Mushrooms belong to the kingdom of Fungi, a group very distinct from plants, animals and bacteria. Fungi lack the most important feature of plants: the ability to use energy from the sun directly through Chlorophyll. Thus, fungi depend on other organisms for food, absorbing nutrients from the organic material in which they live. The living body of the fungus is mycelium made out of a tiny web of threads (or filaments) called hyphae. Under specific conditions, sexually compatible hyphae will fuse and start to form spores. The larger spore producing structures (bigger than about 1 mm) are called mushrooms. In nature this is the most striking part of the organism, but in fact it is just the fruiting body and the major part of the living organism is found under the ground or inside the wood. Mushrooms are not plants, and require different conditions for optimal growth. Plants develop through photosynthesis, a process that converts atmospheric carbon dioxide into carbohydrates, especially cellulose. While sunlight provides an energy source for plants, mushrooms derive all of their energy and growth materials from their growth medium, through biochemical decomposition processes. This does not mean that light is an unnecessary requirement, since some fungi use light as a signal for fruiting. However, all the materials for growth must already be present in the growth medium. Mushrooms grow well at relative humidity levels of around 95-100%, and substrate moisture levels of 50 to 75%.Instead of seeds, mushrooms reproduce sexually during underground growth, and asexually through spores. The biotechnological advances that will help ensure mushroom cultivation without pollution and provide mushroom products with health enhancers and get more reliable yields, then improve substrate utilization and control disease more effectively. Either of these can be contaminated with airborne microorganisms, which will interfere with mushroom growth and prevent a healthy crop.
Fungi depend on other organisms for their food. Three modes of living can be recognised Saprophytes degrading already dead material , Symbionts living together with other organisms (especially trees) in a close, mutually beneficial relationship , Parasites living at the expense of other organisms.
Saprophytic fungi need organic matter to decompose. In nature they will grow on fallen leaves, animal droppings, or stumps of dead wood. Some are specialized in breaking down the hairs of mammals, while
others may decompose bird's feathers. Saprophytes decompose the complex organic structures left behind by plants and animals. And in the natural run of things, plants or animals regain access to minerals and other nutrients present in the substrate. Oyster mushrooms degrade dead wood in nature. They can be cultivated on a wide range of ligno-cellulose waste materials. The mode of living has nothing to do with edibility both edible and poisonous mushrooms can be found in all three groups.
Mushrooms are good cash crop; they are rather easy to grow and are brimming with protein, B vitamins and minerals. Mushrooms have been valued throughout the world as both food and medicine for thousands of years. They are rich source of nutrition and form a major chunk of health foods. Fats occur in mushrooms in minor amounts, especially compared with protein and carbohydrate and the fatty fraction consists predominantly of unsaturated fatty acids such as linoleic acid, they may be the perfect food for maintaining a healthy heart and cardiovascular system
The two most commonly grown species of mushroom in India are white button mushroom and oyster mushroom .most of the production of white button in our country is seasonal .The cultivation is done in conventional method. However, in recent years, yield of mushroom has increased as a result of introduction of improved agronomic practices. At present three mushrooms are being cultivated in India; these are Agarics bisporus ,Volvariella vovvacea and Pleurotus sajor-caju .Of these A.bisporus is the most popular and economically sound to grow and extensively cultivated through the world. however due to its low temperature requirement, its cultivation is restricted to the cool climatic areas and to the winter in the plains of northern India In summer ,the paddy straw mushrooms suitable for growing in most part of India, as a kitchen garden crop it is preferred because it is very delicious and nutritious. Oyster mushrooms can grow at moderate temperature ranging from 22 cto 28c.In north India, the climate conditions prevailing during different seasons can be exploited for growing mushroom through out the year.
All mushroom growing techniques require the correct combination of humidity, temperature, substrate (growth medium) and inoculum (spawn or starter culture). Wild harvests, outdoor log inoculation and indoor trays all provide these elements.
Kerala is the leading state in the production of coconuts [Thampan, 1997]. Coir pith, a highly lignocellulosic material, is available in large quantities as a by product of coir industry. Its high lignin concentration coupled with wide C/N ratio doesn’t permit its direct application to soil. Biodegradation of coir pith for converting it into a nutrient rich material for plant growth not only reduces environmental pollution but it is also an eco friendly approach.
The material on which the mycelium of the mushrooms grows is called substrate. Agricultural waste like wood chips/sawdust, sugar cane bagasse, and different types of straw can be used as the main ingredients
in the substrate for oyster mushrooms. The properties of a substrate determine which mushrooms and microbe can grow in it. The more selective it is, the better the substrate meets the demands of a specific mushroom and the less suitable it is for others. After mixing and adding certain supplements, the substrate undergoes a heat treatment to give the desired mushroom mycelium an environment with few competitors.
