**2.7.5 Substrate incubation**

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

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

production and the quality of mushrooms (Zadrazil and Grabbe, 1983).

processes that trigger the induction (Przybylowicz and Donoghue, 1990).

temperature depends on the strain (Przybylowicz and Donoghue, 1990).

shock, using water immersion only.

mushrooms are ready to be collected.

removing the bag after water drainage.

relative humidity varies with the location of cultivation.

temperature variation, others produce more when subjected to thermal shock.

Fries Mushroom from the Amazon Region Cultivated in Sawdust Supplemented with Soy Bran 243

fruiting body (Bononi et al., 1999). On the cultivation of mushrooms, it is used to stimulate or speed their formation. During the induction phase and the production of mushrooms, physical factors such as temperature, lighting, gas exchange, water availability in the compost, relative humidity and the methods of induction are aspects that influence the

Sudden changes in temperature usually cause induction of primordia. However, there are differences according to the strains (Przybylowicz and Donoghue, 1990). Low temperatures may indirectly induce fruiting in strains of shiitake, because of the reduction of metabolic activity, reducing therefore the available nutrients, leading to a condition of "stress". On the other hand, in other fungi, temperature can have a direct effect, favoring specific metabolic

Hawker (1966), cited by (Przybylowicz and Donoghue, 1990) reports that studies with various fungi showed that reducing sugars readily available on the substrate (end of vegetative growth) favors the fruiting. During the entire cycle of fruiting, the primordia phase is the most sensitive to environmental changes. The moisture content of the substrate, temperature and relative humidity are important in this process. On the cultivation of shiitake in logs, moisture for primordia induction should be around 55-65% and the

There are several artificial induction mechanisms of primordia. It can be done by changing the temperature of incubation (± 250 C) to lower temperatures (±160 C) in *Pleurotus* cultivation "shimeji" (Eira and Minhoni, 1997). Some strains respond well to this

Marino (2002) in a study about genetic improvement with *Pleurotus ostreatus* aiming the axenic cultivation of strains resistant to heat obtained strains that stood out by their early fruiting and productivity, with two production cycles and without the need for thermal

For the cultivation of shiitake in sawdust, according to Leatham (1985), cited by Przybylowicz and Donoghue (1990), the thermal shock can be done by cooling the cultivation blocks (packed in bags of polypropylene) at a temperature of 50-80 C for five to twelve days or by putting them into cold water (5-160 C) for 12 to 24 hours, packing them later in the fruiting room (160C). After some time primordia will appear at the top of the bags. After the development (3-4 days), according to Eira and Minhoni (1997), the

Additional flushes of fruiting will emerge without the need for new inductions, provided they are kept in conditions of fruiting. Producers can control the flush making synchronized induction by heating the blocks, followed by reduction of temperature or thermal shock. Sprinkling or immersion can also induce the flush (Przybylowicz and Donoghue, 1990). Treatment for production of mushrooms (Eira and Minhoni, 1997) is done by reducing temperature and/or water logging (covering with clean cold water for 2 to 4 hours) and

The thermal shock for *Lentinula edodes* in modified Jun-Cao technology is made by dipping the miceliated substrates in cold or icy water during 7-8 hours. Then, the bags are packed in a shed or green house. When the buttons (primordia) begin to emerge, the plastic bags (high-density polyethylene) are removed, and the substrates are watered twice a day. After spraying, the bags are covered with a plastic for two hours or until the environment is agreeable (Urben et al., 2003). Regional climatic variations need to be considered. The

Guzmán et al. (1993) uses the range 25 to 300 C for the cultivation of several *Pleurotus* species in Mexico. Bononi et al. (1999) report that the ideal temperature for the incubation of these fungi varies between species, but in general it should be kept between 25 and 280 C (the temperature of the compost). The range 25-300 C is also used for the *Pleurotus* cultivation, and 22-25 0 C for *L. edodes* cultivation in Jun-Cao (Urben et al., 2003). Przybylowicz and Donoghue (1990), reported temperatures of 250 C that are ideal for *L. edodes*.

It is important to monitor the temperature during the mycelial race to maintain the optimum temperature for the growth of the mycelium. If there is an excessive rise in temperature (a phenomenon that occurs during metabolic activity of *Pleurotus* and micro-organisms present in the substrate) mycelial growth retardation or even its death may occur. Containers with large amounts of substrate mass are avoided since of heat loss is hampered, and generates an increase in temperature. (Maziero, 1990).

The incubation period is approximately three weeks for *Pleurotus* (Maziero, 1990; Bononi et al., 1999). At low temperatures, such as 4-50 C, the mycelium of most species ceases its activity, entering "latency", and at temperatures over 35-400 C can be lethal to certain species (Bononi et al., 1999). To avoid excessive internal temperature of the substrate during the incubation period, Bononi et al. (1999) recommend keeping the room temperature between 20-220 C, and avoid to clutter the bags of the substrates.

