**2.4.4 Vitamins**

Vitamins play an important role in the metabolism of fungi, acting as coenzymes. Fungi are capable of producing sufficient quantities of most of the vitamins they need (Miles and Chang, 1997).

Maziero (1990), in some studies testing several vitamins (Vitamin C, folic acid, calcium pantothenate, niacin, pyridoxine, riboflavin and thiamine) in relation to the mycelial growth of *Pleurotus*, observed a better growth of the mycelium on all vitamins tested, but the best result was to thiamine. Kurtzman and Zadrazil (1989) say that there is no need for the addition of thiamine or other vitamins in "not sterile" substrates, because the other present organisms will normally synthesize them. Molena, (1986) experimented various combinations of vitamins, but their high cost did not compensate for the increased production of mushrooms. (Eira and Minhoni, 1991), report that the vitamins and other growth factors are normally excreted by many microorganisms that live in synthrofy during composting, pasteurization and incubation of the substrate, therefore there was no need of vitamin supplements.

#### **2.5 Physical factors**

The growth and development of the fungus are not affected only by nutritional factors, but also by physical factors such as temperature, humidity, light, aeration and gravity. There is a range that varies from minimum, maximum, and optimum growth in relation to these physical factors. Certainly these factors are influenced by other factors such as nutrition, medium conditions, genetic characteristics of the strain and mycelial growth stage (Miles and Chang, 1997).

#### **2.5.1 Temperature**

234 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

suggested the hypothesis that the spores of nitrogen fixing bacteria are stimulated to develop during the process of pasteurization of the substrate, generating bacteria

Care should be taken to avoid excessive nitrogen supplements, which can inhibit the development of the fungus. Montini (2001) reports that tested substrates with high concentrations of cereal bran inhibited the formation of the mushroom and consequently, the number of cultivated mushrooms *Lentinula edodes* in axenic conditions (cultivation with substrate sterilized and under controlled environmental conditions). In Taiwan, the substrate for cultivation of *Pleurotus* mushroom is prepared with 84% of sawdust, 5% of rice bran, 5% wheat straw, 3% soya bran and 3% calcium oxide (Przybylowicz and Donoghue,

In general, the mineral elements necessary for the fructification of the mushroom are the same as those required by any cultivated plant, which are major elements and microelements (Molena, 1986). Phosphorus, potassium, magnesium and sulphur are major nutrients needed for the growth of various fungi (Miles and Chang, 1997). Molena, (1986), cites the calcium as one of these elements. In addition to increased growth of mycelium, some minerals such as sodium chloride, magnesium, and calcium also stimulate the early

Among the more studied microelements (trace elements) and essential for the growth of many species of fungus are: iron, zinc, aluminium, manganese, copper, chrome and molybdenum (Molena, 1986; Miles and Chang, 1997). Experimentally, it is not easy to determine the required quantity of these elements because the element under test may be present in sufficient quantities in an impure form in any ingredient of the cultivation medium or may have been introduced through the inoculum. These elements are

Vitamins play an important role in the metabolism of fungi, acting as coenzymes. Fungi are capable of producing sufficient quantities of most of the vitamins they need (Miles and

Maziero (1990), in some studies testing several vitamins (Vitamin C, folic acid, calcium pantothenate, niacin, pyridoxine, riboflavin and thiamine) in relation to the mycelial growth of *Pleurotus*, observed a better growth of the mycelium on all vitamins tested, but the best result was to thiamine. Kurtzman and Zadrazil (1989) say that there is no need for the addition of thiamine or other vitamins in "not sterile" substrates, because the other present organisms will normally synthesize them. Molena, (1986) experimented various combinations of vitamins, but their high cost did not compensate for the increased production of mushrooms. (Eira and Minhoni, 1991), report that the vitamins and other growth factors are normally excreted by many microorganisms that live in synthrofy during composting, pasteurization and incubation of the substrate, therefore there was no need of

The growth and development of the fungus are not affected only by nutritional factors, but also by physical factors such as temperature, humidity, light, aeration and gravity. There is a

formation of fruiting bodies (Kurtman and Zadrazil, 1989).

constituents or enzyme activators (Miles and Chang, 1997).

responsible for the nitrogen fixation.