A number of fungi like Pleurotus sajor-caju were found to be potent degraders of coir pith [Savithri and Khan, 1994]. Pleurotus spp have been successfully cultivated on aquatic weeds such as water hyacinth [Murugesan et al., 1995] and Elio charis punt gene [Subarhan et al., 1993]. Deka et al., (1994) studied the feasibility of cultivation of P.florida, P.sajor caju on paddy straw and bamboo leaves. Siva Prakasum and Kandaswamy (1981) and Geetha (1994) have reported that the yield of sporophores was positively correlated with the cellulose content and also with the cellulose lignin ratio.
Mushroom production converts raw natural ingredients into mushroom tissue, most notably the carbohydrate chitin. An ideal substrate will contain enough nitrogen and carbohydrate for rapid mushroom growth.
REVIEW OF LITERATURE
Oyster mushrooms, Pleurotus spp, are edible fungi popularly known as wood fungi. Pleurotus is a genus of gilled mushrooms which includes one of the most widely eaten mushrooms, P. ostreatus. Species of Pleurotus may be called oyster, abalone, or tree mushrooms, and are some of the most commonly cultivated edible mushrooms in the world. Pleurotus fungi have been used in mycoremediation of pollutants such as petroleum and polycyclic aromatic hydrocarbons. The genus was established by Fries in 1821. There are a number of spp under this genus. According to Pegles (1970) and Singer (1986) this genus is known to contain 50 spp, of these about 25 spp are known to occur in India.
It is an 'oyster mushroom'. A number of different species are grown including, Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus cystidus, Pleurotus cystidus, Pleurotus citrinopileatus and Pleurotus flabellatus. This mushroom is cultivated on a wide range of plant wastes (cereal straw, sawdust, bagasse, waste cotton) often enclosed by plastic bags. Pleurotus mushrooms are the second most important mushrooms in production in the world, 25% of total world production of cultivated mushrooms. Pleurotus mushrooms are world-wide, China is the major producer.
Plate 1 , 2 and 3 shows the Photos of Pleurotus spp
P.sajor-caju
Spawn
The mycelium of mushroom growing in its substrate and prepared for the purpose of mushroom production is called spawn [Chang, 1982]. Kinden (1932, 1934) was the first to introduce grain spawn in the cultivation of mushrooms. Different kinds of grains viz., wheat, rye, millets etc can be used as spawn substrates [Kotwaliwale et al., (1991)]. The complete procedure of spawn production involves preparation of the medium, filling the test tubes or Petri dishes and sterilizing them, and the process of inoculating larger containers with this culture.
Mother spawn can be used to inoculate either grain spawn or a second generation of mother spawn. In simple laboratories, grain mother spawn should not be used to inoculate another generation of grain mother spawn because the risk of contamination and degeneration will be too high. The main advantage of grain is that it is very nutritious for fungi and forms kernels easily. The kernels can easily be dispersed in the substrate. The main disadvantage is that it provides an optimal substrate for other organisms too. The chances of contamination are therefore much higher compared to sawdust spawn.
Enzymology
Extra cellular enzymes are those enzymes produced within the cells and then liberated into the external environment to carryout the function of cultivation of the nutrients in the substrate. The rate of extra cellular enzymes is the production and activity of the mushrooms. Since, only by their production and activity can the mycelium grow and produce mushroom fruit bodies. The extra cellular enzymes are produced to degrade the large insoluble molecules of the substrates into small soluble molecules which the mycelium can utilize (Wood, 1990).
Fungal laccases form an important group of enzymes, as they are involved in the degradation of lignin and in removal of potentially toxic compounds (Thurston, 1994).For detecting laccase, some assay methods including HPLC method, manometry, order spectrum method and spectrophotometry are involved (Zhu et al., 2006).The extra cellular enzymes of Pleurotus spp play a major role in the degradation of structural elements such as cellulose, hemi cellulose, lignin and pectin present in the natural substrates. The enzymes most frequently associated with cellulose degradation are cellulases and those associated with lignin degradation are laccase.
Laccase
Laccase enzyme typically contains 15–30% carbohydrate. It has an acidic isoelectric point and has a molecule mass of 60–90 kDa. Laccases are the model enzymes for multi-copper oxidases and participate in cross-linking of monomers, degradation of polymers, and ring cleavage of aromatic compounds. For catalyzing the oxidation of non-phenolic substrates, laccase requires the presence of a mediator in the medium. A mediator is a small molecule that behaves like an ‘electron shuttle’ between laccase and substrate and these small molecular-mass compounds are converted into stable radicals by means of enzymatic oxidation.
