Title of invention:

Chemical Sterilization Method of Mushroom Spawn Production

Field of invention:

This invention relates to mushroom spawn production by adopting an easy cheap and efficient method involving chemical method of sterilization. Mushroom spawn is the mushroom mycelium grown on a given substrate. It serves as the planting material in mushroom cultivation and is commonly called the mushroom seed.

Background of invention with regard to the drawback associated with known art

Mushrooms have been attracting attention of mankind from time immemorial. The Indian mushroom industry has come a long way since 1950 when the first experiments were conducted on this novel protein source. Edible mushroom is nothing but the fruiting body of a heterotropic fimgus which is rich in proteins, vitamins, fibres, essential amino acids and is low in fats and carbohydrates. They also have several medicinal properties. The awareness about mushroom has been increasing over the years. India has a large domestic market for mushrooms. The main advantage of mushroom production is the fact that it does not require any arable land and can be cultivated on waste and unproductive land. In India large numbers of farmers are going in for mushroom farming especially women.

India has been bestowed with a wide range of climate like tropical, subtropical and temperate and hence is suitable for the cultivation of a wide variety of mushrooms like Agaricus sp.(button mushroom), Pleurotus.sp.(Oyster mushroom), Volvariella sp.(Paddy straw mushroom), and Calocybe indica (milky mushroom). Despite all favorable conditions and the fact that mushroom farming is a highly remunerative enterprise with quick returns in a short time it is not spreading fast, we are lagging behind in production with an annual production of about 70,000 tonnes compared to 5 million tonnes of world production. Agricultural and industrial survey 2006 reported that this was due to some inherent problems most important among them being non availability of quality spawn and also nonavilablity in time, this not only affected mushroom yield and quality but also led to diseases and pests that wiped out the entire cultivation. The reason why the fanners are not going in for their own spawn production is the high initial investment required for spawn production as it requires costly equipments like autoclave, laminar flow or sterile room etc.

Mushroom spawn serves as the planting material in mushroom cultivation and use of spawn is three fold. It is used for mushroom production, spent waste after harvest of mushroom is a good nutrient feed for live-stock and also a good manure for soil and spawn is also used for composting coir pith and other lignocellulosic waste. Most of research is on increasing mushroom yield and quality through genetic research and have also produced a series of machineries like grain cleaner; grain boiler, boiled grain and chalk powder mixer, grain filler and spawn inoculators etc but these only increased the cost of establishing a spawn unit. In the existing method to establish spawn production laboratory costly equipments like autoclave and laminar flow were required. The substrate for spawn production like cholam grains were filled in heat resistant polypropylene bags and steam sterilized at high temperature and pressure using an autoclave and the inoculation is done in sterile chamber using laminar-flow chamber and after inoculation they are incubated in sterile room free from contamination.

The unique feature of this new invention is that it is an easy low cost technology of spawn production in the scientific way using easily available materials. Here the principle of chemical

Sterilization is used instead of steam and heat sterilization that requires costly equipments. In the new method the substrate, rice bran are filled in ordinary plastic bags and a sterilant chemical is added to them which protects the substrate from contamination and then inoculation is done in ordinary room ( sterile chamber not necessary) and the inoculated packets are incubated in ordinary rooms. Hence a technology was developed for cheap and easy production of quality spawn.

Object of invention: The objective of the invention is to identify a simple and easy method of spawn production using the principle of chemical sterilization. By Mushroom spawn we mean the mushroom mycelium grown on a given substrate. It serves as the planting material in mushroom cultivation and is commonly called the mushroom seed. By mushroom we mean the various species of the genus Agaricus, Pleurotus and Calocybe .

Statement of invention:

The new technology of spawn production developed uses the principle of chemical sterilization instead of steam and heat sterilization that has been used so far. Among the different chemicals tested it was found that Vinperoxide was found to be the best and safe chemical sterilant and experiments showed that there was no residual effect in mushroom produced. And among the different substrates tested rice bran was the best.

Detailed description of the invention

This invention relates to mushroom spawn production by adopting an easy cheap and efficient method involving chemical method of sterilization. The process involved is depicted below.