1990).

**2.4.3 Mineral salts** 

**2.4.4 Vitamins** 

Chang, 1997).

vitamin supplements.

**2.5 Physical factors** 

The influence of temperature on mycelial growth and production of fruiting bodies is dependent on the species and strains in question, i.e. there is an ideal temperature for the proper development of the metabolism of the fungus, which is a characteristic of each strain. Nonetheless, there is an interval that varies between 10-40 C, which must be respected, because exceeding these limits, it is going to cause the death of the mycelium (Maziero, 1990). The optimum temperature for growth also varies with the purpose of cultivation. So, the ideal temperature to produce the fruiting body (Miles and Chang, 1997) is different from that intended for the production of metabolic products such as those intended for medicinal compounds as polysaccharides/polypeptides immune-regulatory compounds (PSPC). Temperature extremes are important in determining the survival and dispersion of species in nature (Miles and Chang, 1997).

Kaufer (1935), cited by Maziero (1990) cultivated *Pleurotus corticatus* in laboratory and according to their results, the ideal temperature for the growth of mycelium was 27C. Clock et al., (1959) according to Maziero (1990), obtained a good growth of mycelium of *P. ostreatus* in the range of 22-31 C. At 37 C the mycelium still was able to grow, but abnormally, while at 17 C no growth was observed. The lethal temperature for *P.* "florida", *P. ostreatus* and *P. eringii* is 40 C when exposed to more than 24 hours (Zadrazil, 1978). Maziero (1990) studying different strains of *Pleurorus* observed that the better mycelial growth happened between 25 and 30 C.

In relation to the emergence of primordia, Block et al. (1959) cited by Maziero (1990) report that the strain of *P. ostreatus* fructified at a 26 C, however at 31 C, although the fruiting body continues to develop, there was no emergence of primordia. For Kurtzman and Zadrazil (1989), the authors must have used in their work, a strain of *P. "florida*" since the fruiting temperature at 26 C is very high, being more appropriate for *P. "florida"*.

Eira and Minhoni (1997) report that the control of temperature in a cultivation chamber is decisive for a good harvest. For a good growth of the mycelial mass on substrate cultivation, the ideal temperature for *Pleurotus* spp should be between 24 and 26 C. After that the primordia initiation and growth phases start, when the temperature inside the cultivation chamber must be between 15 and 240C, considering that, the lowest are ideal for cultivation of shimeji or *Pleurotus* spp strains that are more demanding and also minimizes the incidence of pests and diseases. According to the same authors, some strains of hiratake usually fructify in hot weather (up to 30 C). For the most demanding strains, temperature and relative humidity control in the chamber of cultivation can be achieved with an automated central air-conditioning associated with a ventilation system, to ensure the ideal climatic conditions for the development of the mushroom.

#### **2.5.2 Moisture**

Most fungi require high moisture content. Guzmán et al. (1993) report that fungi have an optimum growth on substrates with 70 to 80% humidity. Urben et al. (2003) cite a good humidity range for *Lentinula edodes* cultivated with Jun-Cao technique between 55-70%. It

Productivity and Nutritional Composition of *Lentinus strigosus* (Schwinitz)

use plastic bags in the green house to cover the mushrooms during the day.

However, it does not cause damage to growth. It is a self-regulated system.

**2.6 Chemical factors 2.6.1 Gaseous exchanges** 

of fruiting body oxygen is essential.