The incubation period is variable, because the development of mycelium occurs within variable time, according to the type of the inoculum, the quality of the compost and conditions of the cultivation chamber, but it generally oscillates between 20 and 30 days for *Pleurotus* (Eira and Minhoni, 1997). Urben et al. (2003) report 20-45 days for the total development of the mycelium with Jun-Cao technique. For the cultivation of shiitake in logs, Eira and Minhoni (1997) report that, after two to three months from log incubation, there is already a significant mycelial growth, which can be indicated by a yellow color in the region of the inoculated holes and the region around those holes become soft. In natural conditions of cultivation on logs, this period of maturity of the mycelium for mycelia production, ranges from six months to a year (Przybylowicz and Donoghue, 1990).

During the colonization of the substrate in the cultivation of *Lentinula edodes* using sawdust enriched with rice bran, packed in plastic bags, Bononi et al. (1999) recommend cycles of alternating light and dark, with at least 8 hours of light per day during a period of four to six weeks. The wavelengths between 370 to 420nm and light intensity between 180 to 500 lux are more efficient during the process of colonization, and it can be achieved with cold fluorescent lamps (Przybylowicz and Donoghue, 1990). According to Bononi et al. (1999), after the total colonization of the substrate, the plastic bags are cut and the surface of mycelium begins to turn into a brown skin. The air humidity must be maintained around 80 to 90% and between 40 to 50 days after the opening of the bags the production starts, after the induction of primordia through thermal shock for 24 to 48 hours at 100 C.

At the end of the mycelial race for shitake there is a period of mycelial stability or mycelium maturation, which lasts until the hardening and darkening of the mycelial skin that becomes brownish grey (Chang and Miles, 1989). The formation of mycelial cover is very important because it acts as a barrier to moisture loss, being also a defense against contaminants, resulting from the oxidation of poliphenol oxidase, a reaction to light and oxygen (Przybylowicz and Donoghue, 1990).

#### **2.7.6 Induction of primordia, fruiting and harvesting**

The induction of the primordia occurs naturally in nature. The sudden change of external physical conditions stimulates primordia formation, which will develop, forming the

Guzmán et al. (1993) uses the range 25 to 300 C for the cultivation of several *Pleurotus* species in Mexico. Bononi et al. (1999) report that the ideal temperature for the incubation of these fungi varies between species, but in general it should be kept between 25 and 280 C (the temperature of the compost). The range 25-300 C is also used for the *Pleurotus* cultivation, and 22-25 0 C for *L. edodes* cultivation in Jun-Cao (Urben et al., 2003). Przybylowicz and

It is important to monitor the temperature during the mycelial race to maintain the optimum temperature for the growth of the mycelium. If there is an excessive rise in temperature (a phenomenon that occurs during metabolic activity of *Pleurotus* and micro-organisms present in the substrate) mycelial growth retardation or even its death may occur. Containers with large amounts of substrate mass are avoided since of heat loss is hampered, and generates

The incubation period is approximately three weeks for *Pleurotus* (Maziero, 1990; Bononi et al., 1999). At low temperatures, such as 4-50 C, the mycelium of most species ceases its activity, entering "latency", and at temperatures over 35-400 C can be lethal to certain species (Bononi et al., 1999). To avoid excessive internal temperature of the substrate during the incubation period, Bononi et al. (1999) recommend keeping the room temperature between

The incubation period is variable, because the development of mycelium occurs within variable time, according to the type of the inoculum, the quality of the compost and conditions of the cultivation chamber, but it generally oscillates between 20 and 30 days for *Pleurotus* (Eira and Minhoni, 1997). Urben et al. (2003) report 20-45 days for the total development of the mycelium with Jun-Cao technique. For the cultivation of shiitake in logs, Eira and Minhoni (1997) report that, after two to three months from log incubation, there is already a significant mycelial growth, which can be indicated by a yellow color in the region of the inoculated holes and the region around those holes become soft. In natural conditions of cultivation on logs, this period of maturity of the mycelium for mycelia production,

During the colonization of the substrate in the cultivation of *Lentinula edodes* using sawdust enriched with rice bran, packed in plastic bags, Bononi et al. (1999) recommend cycles of alternating light and dark, with at least 8 hours of light per day during a period of four to six weeks. The wavelengths between 370 to 420nm and light intensity between 180 to 500 lux are more efficient during the process of colonization, and it can be achieved with cold fluorescent lamps (Przybylowicz and Donoghue, 1990). According to Bononi et al. (1999), after the total colonization of the substrate, the plastic bags are cut and the surface of mycelium begins to turn into a brown skin. The air humidity must be maintained around 80 to 90% and between 40 to 50 days after the opening of the bags the production starts, after

At the end of the mycelial race for shitake there is a period of mycelial stability or mycelium maturation, which lasts until the hardening and darkening of the mycelial skin that becomes brownish grey (Chang and Miles, 1989). The formation of mycelial cover is very important because it acts as a barrier to moisture loss, being also a defense against contaminants, resulting from the oxidation of poliphenol oxidase, a reaction to light and oxygen

The induction of the primordia occurs naturally in nature. The sudden change of external physical conditions stimulates primordia formation, which will develop, forming the

Donoghue (1990), reported temperatures of 250 C that are ideal for *L. edodes*.

an increase in temperature. (Maziero, 1990).