The most efficient lignin degrading oyster mushrooms [Zadrazil and Dube, 1992] has been reported to produce high level of laccase and acyl alcohol oxidase on their substrates [Kirk and Farell, 1987]. Reddy (1985) reported the capacity of Pleurotus spp to produce laccase and degrade the part of lignin and cellulose present in their substrates. Jabionsky (1984) cultivated P.florida on ground maize and observed high laccase activity. Assay of laccase during different stages of growth of Pleurotus spp on paddy straw showed that laccase production reached the maximum after 24 days of incubation and defined thereafter.
Lignin degradation
Discovery of novel laccases with different substrate specificities and improved stabilities is important for industrial applications. Microbes that produce laccases have been screened for either on solid media containing coloured indicator compounds that enable the visual detection of laccase production.
Lignin degradation is important in the global recycling of carbon because of the least abundance of lignin in the atmosphere and also because it is an important factor delimiting the degradation of cellulose and other poly saccharides [Krick and Farell, 1987]. All fungi capable of degrading lignin are known as white rot fungi. Pleurotus is a white rot fungus capable of degrading ability of Pleurotus spp [Ibrahim and Pearce, 1980 and Nizkovskaya et al., 1984].
METHODOLOGY
1. Isolation and maintenance of pure cultures of Pleurotus spp.
Isolation and maintenance of pure cultures of Pleurotus spp viz., Pleurotus florida, Pleurotus sajor-caju and Pleurotus eous were developed at the biotechnology lab in Mar Ivanios College by adopting tissue culture methods[Scarcye 1995].
Mycelium, or actively growing mushroom culture, is placed on growth substrate to seed or introduce mushrooms to grow on a substrate. This is also known as inoculation, spawning or adding spawn. Its main advantages are to reduce chances of contamination while giving mushrooms a firm beginning. Spores are another inoculation option, but are less developed than established mycelium. Since they are also contaminated easily, they are only manipulated in laboratory conditions with laminar flow cabinet.
Tissue from the junction of pileus and stipe of sporocarp was scooped out and surface sterilized by placing in 95% ethyl alcohol for one minute, after that the PDA slants were prepared, autoclaved for 15 minutes at 120°C. Then inoculation of mycelium under the laminar flow in PDA slants and incubated at room temperature for 7-18 days. Following sub culturing it is maintained on PDA slants.
2. Spawn production
The mycelium will colonise the substrate and use the available nutrients. This is commonly referred to as the spawn run. When some nutrients run out, or when the weather changes, the mycelium will reach a different phase: the reproductive stage. A temperature of about 25 °C is optimal for the spawn run of most species. The environment can also enhance the growth of the desired mycelium: a high CO2 concentration is favorable for mycelial growth (but not for cropping). During spawn run stage the mycelium will grow through the substrate. The spawn run time is different for each species and depends on the size of the bag, amount of spawn, the strain used and the temperature.
Rice grain spawn of the three Pleurotus spp was prepared adopting the method described by Siva Prakasam (1980). Rice grains were boiled in water and are half boiled. After draining excess water, it was mixed with calcium carbonate at the rate of 50g per kg of rice grains to prevent adhesion of grains and for optimizing the pH for spawn. Bottles of 750ml capacity were filled with the grains to 2/3 of its capacity, plugged with cotton and autoclaved at 1.05kg cm-2 for 2 hours. The substrate should have cooled down (whether pasteurised by steam or by immersion in hot water) to 30 C. The spawn (3% to 8% of the weight of the substrate) can be mixed in with when filling the bags. Or a layer of substrate can be topped with some spawn, layer by layer.
Inoculation of the grains with pure cultures of pleurotus spp was carried out and incubated at room temperature. The nature of growth and time taken for completing mycelium colonization of the grains was recorded. The spawn thus prepared was utilized for laying out mushroom beds.
3. Preparation of mushroom beds
Preparation of bags : Use 60x30 cm polythene bags (both side open).Tie one end of bag, put two holes of 1 cm diameter in the middle.Put handful of cooked straw in the bag to a height of 5 cm; sprinkle about 25 g of spawn. Layer the straw to 25 cm height. Repeat the process to get four layers of spawn and 5 layers of straw. Tie the mouth and arrange beds in tiers in the spawn running room. After 15-20 days, cut and remove the polythene bag and transfer the beds to cropping room. Keep the beds moist by periodical spraying with water.