**2.6.2 pH** 

Fries Mushroom from the Amazon Region Cultivated in Sawdust Supplemented with Soy Bran 237

Urben et al. (2003) report that the light affects the growth of mycelium and spores of *Lentinula edodes*, and therefore, it needs a dark environment for its development. Under a light intensity of 50 to 270 lux and a suitable temperature, the mycelium forms a membranous brown layer for substrates made with Jun-Cao and sawdust. According to the same authors, for the formation of the fruiting body (mushroom), little diffused light is necessary. On the contrary, in a very bright environment the fruiting body becomes pale with a long stipe and a deformed pileus. In very bright environments the authors advise to

Requirements during the growth phases of the vegetative mycelium of a fungus are different from those during the fruiting stage. The rate of CO2 that occurs naturally inside a trunk colonized by *Pleurotus* in the forest will surely be higher than the rate of fruiting.

Zadrazil (1975), studied various *pleurotus* species, relating to the effect CO2, and noted that all studied species grew faster in higher concentrations of CO2, limited to approximately 22%. The good performance of these strains in high rates of CO2 demonstrates their significant competitive advantage against other microorganisms which do not grow or do not survive in such conditions, especially if the substrate is colonized in not axenic conditions. On the other hand, high concentrations cause a deformation of the fruiting body, being similar to that which occurs when there is light deficiency in the development of the fruiting body. The stipe grows sharply and the pileus stays reduced, similar to the process of etiolation in plants (Zadrazil, 1978). Oxygen also influences the growth of mycelium. Despite the fact that *Pleurotus* mycelium develops in semi-anaerobic conditions, a certain rate of O2 is required, otherwise, the growth will be nil (Zadralil, 1978). For the development

Adequate ventilation is essential to reduce the carbon dioxide content (generated during the development stages of the fungus) to a desirable level in the mushroom production phase. Concentrations above 2% may cause delays in the mycelial growth and, consequently, decrease productivity (Eira and minhoni, 1997). Concentrations of CO2 below 0.2% are considered optimum for development. During a peak of growth, the ventilation must be intense and constant, since large quantities of mushrooms in rapid growth give off large amounts of CO2 (Eira and minhoni, 1997). The same authors reported that in cultivations carried out in The Mushrooms Module of the Faculty of Agricultural Sciences of the "Universidade Estadual Paulista" (FCA/UNESP) it is possible to cultivate strains that usually demand cold weather, provided that climatic chambers for thermal shock are used.

Its importance is primarily related to the metabolism of nutrients. Most mushrooms have a good development with pH levels between 6.5 and 7, but there are variations according to the species and strains (Miles and Chang, 1997). The microbiota present in the substrate, according to Zadrazil and Grabbe, (1983), is distinctly influenced by the initial pH level: values below 7.0 usually are good for the development of the mushroom mycelium, but most fungi can develop at pH levels above 7.0. Urben et al. (2003), reporting about the cultivation of *Lentinula edodes* by Jun-Cao technique, stated that the mycelium can grow in pH levels between 3.0 to 6.5, while the ideal range is between 4.0 and 5.5. However, the pH

has to be taken into consideration, not only the moisture content of the substrate, but also the relative humidity of the air. It should also be taken into account that the mushrooms are composed of approximately 90% water, therefore, water is very important to its development, besides the fact that they do not have special structures to protect themselves against water loss, since they lose water easily to the environment, mainly the vegetative mycelium (Maziero, 1990).

There is an optimum water content, both in the compost and in the air. Low relative air humidity causes the mushroom to lose water to the environment, which can even prevent it from growing properly. The outer layers of the mushroom begin to dry and yellow. This way, there is a loss of quality or a loss of production. Low air humidity also causes the compost to lose moisture to the environment, reducing the availability of water for the formation of the mushroom. In the case of *Pleurotus*, if the superficial mycelium of the compost suffers a very intense dryness, it dies and the primordia are aborted (Eira and Minhoni, 1997; Bononi et al., 1999). The relative humidity of the production room is around 80-90% and can be maintained that way by waterproof walls and by sprinkling water (Eira and Minhoni, 1997). There are highly sophisticated systems of cultivation on a commercial scale in Europe, Canada, United States and Japan, where patterns of moisture, temperature, O2 and CO2 are monitored by computers. Currently there are automated systems in South and Southeast of Brazil, but not as much as in those countries. In rustic cultivations in Brazil, it is customary to keep the floor and sides of the cultivation shed damp, so that normal evaporation maintains the relative humidity the air. We consider that, in addition to other factors already mentioned, the humidity is the key factor in the cultivation process of edible mushrooms.