Isolation of the fungus from fresh mushrooms in flat bottles

V Cooking the substrate viz., rice bran in a cooker

V Cooling and adding Calcium carbonate @ 10% directly to cooker

Filling the substrate in ordinary packets 4X U" size

Adding sterilant chemical to the substrate @ 5ml/packet

Mixing the chemical for unifonn distribution hinoculating lyceum from mother spawn to above packet in ordinary room

Incubating the packets for growth in ordinary conditions

Identification of Suitable chemical Sterilant for growth of Mushroom Fungus

Chemicals have been widely used as sterilants in hospitals and other laboratories for several purposes. Different chemicals have different mode of action. Some of them kill microorganisms while some are micostatic preventing the growth of micro organisms while some of them like hydrogen peroxide acts on the wall of the organisms. Since mushrooms are used for edible purpose the chemical sterilant should be safe, hence in this study few easily available, cheap and safe chemicals were tested like vinegar, hydrogen peroxide, lime juice, ethanol and chloramphenicol.

An experiment was conducted to test the efficacy of these chemicals sterilants. One ml of unsterilized soil solution was added to each petri plates of 10 cm diameter, to this one ml of the above chemicals were added and to them potato dextrose agar medium was added and after cooling the petri plates were inoculated with Mushroom mycelium (Pleurotus sp) and incubated for 10 days. A control was maintained in which the soil solution without chemical was plated. It was found that in the case of vinegar and lime juice there was good growth of Pleurotus but contamination ranged from 10 to 25 percent (Fig:lA). While in the case of hydrogen peroxide the contaminants and other spores were inhibited but mycelium of mushroom (Pleurotus) grew well (Fig: IB ), this may be because hydrogen peroxide is reported to kill fungal spores but allows established mycelium to grow, but at later stage water deposits were observed within the petri dish and contamination occurred. This may be because hydrogen peroxide is degraded easily to oxygen and water. Ethanol and chloramphenicol completly inhibited the growth of all spores including Pleurotus sp. (Fig:lC&D) wheras in control there was large number of organisms growing. It was found that both hydrogen peroxide and vinegar (acetic acid) were very good and easily available safe chemical sterilants but the substrate used like cholam grains produce peroxide degrading enzymes that degraded hydrogen peroxide. Hence in this study Vinperoxide or a mixture of hydrogen peroxide + acetic acid was used as they were more stable.

Isolation of mother culture

In this method the mother culture is isolated from the mushroom into an easily available and cheap flat bottle containing potato dextrose agar medium unlike existing method where petri plates are used (Fig2). The narrow mouth of the bottle prevents contamination and the large surface area of the flat bottle allows easy growth of the fungus.

The substrate used in this method is rice bran. Rice bran was selected because it was cheap, had high bulk and high C:N ratio and the fact that it is not easily prone to contamination unlike cholam used in the existing method which has several seed borne organisms. An experiment was conducted to identify suitable substrate for the growth of spawn using new method. The experimental design followed was CRD and replication maintained was five. The different substrate tested was cholam grains, maize seeds, red rice, and rice bran. This substrate was tested under old and new method of spawn production. Results showed that the growth reached 100 per cent in all the substrates by 30 days (Table 1) but in case of cholam the contamination was high (25 and 50 per cent) under old and new method of production (Fig:3). This is because cholam is prone to head mould disease which is caused by a complex of fungi. In case of rice bran the contamination was low.

Preparation of Spawn

In the new method the substrate namely rice bran is first cooked in a pressure cooker unlike boiling the cholam grains or other substrates in open vessel in existing method (Fig:4) and after cooling Calcium carbonate 10 per cent was added directly to the cooker and mixed thoroughly (Fig:5). The substrate mixed with calcium carbonate is then filled in ordinary plastic packets 4X11 "size. To these packets of substrate the sterilant chemical is added @ 5ml per packet (Fig:6) and mixed thoroughly for uniform distribution unlike the existing method in which the packets are steam sterilized at high pressure in an autoclave (Fig: 7).

The substrate is then inoculated with the mother culture of the mushroom fungus in an ordinary room imlike the existing method in which the inoculation needs to be done in a sterile chamber like the Laminar flow (Fig: 8) and then a plastic ring is introduced into the mouth of the packet and plugged with cotton. Finally the packets are incubated in ordinary room for growth (Fig:9). In this case as the chemical sterilant gives protection against any infection that can occur during incubation the packets need not be incubated in sterile conditions 2.