In the bed system mushrooms are grown in wooden receptacles which are approximately 65" wide×60 foot long. These receptacles may be wider or narrower than 65" and/or they can be shorter or longer than 60 feet long. The depth of these receptacles is generally from 6" to 8" deep, however, they can be any suitable size. In what is referred to as a mushroom house these receptacles are usually stacked in four tiers, In each of these tiers six receptacles are usually stacked in a superimposed arrangement so that one mushroom house contains 4 tiers and 24 receptacles.
In the tray system mushrooms are also grown in wooden receptacles. These receptacles or trays can be square or rectangular, however, the most popular size is 48" wide×72" long×8" deep. In a farm using the tray system approximately 220 of these trays are put into one growing room. The trays are generally stacked four to five trays high and are arranged in eleven rows four stacks to a row. A typical mushroom tray is about 175 cm. long by 120 cm. wide with the tray height being 17.5 cm. and the corner post extending upwardly from the tray sides a further distance of about 12.5-15 cm.
Mushrooms are grown on decaying organic material. This material can be horse manure, hay, cereal straw or a variety of other vegetable wastes. The breakdown of this organic material is accelerated by the mushroom grower by composting. The purpose of composting is to convert the crude, often variable, raw material into a medium rich in nutrition which is specific for the growth of mushrooms. Three different substrates were tried for mushroom production viz., paddy straw, non retted coir pith [obtained by manually extracting the mesocrap] and retted coir pith. Mushroom beds were prepared following the method described by Baskaran et al., (1978) in the case of paddy straw or non retted coir pith as the substrate and the method described by Theradinani and Marimuthu (1991) was adopted for the preparation of beds using retted coir pith as the substrate.
In the case of paddy straw and non retted coir pith, substrates were kept in water overnight, boiled for half an hour, drained and dried. In perforated polythene bags of size 60×30cm, 3 layers of substrate [each of 5 to 8cm thickness] was placed with spawning of each layer. 100gm spawn per mushroom beds was used. The bags were then tied and incubated in the dark for 15 days, after which they were opened and transferred to cropping room were adequate ventilation and moisture was maintained.
In the case of retted coir pith, well perforated polythene bags of size 30cm was first filled with 0.5kg of the substrate and inoculated with pleurotus spawn [at the rate of 100g per kg of the substrate] uniformly over surface and covered with another layer of 0.5kg of coir pith. This process was repeated till the bag was filled. The bag was tied and incubated in a room where adequate moisture and ventilation were present.
The nature and rate of growth and yield characteristics in all the substrates were recorded. Samples were drawn at 10, 20 and 30 days interval to study laccase production and also to estimate the laccase content.
4. Cultivation of oyster mushroom
Cultivation of Pleurotus spp on their natural habitat was first described at the beginning of 20th century [ Falck, 1917] and on a saw dust-cereal mixture by Kaufert (1935). The foundation for the industrial production of Pleurotus on different substrates was laid by several workers [Kalberes and Vogel, 1974; Zadrazil, 1974 and Kurtz man, 1979].
Pleurotus spp have been successfully cultivated of different agricultural wastes such as mixture of coconut fiber and coffee pulp [Bernabe et al., 1993], kidney bean stubbles [Sobal et al., 1993], sugarcane bagasse [Shi, 1994] and cotton wastes [Haq et al., 1994].
5. Enzyme Assay
Mycelium is used as the source of extra cellular enzyme. To detect intra cellular laccase, grind the mycelium in 4ml of cold 0.1M phosphate buffer pH in a pre-chilled mortar with pestle. Centrifuge the homogenate at 9000rpm for 24min to remove cell debris. Use supernatant as enzyme source.
Pipette 4ml of sodium phosphate buffer, pH 6 containing guaiacol into test tubes and equilibrate at 25°C. Add 0.1ml of the enzyme source. After 5min, determine the absorbance at 470nm. Express the results as enzyme units. Maintain control with heat killed enzymes.
6. Assay of laccase
Laccase production by Pleurotus spp in different substrates at different time intervals was assayed using the method described by Frochner and Eriksson (1974).
4ml sodium phosphate butter, pH 6 containing 10mM guaiacal was added to the test tubes. Enzyme source of 0.1ml was added to this and the mixture incubated for 5 min. The absorbance was obtained at 470nm in spectrophotometer. Sodium phosphate buffer served as control. The activity of laccase was expressed in terms of enzyme units. [1 unit the change in absorbance of 0.01 per minute
RESULT AND DISCUSSIONS
1. Isolation and maintenance of pure cultures of Pleurotus spp:
Pure cultures of three Pleurotus spp viz., P.sajor-caju, P.florida and P. eous were obtained from the sporocarp by tissue culture method. The cultures were maintained on PDA slant.