#### **2.5.3 Lighting**

Even though it is not a photosynthesizer organism, luminosity is essential to many species of fungi. It can retard the primordia formation in some species while in others, it is essential for fruiting. For *Pleurotus* and *Lentinus* cultivation, as well as for many other edible fungi, there must be some light to induce the formation of primordia and also for the normal development of fruiting bodies. The recommended luminosity for *Pleurotus*, after the incubation period and the opening of the cultivation bags is 2000 lux/hour, 12 hours a day (Bononi et al., 1999). Nevertheless, it can vary according to the mushroom species.

Miles and Chang (1997) mention that ultraviolet light in the range from 200 to 300nm affects the growth of the fungus, it can be lethal or induce mutation, since this wavelength is absorbed by the DNA. The authors report that the effects of ultraviolet light can be reversible by the photo reactivation process, provided that these mycelia are exposed to visible light at a wavelength between 360 and 420nm.

For Przybylowicz and Donoghue (1990), shiitake mushroom needs light in both stages: vegetative growth and fruiting. Light exposure during vegetative growth, according to Ishikawa (1967) cited by Przybylowicz and Donoghue (1990), is a prerequisite for the fruiting stage. The duration is not well defined. However, Przybylowicz and Donoghue (1990) suggest that a brief exposure of 20 minutes per day can be enough. For these authors, the growth of shiitake responds well to a range between 180-940 lux, with an optimum value of 500 lux. Rajarathnan and Bano (1987), cited by Eira and Minhoni (1997), stated that the presence of light is required for the formation of fruiting bodies. However, there may be changes in the color of the pileus, where *Pleurotus* species can change from white to opaque and dark color in the presence of light, due to the release of fenoloxidases that oxidize phenol and form melanoidins.

Urben et al. (2003) report that the light affects the growth of mycelium and spores of *Lentinula edodes*, and therefore, it needs a dark environment for its development. Under a light intensity of 50 to 270 lux and a suitable temperature, the mycelium forms a membranous brown layer for substrates made with Jun-Cao and sawdust. According to the same authors, for the formation of the fruiting body (mushroom), little diffused light is necessary. On the contrary, in a very bright environment the fruiting body becomes pale with a long stipe and a deformed pileus. In very bright environments the authors advise to use plastic bags in the green house to cover the mushrooms during the day.

#### **2.6 Chemical factors**

236 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

has to be taken into consideration, not only the moisture content of the substrate, but also the relative humidity of the air. It should also be taken into account that the mushrooms are composed of approximately 90% water, therefore, water is very important to its development, besides the fact that they do not have special structures to protect themselves against water loss, since they lose water easily to the environment, mainly the vegetative

There is an optimum water content, both in the compost and in the air. Low relative air humidity causes the mushroom to lose water to the environment, which can even prevent it from growing properly. The outer layers of the mushroom begin to dry and yellow. This way, there is a loss of quality or a loss of production. Low air humidity also causes the compost to lose moisture to the environment, reducing the availability of water for the formation of the mushroom. In the case of *Pleurotus*, if the superficial mycelium of the compost suffers a very intense dryness, it dies and the primordia are aborted (Eira and Minhoni, 1997; Bononi et al., 1999). The relative humidity of the production room is around 80-90% and can be maintained that way by waterproof walls and by sprinkling water (Eira and Minhoni, 1997). There are highly sophisticated systems of cultivation on a commercial scale in Europe, Canada, United States and Japan, where patterns of moisture, temperature, O2 and CO2 are monitored by computers. Currently there are automated systems in South and Southeast of Brazil, but not as much as in those countries. In rustic cultivations in Brazil, it is customary to keep the floor and sides of the cultivation shed damp, so that normal evaporation maintains the relative humidity the air. We consider that, in addition to other factors already mentioned, the humidity is the key factor in the cultivation process of edible