(Przybylowicz and Donoghue, 1990).

**2.7.6 Induction of primordia, fruiting and harvesting** 

20-220 C, and avoid to clutter the bags of the substrates.

ranges from six months to a year (Przybylowicz and Donoghue, 1990).

the induction of primordia through thermal shock for 24 to 48 hours at 100 C.

fruiting body (Bononi et al., 1999). On the cultivation of mushrooms, it is used to stimulate or speed their formation. During the induction phase and the production of mushrooms, physical factors such as temperature, lighting, gas exchange, water availability in the compost, relative humidity and the methods of induction are aspects that influence the production and the quality of mushrooms (Zadrazil and Grabbe, 1983).

Sudden changes in temperature usually cause induction of primordia. However, there are differences according to the strains (Przybylowicz and Donoghue, 1990). Low temperatures may indirectly induce fruiting in strains of shiitake, because of the reduction of metabolic activity, reducing therefore the available nutrients, leading to a condition of "stress". On the other hand, in other fungi, temperature can have a direct effect, favoring specific metabolic processes that trigger the induction (Przybylowicz and Donoghue, 1990).

Hawker (1966), cited by (Przybylowicz and Donoghue, 1990) reports that studies with various fungi showed that reducing sugars readily available on the substrate (end of vegetative growth) favors the fruiting. During the entire cycle of fruiting, the primordia phase is the most sensitive to environmental changes. The moisture content of the substrate, temperature and relative humidity are important in this process. On the cultivation of shiitake in logs, moisture for primordia induction should be around 55-65% and the temperature depends on the strain (Przybylowicz and Donoghue, 1990).

There are several artificial induction mechanisms of primordia. It can be done by changing the temperature of incubation (± 250 C) to lower temperatures (±160 C) in *Pleurotus* cultivation "shimeji" (Eira and Minhoni, 1997). Some strains respond well to this temperature variation, others produce more when subjected to thermal shock.

Marino (2002) in a study about genetic improvement with *Pleurotus ostreatus* aiming the axenic cultivation of strains resistant to heat obtained strains that stood out by their early fruiting and productivity, with two production cycles and without the need for thermal shock, using water immersion only.

For the cultivation of shiitake in sawdust, according to Leatham (1985), cited by Przybylowicz and Donoghue (1990), the thermal shock can be done by cooling the cultivation blocks (packed in bags of polypropylene) at a temperature of 50-80 C for five to twelve days or by putting them into cold water (5-160 C) for 12 to 24 hours, packing them later in the fruiting room (160C). After some time primordia will appear at the top of the bags. After the development (3-4 days), according to Eira and Minhoni (1997), the mushrooms are ready to be collected.

Additional flushes of fruiting will emerge without the need for new inductions, provided they are kept in conditions of fruiting. Producers can control the flush making synchronized induction by heating the blocks, followed by reduction of temperature or thermal shock. Sprinkling or immersion can also induce the flush (Przybylowicz and Donoghue, 1990).

Treatment for production of mushrooms (Eira and Minhoni, 1997) is done by reducing temperature and/or water logging (covering with clean cold water for 2 to 4 hours) and removing the bag after water drainage.

The thermal shock for *Lentinula edodes* in modified Jun-Cao technology is made by dipping the miceliated substrates in cold or icy water during 7-8 hours. Then, the bags are packed in a shed or green house. When the buttons (primordia) begin to emerge, the plastic bags (high-density polyethylene) are removed, and the substrates are watered twice a day. After spraying, the bags are covered with a plastic for two hours or until the environment is agreeable (Urben et al., 2003). Regional climatic variations need to be considered. The relative humidity varies with the location of cultivation.

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

cultivation substrates for the production of *L. strigosus* mushrooms

**3.3 Preparation of cultivation substrate and processing** 

(LOM).

