**2.7.2 Substrate cultivation**

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

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

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

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

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

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,

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

et al., 2003).

and Donoghue, 1990)

covered here.

walls (Maziero, 1990).

Eira and Montini, 1997).

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

**2.7.1 Obtaining primary matrix and spawn production** 

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 technique.

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 production of this mushroom.

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; Stames, 2000).

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 substrate (Stames and Chilton, 1993)

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

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

sheets or in special containers (Maziero, 1990).

cooling of substrate that is around 300 C, in aseptic place.

(Itaavara, 1993), cited by Gonçalves (2002).

a temperature between 22 to 250 C for *Pleurotus* (Maziero, 1990).

**2.7.5 Substrate incubation** 

**2.7.4 Inoculation of the substrate** 

Fries Mushroom from the Amazon Region Cultivated in Sawdust Supplemented with Soy Bran 241

of microorganisms, falls below 45 to 500 C (Eira and Minhoni, 1997). The compost is then introduced in the pasteurization chamber. The pasteurization temperature for *Pleurotus* is more severe than for *Agaricus*, being raised to 750C during the first 6 hours. After cutting the steam, the temperature falls to 40 to 450 C, maintaining a constant ventilation to cool the compost and then proceeding to the inoculation process (Eira and Minhoni, 1997). Depending on the type of cultivation, the substrate to be inoculated can be packed in plastic bags, put in wooden or plastic boxes, shelves or "bed", pressed blocks covered with plastic

The sterilization process (used in axenic cultivation) or pasteurization (when working with composted natural substrate) is followed by the inoculation of the substrate, which is made immediately after the cooling of the substrate in aseptic conditions (laminar flow chamber) in case of cultivation in totally axenic conditions. Under these conditions, the substrate to be inoculated is autoclaved at 1210 C for 2 to 4 hours (Eira and Minhoni, 1997). For cultivation under natural conditions (not axenic), the substrate is inoculated after pasteurization and

There are several types of inoculum ("seed"): with grains, grains with sawdust, and less used liquid inoculum. There are still those that are made of small wooden dowels (wooden rods inoculated with fungus, used for the cultivation of shiitake in logs). Inoculum of sawdust/grain is used for *Pleurotus* and *Lentinus* cultivation, when using sawdust for cultivation. It is important that the inoculum is the same sawdust from the cultivation substrate. The quantity used for the cultivation substrate is also variable. It is usually 0.5 to 5% (v/v) (Chang and Miles, 1997). (Zadrazil and Grabbe, 1983), recommend 0.5 to 5% of wet substrate. Urben et al. (2003), recommend 0.5 to 5% of the wet weight of the substrate for the *Pleurotus* cultivation when using the Jun-Cao technique. Gonçalves (2002) studying the effect of mycelial fragmentation in order to obtain inoculants in suspension (liquid fermentation) for cultivation of shiitake in axenic cultivation, found that inoculants fragmented up to 10 seconds provided greater biological productivity and efficiency in comparison with usual solid inoculants. The author reports that cultivation using liquid inoculants has the advantage of reducing the time for fruiting. However, it has the disadvantage of having predisposition for degeneration and mutation after successive crops

Incubation period, also known as the "mycelial race", is the development of the vegetative mycelium on the substrate (Przybylowicz and Donoghue, 1990). It is the mechanism in which the mycelium of the fungus, through an enzymatic process, digests the substrate and stores reserves for fruiting. During this process the mycelium develops and colonizes the whole compost, forming a compact white mass. It is a complex process, characterized by intense biological activity in which molecules of cellulose, hemicellulose and lignin of the compost are attacked by fungal enzymes such as cellulase and lacase that reduce these molecules to phenols and simple sugars which are more easily assimilated. This enzymatic activity lasts from the beginning of colonization until the production of mushrooms, but during the period of growth of the mycelium production it is greater (Bononi et al., 1999). Incubation usually occurs in a room that can be dark or not, depending on the light requirement of the fungus, at

sawdust and it is therefore necessary to make a mixture of the two kinds of sawdust (Przybylowicz and Donoghue, 1990). Many conifers contain resin and phenolic compounds which inhibit the growth of the fungus. These compounds must be degraded or removed before using this kind of sawdust, or it can be changed with the addition of sodium carbonate to remove these compounds (Przybylowicz and Donoghue, 1990).