Even though it is not a photosynthesizer organism, luminosity is essential to many species of fungi. It can retard the primordia formation in some species while in others, it is essential for fruiting. For *Pleurotus* and *Lentinus* cultivation, as well as for many other edible fungi, there must be some light to induce the formation of primordia and also for the normal development of fruiting bodies. The recommended luminosity for *Pleurotus*, after the incubation period and the opening of the cultivation bags is 2000 lux/hour, 12 hours a day

Miles and Chang (1997) mention that ultraviolet light in the range from 200 to 300nm affects the growth of the fungus, it can be lethal or induce mutation, since this wavelength is absorbed by the DNA. The authors report that the effects of ultraviolet light can be reversible by the photo reactivation process, provided that these mycelia are exposed to

For Przybylowicz and Donoghue (1990), shiitake mushroom needs light in both stages: vegetative growth and fruiting. Light exposure during vegetative growth, according to Ishikawa (1967) cited by Przybylowicz and Donoghue (1990), is a prerequisite for the fruiting stage. The duration is not well defined. However, Przybylowicz and Donoghue (1990) suggest that a brief exposure of 20 minutes per day can be enough. For these authors, the growth of shiitake responds well to a range between 180-940 lux, with an optimum value of 500 lux. Rajarathnan and Bano (1987), cited by Eira and Minhoni (1997), stated that the presence of light is required for the formation of fruiting bodies. However, there may be changes in the color of the pileus, where *Pleurotus* species can change from white to opaque and dark color in the presence of light, due to the release of fenoloxidases that oxidize

(Bononi et al., 1999). Nevertheless, it can vary according to the mushroom species.

visible light at a wavelength between 360 and 420nm.

phenol and form melanoidins.

mycelium (Maziero, 1990).

mushrooms.

**2.5.3 Lighting** 

#### **2.6.1 Gaseous exchanges**

Requirements during the growth phases of the vegetative mycelium of a fungus are different from those during the fruiting stage. The rate of CO2 that occurs naturally inside a trunk colonized by *Pleurotus* in the forest will surely be higher than the rate of fruiting. However, it does not cause damage to growth. It is a self-regulated system.

Zadrazil (1975), studied various *pleurotus* species, relating to the effect CO2, and noted that all studied species grew faster in higher concentrations of CO2, limited to approximately 22%. The good performance of these strains in high rates of CO2 demonstrates their significant competitive advantage against other microorganisms which do not grow or do not survive in such conditions, especially if the substrate is colonized in not axenic conditions. On the other hand, high concentrations cause a deformation of the fruiting body, being similar to that which occurs when there is light deficiency in the development of the fruiting body. The stipe grows sharply and the pileus stays reduced, similar to the process of etiolation in plants (Zadrazil, 1978). Oxygen also influences the growth of mycelium. Despite the fact that *Pleurotus* mycelium develops in semi-anaerobic conditions, a certain rate of O2 is required, otherwise, the growth will be nil (Zadralil, 1978). For the development of fruiting body oxygen is essential.

Adequate ventilation is essential to reduce the carbon dioxide content (generated during the development stages of the fungus) to a desirable level in the mushroom production phase. Concentrations above 2% may cause delays in the mycelial growth and, consequently, decrease productivity (Eira and minhoni, 1997). Concentrations of CO2 below 0.2% are considered optimum for development. During a peak of growth, the ventilation must be intense and constant, since large quantities of mushrooms in rapid growth give off large amounts of CO2 (Eira and minhoni, 1997). The same authors reported that in cultivations carried out in The Mushrooms Module of the Faculty of Agricultural Sciences of the "Universidade Estadual Paulista" (FCA/UNESP) it is possible to cultivate strains that usually demand cold weather, provided that climatic chambers for thermal shock are used.