TABLE :1 Testing different substrates for growth of spawn under old and New Method % growth

Standardization of dose of Vinperoxide(Hydrogen Peroxide + Vinigar)
It was found that both hydrogen peroxide and vinegar (acetic acid) were very good and easily available chemical sterilants but the substrate used like cholam grains produce peroxide degrading enzymes that degraded hydrogen peroxide. Hence in this study Vinperoxideor a mixture of hydrogen peroxide + acetic acid was used as they were more stable. An experiment was conducted to standardize the dose of Vinperoxide(hydrogen Peroxide + acetic acid or vinegar) added to spawn. The experimental design followed was CRD and replication maintained was five. The different concentrations of hydrogen peroxide tested were 3 per cent, 6 per cent and 10 per cent and the different doses tested were 5ml and 10ml. For comparison a standard ie existing method was used. The percent growth of the fungus at different intervals, the percent contamination and mycelia fresh weight was recorded (Table 2). It was found that when the concentration of hydrogen peroxide was increased the percentage growth decreased. This may be because at high concentration it inhibited even Pleurotus growth and when the dose of Vinperoxide was increased to 10ml there was inhibition of growth of spawn at later stage and moreover there was increase in water content of the spawn

This may be because of the degradation of hydrogen peroxide into oxygen and water. Maximum mycelia fresh weight or growth of spawn was seen when 3 per cent hydrogen peroxide+Vinegar @ 5ml was added this was on par with old method of spawn production. Hence a concentration of Vinperoxide containing (3 per cent hydrogen peroxide+ vinegar ) @ 5ml was found to give 100 per cent growth and maximum mycelial yield . Steps followed in two different methods are furnished in Table 3.

TABLE :2 Standardization of dose of Vinperoxide(Hydrogen Peroxide + Vinegar)to be used in New method of spawn production

Table 3. Difference between existing and New Method of Spawn Production

Existing Method I New Method

Pleurotus sp is isolated from fruiting body or Pleurotus sp is isolated from fruiting body mushroom and multiplied in Petri plates or mushroom and multiplied in easily available flat bottles

The substrate for spawn production viz., Sorghum The substrate for spawn production viz., grains are boiled and then mixed with 10% calcium rice bran is cooked in cooker and mixed carbonate and spread in shade for drying with 10% Calcivun Carbonate directly

They are packetted in heat resistant non degradable They are packetted in ordinary degradable polypropylene bags @ 200-250g/packet plugged with plastic packets @ 200-250g/packet cotton

They are then sterilized at 201b pressure for 15-20 min The sterilant chemical is added to the using an autoclave packets @ 5ml/packet and mixed thoroughly

After cooling the grains are inoculated with mycelium The packets are then inoculated with of Pleurotus sp under aseptic conditions using a mycelium of Pleurotus sp inordinary room laminar flow chamber or in aseptic culture room (Sterile conditions not necessary) and plugged with cotton The packets are then incubated in aseptic conditions. The packets are then incubated in ordinary room (Sterile conditions not necessary)

Advantage of the New Technology

• Isolation and culturing can be done in ordinary bottles compared to Petri dishes that costs Rs 100/dish

• No need for any costly equipments like autoclave for sterilization hence cost of establishment low

• No need for sterile chamber/room like laminar flow for inoculation as it can be done in any room

• Prevents environmental pollution by using only degradable plastic packets

• Can be started in small scale of even few packets

• Substrate used is rice bran is not only cheap and bulky but is scientifically better because of its high C:N ratio

• Method easy and less time consuming

Summary of invention

Pleurotus sp is isolated from fruiting body or mushroom and multiplied in easily available flat bottles. The substrate for spawn production viz., rice bran is cooked in cooker and mixed with 10 per cent Calcium Carbonate. They are packetted in ordinary degradable plastic packets. The sterilant chemical, Vinperoxide is added to the packets @ 5ml/packet and mixed thoroughly. The packets are then inoculated with mycelium of Pleurotus sp in ordinary room (Sterile conditions not necessary) and plugged with cotton. The packets are then incubated in ordinary room (Sterile conditions not necessary). The unique feature of the technology developed is that it is an easy low cost technology of spawn production in the scientific way using easily available materials. Here the principle of chemical sterilization is used instead of steam and heat sterilization that requires costly equipments and that has been used so far. Among the different chemicals tested it was found that Vinperoxide was found to be the best and safe chemical sterilant and experiments showed that there was no residual effect in mushroom produced. And among the different substrates tested rice bran was the best. Hence a technology was developed for cheap and easy production of quality spawn.

Claims

Our main claim is Chemical Sterilization Method of Mushroom Spawn Production using Vinperoxide and our subsidiary claims are

(i) Vinperoxide is a mixture of equal volume of Hydrogen Peroxide and Vinegar as a chemical sterilant (3 per cent hydrogen peroxide+ vinegar) @ 5ml.

(ii) Use of rice bran as a substrate and boiling of rice bran using pressure cooker,

(iii) Flat bottle containing potato dextrose agar medium for isolating mother culture, which has narrow mouth to prevent contamination and the large surface area to allow easy growth of the fungus.

(iv) Degradable plastic packets measuring 4x1 T'size for culturing spawn in rice bran substrate