wood-rotting fungi. Each of these steps modifies the substrate physico-chemically and it has been found that the thus-modified substrate is useful in other applications. For example, the modified substrate is useful as a compost and as an animal feed. Accordingly, according to a sixth aspect of this invention there is provided a modified substrate for use in an application such as a compost or animal feed, said substrate having been modified by depletion of soluble nitrogen and carbon sources followed by heat treatment in order to achieve a minimal microbial loading and having been used as a substrate for the cultivation of wood-rotting fungi.

Best and Other Modes for Carrying Out the Invention The following describes one embodiment of the invention which should not be construed as limiting on the scope thereof.

A substrate was developed for growing shiitake mushroom. In this method, fresh spent coffee grounds were used. The spent coffee grounds contained 60-70% moisture and about 30-40% of dry weight.

These spent coffee grounds were then heat treated according to protocol (a) described above i. e. day 1: 50°C; day 2: 55°C; day 3: 60°C and day 4: 65°C.

Spawn was then thoroughly mixed with the substrate.

The spawned substrate was placed either in small plastic bags which were thoroughly perforated or in trays covered with a moisture barrier. Mycelial growth was well established after 2 weeks of spawn run at 20°C. About six weeks of spawn run was required before the blocks were ready for fruiting, as indicated by the excretion of brown exudate. The blocks were opened and placed in a room at 25-30°C for one week before the temperature was lowered to 16-18°C to induce fruiting. The first harvest of shiitake occurred in about 10 to 14 days after the bags were opened.

The shiitake blocks were allowed to rest for about 2 weeks before cold shock at 10-15°C.

The method of substrate preparation, spawn run and fruiting are well suited for Agaricus mushroom growers without much modification of the equipment and mushroom house facilities.

The invention is further illustrated by way of the following examples which are not to be construed as limiting on the scope of the invention thereof.

Example 1 Samples of fresh spent coffee grounds were placed in trays and a steam/air-mixture was introduced to raise the substrate temperature to 50°C. The daily increase of temperature was 5°C until the fourth day when the final temperature was 65°C. After cooling to 20-25°C, the substrate was ready for spawning.

Example 2 A strain of L. edodes (L96) available from the Department of Microbiology, University of Sydney, was cultivated on the heat treated substrate prepared by the process of this invention. The heat treated substrate described above was thoroughly mixed with millet spawn (about 10%-15% of the dry weight of the substrate) and the bags containing the spawned substrate were incubated at 20°C for 2 months when brown exudate appeared. The bags were further incubated for one week, before the temperature of the room was dropped to 16-18°C for the fruiting period.

The first harvest occurred after about 10 days. The blocks were left standing with no watering for about 10 days when they were immersed in cold water (10-15°C) for 24

hours. The second flush occurred after about 10 days. The cold shock procedure was repeated until after three or four months when the yield dropped considerably.

The conversion efficiencies are expressed as ofmushroomsWetweight x 100%<BR> <BR> ofsubstrateDryweight The average conversion efficiency of the first two flushes ranges from about 40-50%.

Example 3-Control Cultivation-No Spawn A control was set up with no spawn added. Slight contamination was observed after one month of incubation at 20°C. In practice, mycelial growth was well established two weeks after spawning, and this excluded contamination by Trichoderma. Even though these observations to detect contamination, were made only for one month, one of skill in this art will realise that if the Trichoderma did not appear after one month, then it would not appear at three months as in Example 2.

Example 4-Purposeful Addition of Trichoderma A further control was set up with no spawn added but using Trichoderma as the inoculum. It was found that there was no contamination with Trichoderrna until after approximately one month. Similar comments apply to this Example as applied to Example 3 in relation to the observation time being 1 month.

Industrial Applicability It should be clear that the present invention will find wide applicability in the mushroom cultivation industry and particularly in the specialty mushroom cultivation industry where the normal substrate is sawdust.

The process of this invention is also suitable for the cultivation of medicinal mushrooms where the normal habitat is logs of trees or sawdust. Bioactive agent (s) may be extracted directly from the spawn run substrate.