Various types of substrates have been used for the production of edible fungi (Guzmán and Martinez; 1986; Guzmán et al., 1993; Maziero, 1990; Bononi et al., 1999; Eira and minhoni, 1997; Miles and Chang, 1997; Stames and Chilton, 1983; Stames, 1993; Urben, 2001; Urben et al., 2003; Zhanhua and Zhanxi, 2001). The most used are: sawdust, wheat straw, corn, rice; corn cobs, sugar cane bagasse, various grasses, supplemented with cereal grain or bran. The choice of one or more residues as supplement to sawdust will depend, among other factors, on cost and availability of these materials (Maziero, 1990; Eira and Minhoni, 1997; Eira and Montini, 1997; Guzmán et al., 1993; Urben et al., 2003; Stames and Chilton, 1983; Stames, 1993).

When using the bagasse of sugar cane it is important to make sure that this residue is not very old, which can reduce productivity. However, the fresh ground bagasse is rich in carbohydrates, allowing other competitors or pathogens to colonize the substrate more quickly. To avoid this problem, the residue should be pre-treated through a process of fermentation or washing (Kurtzman and Zadrazil (1989)

Japanese producers of *Flammulina Velutipes*, *Auricularia* and *Pleurotus ostreatus* use a standard formula with a ratio 4:1 of sawdust and bran respectively, where the sawdust is aged for one year, with the purpose to improve the water retention capacity. An immersion of sawdust in water before mixing with the bran is an effective way used by these producers to achieve an optimum of 60% humidity (Samets and Chilton, 1983). This method, according to Lizuka and Takeuchi (1978) cited by Przybylowicz and Donoghue (1990) is widely used in Asia. In the United States, 80% of sawdust, 10% bran and 10% grain (usually wheat or millet). In Taiwan, the substrate for the cultivation of shiitake is done with 84% of sawdust, 5% of rice bran, 5% wheat straw, 3% soy bran and 3% calcium oxide (Przybylowicz and Donoghue, 1990). The substrate formulations have become unlimited in terms of raw material and agro-industrial residues (Stames, 1993).

Currently, studies are trying to develop a technology that allows the cultivation of edible mushrooms in substrates of low cost and easily available. Perhaps this explains why in Brazil the largest edible fungi producing region is located in São Paulo, where there is a big sugar and alcohol production. This bagasse comes from sugar cane mills, it is homogeneous, it has a fibrous characteristic, and when it is pressed allows aeration for mycelial growth (Rossi, 1999). The sawdust is also a material in abundance in the Amazon region, because of its timber industry.

#### **2.7.3 Pasteurization/sterilization**

The pasteurization process is a heat treatment given to the compost for the removal of possible organisms that could compete with the fungus to be cultivated (Maziero, 1990). It can be done in a natural way, in a pasteurizing tunnel or room without heated steam, using only the thermogenesis, with the control of the air that gets in and out in order to control the temperature inside the room, or it can be made with heated steam produced by boilers heated with firewood, diesel or gas.

Pasteurization for the cultivation of lignicol fungi as *Pleurotus*, occurs when after the revolving process of the compost, the temperature of thermogensis, produced by the action of microorganisms, falls below 45 to 500 C (Eira and Minhoni, 1997). The compost is then introduced in the pasteurization chamber. The pasteurization temperature for *Pleurotus* is more severe than for *Agaricus*, being raised to 750C during the first 6 hours. After cutting the steam, the temperature falls to 40 to 450 C, maintaining a constant ventilation to cool the compost and then proceeding to the inoculation process (Eira and Minhoni, 1997). Depending on the type of cultivation, the substrate to be inoculated can be packed in plastic bags, put in wooden or plastic boxes, shelves or "bed", pressed blocks covered with plastic sheets or in special containers (Maziero, 1990).

#### **2.7.4 Inoculation of the substrate**

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

sawdust and it is therefore necessary to make a mixture of the two kinds of sawdust (Przybylowicz and Donoghue, 1990). Many conifers contain resin and phenolic compounds which inhibit the growth of the fungus. These compounds must be degraded or removed before using this kind of sawdust, or it can be changed with the addition of sodium

Various types of substrates have been used for the production of edible fungi (Guzmán and Martinez; 1986; Guzmán et al., 1993; Maziero, 1990; Bononi et al., 1999; Eira and minhoni, 1997; Miles and Chang, 1997; Stames and Chilton, 1983; Stames, 1993; Urben, 2001; Urben et al., 2003; Zhanhua and Zhanxi, 2001). The most used are: sawdust, wheat straw, corn, rice; corn cobs, sugar cane bagasse, various grasses, supplemented with cereal grain or bran. The choice of one or more residues as supplement to sawdust will depend, among other factors, on cost and availability of these materials (Maziero, 1990; Eira and Minhoni, 1997; Eira and Montini, 1997; Guzmán et al., 1993; Urben et al., 2003; Stames and Chilton, 1983; Stames,