#### **2.6.2 pH**

Its importance is primarily related to the metabolism of nutrients. Most mushrooms have a good development with pH levels between 6.5 and 7, but there are variations according to the species and strains (Miles and Chang, 1997). The microbiota present in the substrate, according to Zadrazil and Grabbe, (1983), is distinctly influenced by the initial pH level: values below 7.0 usually are good for the development of the mushroom mycelium, but most fungi can develop at pH levels above 7.0. Urben et al. (2003), reporting about the cultivation of *Lentinula edodes* by Jun-Cao technique, stated that the mycelium can grow in pH levels between 3.0 to 6.5, while the ideal range is between 4.0 and 5.5. However, the pH

Productivity and Nutritional Composition of *Lentinus strigosus* (Schwinitz)

reducing the possibility of contamination.

**2.7.2 Substrate cultivation** 

production of this mushroom.

substrate (Stames and Chilton, 1993)

technique.

Stames, 2000).

Fries Mushroom from the Amazon Region Cultivated in Sawdust Supplemented with Soy Bran 239

or with sawdust only. In this case "spawn" or "seed" is the substrate colonized by mushroom mycelium, with the goal of facilitating the distribution of the inoculum in different points of cultivation, thereby contributing to a more uniform and rapid colonization of the substrate,

Currently there is a growing tendency to use agro-industrial residues for the cultivation of edible and medicinal fungi. However, traditional methods are still being used like the cultivation of *Lentinula edodes* (shiitake), by some Japanese and Chinese farmers, using oak and hazel logs, although the cultivation in cylindrical tubes (in polypropylene or high density polyethylene-HDPE bags) with enriched sawdust is the most widely used

The technique for the production in sawdust was developed mainly in Japan. Other countries like the Netherlands and the United States are also using this method for the production of *Lentinula edodes* (shiitake), on a large-scale (Bononi at al., 1999). In Brazil the traditional cultivation is done normally on eucalyptus logs and it may also be grown on logs of avocado, mango, walnut, hazel and oak (the last two being widely used for cultivation in Japan) (Eira and Minhoni, 1997). Eucalyptus sawdust is already used in Brazil for the

The material used for production of mushroom has to be preferably a residue, easily available, and produced not far away from the cultivation place to lower the production costs. Care should be taken to observe that the waste should be free of chemicals that could affect the growth of the mycelium and not offering toxicity. If a low productivity residue is used, supplementation has to be made with cereal grains or cereal bran (Eira and Minhoni, 1997; Bononi et al., 1999; Przybylowicz and Donoghue, 1990; Stames and Chilton, 1983;

The supplements contain a mixture of protein, carbohydrate and fat, where the protein is the main source of nitrogen. They contain minerals and vitamins that also influence the growth of the fungus. The addition of these supplements aims mainly to increase the levels of nitrogen and carbohydrates available. Sugars and starch which are readily available carbohydrates, speed up colonization and the consequent degradation of the substrate, reducing the time of fruiting since the mycelium easily converts these carbohydrates in reserve for the fructification, increasing productivity (Przybylowicz and Donoghue, 1990). Other supplements like limestone (CaCO3) must be added to the cultivation medium, to get the correct pH favorable for the growth of the fungus during the last stages of decomposition since there is an increase in acidity caused by the fungus metabolism. Gypsum is widely used in the mushroom industry to improve the physical structure of the compost and to change the pH value, also acting as a source of calcium (Przybylowicz and Donoghue, 1990). The concentration of 5% (in relation to the dry weight of the substrate) is ideal for the cultivation of shiitake in sawdust, improving structure and porosity of the