2. Spawn preparation:
Wheat grain spawns of the three Pleurotus spp was prepared and their rate of growth on wheat grain was evaluated. The three species showed statistically significant differences in the growth if spawn bottles. (Table 1)
P.sajor-caju required the maximum period of 18 days for completion of mycelium run, P.florida 14 days and P.eous 15 days.
Table 1: mycelium growth in spawn bottles
Pleurotus species | Nature of growth | Period required completion of growth |
P.florida | Cottony growth | 5-8 days |
P.sajor-caju | Cottony growth | 18 days |
P.eous | Cottony growth | 14-18 days |
3. Laccase Production:
There was significant difference in the level of laccase production on three different substrates by Pleurotus spp. The maximum level of laccase production was recorded by P.sajor-caju on paddy straw.
Among the different Pleurotus spp tested, P.sajor-caju was the most efficient lignin degrader. Maximum lignin content was found in retted coir pith followed by non retted coir pith and paddy straw.
The control used in laccase was buffer. The quantification of laccase in the enzyme source using spectrophotometer in P.florida, P.sajor-caju and P.eous were 0.127, 0.210, and 0.08 respectively.
Plate 4 and 5 shows the tissue culture of Purotus Mushroom and its spawn.
Plate 6 and 7 showing Mother spawn having the growth of mushroom and bed preparation
SUMMARY
Pleurotus spp are edible fungi popularly known as ‘wood fungi’. Pleurotus spp have been successfully cultivated in different agricultural wastes such as mixture of coconut fiber and coffee pulp, kidney bean and broad bean stubbles, sugar cane bagasse and cotton wastes. Three Pleurotus spp viz., P.florida, P.sajor-caju, P.eous were used here in the laccase assay. The extra cellular enzymes are produced to degrade the large molecules of the substrate into small soluble molecules which the mycelium can utilize.
The importance of this report was, Kerala is the leading state in the production of coconut coir pith, a highly lingo-cellulosic material which is available in large quantities as a by product of coir industries. In conclusion, this study evaluated the maximum level of laccase production was recorded by P.sajor-caju on paddy straw. Among the different Pleurotus spp tested, P.sajor-caju was the most efficient lignin degrader. Maximum lignin content was found in retted coir pith followed by non retted coir pith and paddy straw.
The importance of this report was Mushrooms had long been used for medicinal and food purposes .It is now increasingly recognized that correct diet, controls and modulates many functions of human body and consequently participates in the maintenance of state of good health, necessary to reduce the risk of many diseases. Modern pharmacological research confirms large parts of traditional knowledge regarding the medicinal effects of mushrooms due to their antifungal, antibacterial, antioxidant and antiviral properties, besides being used as functional foods.
In conclusion, this study evaluated Mushroom cultivation very well with sustainable farming and has several advantages , It uses agricultural waste products , A high production per surface area can be obtained , After picking the spent substrate is still a good soil conditioner . These mushrooms are rather easy to grow on a small scale.
CONCLUSION
ASSAY OF LACCASE ENZYME IN PLEUROTUS SPECIES
Assay of Laccase
Trial No. | Volume of PO4 with Guaicol (ml) | Volume of H2O2 (ml) | Volume of enzyme | Incubation for 5’ at room temperature | Volume of enzyme | OD at 450 nm | Difference in OD |
B | - | 1.6 | - | - | - | - | |
C1 | 1.5 | - | - | 0.1 | 0.066 | 0.061 | |
T1 | 1.5 | - | 0.1 | - | 0.127 | - | |
C2 | 1.5 | - | - | 0.1 | 0.0112 | 0.098 | |
T2 | 1.5 | - | 0.1 | - | 0.210 | - | |
C3 | 1.5 | - | - | 0.1 | 0.092 | 0.074 | |
T3 | 1.5 | - | 0.1 | - | 0.161 | - |
Pleurotus florida (T1)
OD units per 5’ = 0.061
OD units per minutes = 0.0122
Pleurotus sajor-caju (T2)
OD units per 5’ = 0.098
OD units per minutes = 0.0196
Pleurotus eous (T3)
OD units per 5’ = 0.07
OD units per minutes =0.014
Specific activity of Laccase in Pleurotus florida = 1.045 units/mg of protein
Specific activity of Laccase in Pleurotus sajor-caju = 1.78 units/mg of protein
Specific activity of Laccase in Pleurotus eous = 1.233 units/mg of protein
RESULT
The Specific activity of laccase enzyme from pleurotus species lies between 1 and 2 units /mg of proteins.
No comments:
Post a Comment
leave your opinion