**3.4 Experimental conditions** 

Fries Mushroom from the Amazon Region Cultivated in Sawdust Supplemented with Soy Bran 245

(primary matrix) that was used as a source of inoculum for the secondary matrix . Mycelial disks, 9 mm in diameter, were removed from the primary matrix and transferred to Petri dishes containing SDA medium (sawdust-dextrose-agar), prepared according to Sales-Campos (2008), named secondary matrix. "Spawn" is the source for inoculation of the cultivation substrate, considered here as a tertiary matrix. This matrix was produced from cajuí sawdust, with humidification of 75%. The pH was corrected to approximately 6.5, by adding CaCO3. Then that substrate was deposited on glass bottles of 500 mL, in 200 g portions, which were autoclaved at 121 °C for 45 minutes. After cooling, the substrate was inoculated with the secondary matrix. The bottles were partially closed, and kept in special chamber with biochemical oxygen demand (BOD) at 25 ± 2 °C until the complete colonization of the substrate by fungus. This matrix served as a source of inoculation for the

The cultivation substrate was prepared from the same residue (cajuí sawdust) as the spawn inoculums. It consisted of 88% of sawdust + 10% of the soy bran as a protein source + 2% of CaCO3, for pH adjustment (6.5). The material was homogenised and humidified to 75%, and packed into bags of high density polyethylene-HDPE (1 kg capacity). Only 500 g of the substrate (wet basis) were put into each bag, with ten repetitions. The substrates were autoclaved at 121 °C for one hour. After that, they were cooled and inoculated with a tertiary matrix under axenical conditions. Each experimental unit (the bag containing the substrate) received 3% of the inoculum in relation to the wet weight of the substrate. They were taken to an incubating chamber until the colonization of the substrate by the fungus. Afterwards, they were transferred to a production chamber. The control samples were also prepared as above, but without inoculation by the fungus. The bags were taken to an oven with air circulation at 55 ± 5 ºC and dried to a constant weight, in order to obtain the dry mass of the initial substrate (DMIS) so that they were used to calculate the productivity, based on the biological efficiency index of substrate (BE) and the loss of organic matter

The experiment was conducted indoors. The bags contained the substrates were incubated in a climatic chamber at the temperature of 25 ± 3 °C, in the absence of light and at around 80-85% humidity, in order to allow substrate colonization until the production of primordia. Then, they were transferred to the production chamber. The temperature was reduced from 25 ºC to 22 ºC to induce primordial emission and to allow the production of basidioma (fruit body of the mushrooms) in a way that it would be as uniform as possible. Light intensity was maintained at 2000 Lux, with a photoperiod of 12 hours per day. The relative humidity was scheduled to 95% during the "fructification. The total period of cultivation was 100 days. After "fructification", the mature mushrooms were collected and weighed, and then oven dried for the determination of moisture, dry mass and chemical analyzes. During cultivation, the variables analyzed were: biological efficiency (BE), and loss of organic matter (LOM). Biological efficiency (used to express the productivity of fungus), was

> \* 100 *FMM BE DMS*

calculated according to Tisdale et al. (2006) and Das and Mukherjee (2007):

In Brazil, the necessary time for the complete development of the shiitake mushroom is not well defined due to climatic variations. The fruiting occurs over a period between three and twelve months after the inoculation, depending on the temperature of the region and the maintenance of moisture in the log (Eira and Minhoni, 1997; Eira and Montini, 1997). To accelerate this process in the cultivation on eucalyptus logs, the authors recommend soaking the miceliated logs for induction, after the incubation period when the first signs of primordia emission (callus or popcorns) which usually appear after 2 to 3 months. Mineral supplementation in water immersion increased the productivity of this mushroom. However, the increase of productivity and the efficiency of energy conversion were only possible in logs well colonized by the fungus (Queiroz, 2002; Eira and Minhoni, 1997; Eira and Montini, 1997).

In relation to water temperature for immersion, there is a controversy, probably because of environmental differences, and observations often without experimental parameters (Eira and Minhoni, 1997; Eira and Montini, 1997). Some Brazilian producers who own cooling bath system report positive results since this system causes a steady temperature differential of 5-100 C. However, experiments performed in the Module of Mushrooms of the Faculty of Agricultural Sciences "Universidade Estadual Paulista" (FCA/UNESP), in Botucatu, São Paulo State, these same authors report that, in regions with mild climate and thermal amplitude greater than 100 C, the use of ice for cooling did not show significant difference in relation to normal bath immersion.

Induction time depends on environmental conditions and age of the logs and the fruiting temperature varies from 5 to 300 C depending on the strain and the spawn used for cultivation. The relative humidity of the location of the logs should be between 80 and 90%. The emergence of primordia will be within two to three days and harvesting can be made after seven to ten days, and in cool seasons the metabolism of the fungus is reduced, increasing the time before harvest (Eira and Minhoni, 1997). The induction bath can be done in stages, depending on the needs of the producer, thus inducing bath of logs can be programmed as a function of demand (Eira and Minhoni, 1997; Eira and Montini, 1997).