When working with primary-decomposer fungi as *Pleurotus* and *Lentinus*, i.e. fungi that degrade the structural elements of the residue, it is important to ensure that the material to be used in cultivation has not undergone decomposition by microorganisms during storage. If it is already degraded, colonization by these fungi will be hampered and the attack of other organisms will be facilitated, causing a reduction in productivity (Maziero, 1990). The sawdust used to prepare the substrate is usually from hardwoods. Sawdusts of conifers are used for *Lentinula* cultivation (shiitake) in areas where there is shortage of hardwood

value between 3.5 and 5.0 is the best for the formation of primordia and development of the fruiting body. For this reason, the pH value should always be monitored when choosing the materials that compose the substrate, the cultivation and the source of water supply (Urben et al., 2003).

The pH is directly linked to the enzymatic reaction of fungus and wood. Each enzyme has its optimum pH value. The pH affects the solubility of the compost which in turn determines its availability to the fungus (Przybylowicz and Donoghue, 1990). The optimum pH value for the wood-rotting fungi *Lentinus* and *Pleurotus* is between 4.5 and 5.5. The pH of the wood is usually 4.5 to 5.0, increasing the acidity with its decomposition. The optimum pH value for fruiting lies between 3.5 to 4.5 (for laboratory culture or artificial medium) and 5.0 for compost with sawdust for *Lentinula edodes* (Przybylowicz and Donoghue, 1990)

#### **2.7 Steps to be followed in the cultivation of mushrooms**

For the cultivation of edible fungi the following steps are generally adopted: obtaining primary matrix, the production of seed or Spawn (matrix that will serve as inoculum for the substrate), preparation of substrate or compost, sterilization or pasteurization (when cultivation is done in natural conditions), inoculation and colonization of substrate, inducing primordia (with thermal or water shock when necessary), fruiting and harvest. The production aspects of primary decomposition fungi like *Pleurotus*, *Lentinula edodes*) will be covered here.

#### **2.7.1 Obtaining primary matrix and spawn production**

For most mushrooms, the production matrix or mycelium follows the same techniques and recommendations for the cultivation of champignon (*Agaricus*), oyster mushroom (*Pleurotus*), shiitake (*Lentinula edodes*) and jewish ear (Auricularia), with some exceptions (Urben et al., 2003). Two distinct steps are fundamental for the preparation of the matrix: obtaining pure inoculum of the fungus and the preparation of the "spawn" or matrix itself.

Obtaining the primary matrix of mushroom can be performed both by sexual or by asexual process. In this work it will be related as an asexual process. It is relative to the mycelial or vegetative phase of the fungus colonizing a previously sterilized nutritional substrate (growth medium). Its production starts by the isolation of a fungus using tiny fragments of a mushroom, placed in sterile culture medium under aseptic conditions. After mycelial growth in the dark, with a temperature of 24 ± 10C (depending on the strain), fragments of this culture (primary matrix) are transferred to the cereal grain or bran or sawdust enriched with bran and incubated for 30 days in the dark at 24 ± 1 0C. This step corresponds to the production of the "seed" or spawn (Molena, 1986; Eira and Minhone, 1997; Eira and Montini, 1997). The main function of the grain is to serve as means of dispersion of mycelium, since it is impossible to handle the mycelium without damaging the fragile structure of the hyphae walls (Maziero, 1990).

Although the most used media to obtain the primary matrix are potato-dextrose-agar and malt extract (Bononi et al., 1999), the sawdust-dextrose-agar (SDA) medium is the most indicated by avoiding the physiological adaptation that can occur when the used culture medium has very different characteristics of production substrate (Eira and Minhoni, 1997, Eira and Montini, 1997).

The current trend is to produce inoculum from the cultivation substrate. When working with sawdust it is possible to produce the inoculum ("seed") with grain mixed with sawdust or with sawdust only. In this case "spawn" or "seed" is the substrate colonized by mushroom mycelium, with the goal of facilitating the distribution of the inoculum in different points of cultivation, thereby contributing to a more uniform and rapid colonization of the substrate, reducing the possibility of contamination.