When using the bagasse of sugar cane it is important to make sure that this residue is not very old, which can reduce productivity. However, the fresh ground bagasse is rich in carbohydrates, allowing other competitors or pathogens to colonize the substrate more quickly. To avoid this problem, the residue should be pre-treated through a process of

Japanese producers of *Flammulina Velutipes*, *Auricularia* and *Pleurotus ostreatus* use a standard formula with a ratio 4:1 of sawdust and bran respectively, where the sawdust is aged for one year, with the purpose to improve the water retention capacity. An immersion of sawdust in water before mixing with the bran is an effective way used by these producers to achieve an optimum of 60% humidity (Samets and Chilton, 1983). This method, according to Lizuka and Takeuchi (1978) cited by Przybylowicz and Donoghue (1990) is widely used in Asia. In the United States, 80% of sawdust, 10% bran and 10% grain (usually wheat or millet). In Taiwan, the substrate for the cultivation of shiitake is done with 84% of sawdust, 5% of rice bran, 5% wheat straw, 3% soy bran and 3% calcium oxide (Przybylowicz and Donoghue, 1990). The substrate formulations have become unlimited in terms of raw

Currently, studies are trying to develop a technology that allows the cultivation of edible mushrooms in substrates of low cost and easily available. Perhaps this explains why in Brazil the largest edible fungi producing region is located in São Paulo, where there is a big sugar and alcohol production. This bagasse comes from sugar cane mills, it is homogeneous, it has a fibrous characteristic, and when it is pressed allows aeration for mycelial growth (Rossi, 1999). The sawdust is also a material in abundance in the Amazon region, because of

The pasteurization process is a heat treatment given to the compost for the removal of possible organisms that could compete with the fungus to be cultivated (Maziero, 1990). It can be done in a natural way, in a pasteurizing tunnel or room without heated steam, using only the thermogenesis, with the control of the air that gets in and out in order to control the temperature inside the room, or it can be made with heated steam produced by boilers

Pasteurization for the cultivation of lignicol fungi as *Pleurotus*, occurs when after the revolving process of the compost, the temperature of thermogensis, produced by the action

carbonate to remove these compounds (Przybylowicz and Donoghue, 1990).

fermentation or washing (Kurtzman and Zadrazil (1989)

material and agro-industrial residues (Stames, 1993).

1993).

its timber industry.

**2.7.3 Pasteurization/sterilization** 

heated with firewood, diesel or gas.

The sterilization process (used in axenic cultivation) or pasteurization (when working with composted natural substrate) is followed by the inoculation of the substrate, which is made immediately after the cooling of the substrate in aseptic conditions (laminar flow chamber) in case of cultivation in totally axenic conditions. Under these conditions, the substrate to be inoculated is autoclaved at 1210 C for 2 to 4 hours (Eira and Minhoni, 1997). For cultivation under natural conditions (not axenic), the substrate is inoculated after pasteurization and cooling of substrate that is around 300 C, in aseptic place.

There are several types of inoculum ("seed"): with grains, grains with sawdust, and less used liquid inoculum. There are still those that are made of small wooden dowels (wooden rods inoculated with fungus, used for the cultivation of shiitake in logs). Inoculum of sawdust/grain is used for *Pleurotus* and *Lentinus* cultivation, when using sawdust for cultivation. It is important that the inoculum is the same sawdust from the cultivation substrate. The quantity used for the cultivation substrate is also variable. It is usually 0.5 to 5% (v/v) (Chang and Miles, 1997). (Zadrazil and Grabbe, 1983), recommend 0.5 to 5% of wet substrate. Urben et al. (2003), recommend 0.5 to 5% of the wet weight of the substrate for the *Pleurotus* cultivation when using the Jun-Cao technique. Gonçalves (2002) studying the effect of mycelial fragmentation in order to obtain inoculants in suspension (liquid fermentation) for cultivation of shiitake in axenic cultivation, found that inoculants fragmented up to 10 seconds provided greater biological productivity and efficiency in comparison with usual solid inoculants. The author reports that cultivation using liquid inoculants has the advantage of reducing the time for fruiting. However, it has the disadvantage of having predisposition for degeneration and mutation after successive crops (Itaavara, 1993), cited by Gonçalves (2002).
