Aspects of mushroom cultivation to obtain polysaccharides in submerged cultivation

Medicinal and edible mushrooms have biological properties that are important in promoting health. In the last decades, an increasing number of studies have explored various aspects of the cultivation and metabolism of various species. Many isolated substances have potential for use in medicine and among them are polysaccharides, which have been shown to have physiological properties such as anti-tumor, immunomodulatory, anti-hypercholesterolemia, antiviral and antiinflammatory. The most common way of obtaining products from mushrooms is through cultivation in a solid medium. However, in this type of cultivation there is a difficulty in controlling the physical and chemical parameters and, consequently, the quality of the product. In this context, the submerged fermentation of the mycelial form of the mushroom has received attention as an alternative for the efficient production of biomass and, therefore, of polysaccharides. Technical problems and forms of cultivation are studied so that the standardization of cultivation is more effective. This review describes some aspects about the submerged fermentation technique in bottles and bioreactors, exploring the two main ones, which are the stirred tank fermentator and the air lift reactors and the species that are in these cultivated systems.


INTRODUCTION
In recent decades, a large number of publications have presented the possibility of using mushrooms in the production of several metabolites of interest of low molar mass (Shu and Lung, 2004;Zou, 2006;Tang et al., 2007) and high molar mass, such as polysaccharides, which are shown as potent pharmacological agents with diverse activities. In addition, mushrooms have been widely consumed for centuries in the East and Europe, given their high nutritional value, with around 2000 edible species recognized and approximately 25 grown on a commercial scale (Jong and Donovick, 1989;Furnali and Godoy, 2007). In its composition, there are essential and non-essential amino acids, carbohydrates, organic acids, minerals, vitamins low lipids and high protein and fiber contents (Ghorai et al., 2009;Kawagoe et al., 2004;Sales-Campos et al., 2011a.
Special attention is given to fungal polysaccharides because they have biological activity as antioxidant, antitumor, hypoglycemic, aintinflammatory and immunomodulatory (Bai et al., 2020;Fan et al., 2007; *Corresponding author. E-mail: lcampestrini@gmail.com. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Zhang et al., 2011;Silveira et al., 2015;Wang et al., 2019). Polysaccharides are metabolites of great interest in the cultivation of basidiomycetes. The molecular arrangement of these polysaccharides is well distributed, but most belong to the groups of β-glucans, whose main chain connections are of the type β-(13) and varying number of β-(16) branches (Seo et al., 2019). However, at least one example of a β-(13) glucan, containing β-(12) and β-(16) branches has already been described (Ruiz-Herrera and Ortiz-Castellanos, 2019).
Traditionally, mushrooms are grown on a solid substrate under controlled temperature and humidity conditions. However, this method requires a long cultivation time and low yield, in addition to the possibility that the culture is contaminated by other microorganisms (El-Enshasy et al., 2010).
In addition to solid-state crops, fungi are grown in liquid media in bottles or bioreactors. This type of cultivation presents a fast, efficient and less expensive alternative form (Wu et al., 2003(Wu et al., , 2004Tang and Zhong, 2002), in which organisms can be obtained in limited physical spaces under conditions controlled and optimized for the production of biomass, exopolysaccharides (EPS) and endopolysaccharides (IPS), simultaneously (Cui et al., 2006;Confortin et al., 2008;Liu and Zhang, 2019;Guillén-Navarro et al., 1998), also showing the ease of separating the mycelium from the culture medium, obtaining products of uniform quality, in addition to the possibility of being cultivated all year round (Leonowicz et al., 1991;Rosado et al., 2002).
Many species are grown in flasks of liquid medium or even in bioreactors, including Lentinus edodes, Ganoderma, lucidum, Schizophyllum commune, Trametes versicolor, Inonotus obliquus and Flammulina velutipes. For the production of polysaccharides, an important step in submerged culture is the type of bioreactor used, since it directly influences the development of the mycelium. When it comes to flasks or stirred tank reactors, even optimized conditions can behave differently (Burns et al., 1994;Maziero et al., 1999;Rosado et al., 2003;Zhang et al., 2003).
Many studies are carried out with the aim of optimizing culture media for the production of fungal metabolites using statistical techniques (Fan et al., 2007;Joo et al., 2004;Assis et al., 2013). Studies, such as the one carried out using the fungus Grifola frondosa, were conducted in order to optimize the production of biomass and exopolysaccharide by response surface techniques (RSM) (Cui et al., 2006).
The bioprocesses used in the cultivation of fungi are carried out under optimal conditions such as temperature, pH, aeration, types of nutrients, pressure, type of reactor used, shear stress, the use of surfactants or defoamers and produced biomass. These processes involve complex and multiple biochemical reactions as well as a particular kinetics for each type of organism grown (Garcia-Ochoa and Gomez, 2009).
Thus, the objective of this work is to review some fungi of interest grown in a submerged manner for the production of polysaccharides, in flasks and bioreactors in agitated and pneumatic tanks.

CULTIVATION IN SUBMERGED FERMENTATION: A NEW PERSPECTIVE OF CULTIVATION
The submerged fermentation process is the process of cultivating microorganisms in liquid culture medium. This type of cultivation has several advantages over solid state fermentation. One of the main advantages is the possibility and ease of homogenization during the process. Homogenization is possible, as the medium is easily mixed, eliminating the differences that are generated as the microorganism grows. The liquid medium allows important factors, such as temperature and pH, to be easily measured and controlled. In addition, the consumption of nutrients is more complete, allowing maximum use of nutrients and maximum growth. These variables can be measured and controlled due to the greater precision of the samples taken during cultivation (Rossi et al., 2002;Elisashvili, 2012). Another advantage associated with submerged fermentation is the speed with which crops are grown, reducing production time and, consequently, the chances of contamination occurring. This is due to the ease with which nutrients are dispersed in the environment, maximizing the contact area between microorganisms and nutrients (Wu et al., 2003).
Cultivation can be carried out in static and agitated flasks, with forced aeration and also in bioreactors. The studies in small flasks aim to evaluate the best cultivation conditions so that the process can be scaled, reducing the risk of failure in the process at the industrial level. The cultivation of filamentous fungi by submerged fermentation is already a widespread practice, with a primary focus on obtaining products from primary or secondary metabolism secreted in the medium such as proteins, enzymes, acids, antibiotics, exopolysaccharides (EPS) intracellular polysaccharides (IPS) and other bioactive, medicinal and/or industrial compounds and also biomass for use as a food supplement (Saeki et al., 2011;Elisashvili, 2012;Vamanu, 2012;Singh et al., 2013).

Cultivation in flasks
The medium used for the cultivation of microorganisms must contain in a balanced way all the elements and nutrients for the synthesis of cellular substances and for the production of metabolites. Chemically defined culture media are used in laboratory research, but on an industrial scale, for economic reasons, complex substrates of variable composition are used, by-products of other industries. These substrates must be adapted for cultivation by means of extraction, supplementation and balancing the composition of the medium in order to increase the production yield of the selected microorganism, using screening experiments prior to cultivation in bioreactors or even aiming at the production of polysaccharides in the plants themselves (Crueger and Crueger, 1990).
Submerged fermentation techniques are widely developed to be applied to most medicinal mushrooms for the purpose of mycelium propagation (Smith et al., 2002). Factors such as nutrients (macronutrients and micronutrients), temperature, pH and agitation are factors that can influence the growth rate of macromycetes, the biomass yield and the production of metabolites (Sales-Campos et al., 2011b). Thus, it is essential to optimize the parameters in advance and to maintain them during the cultivation period (Singh et al., 2013).
Fungi of the genus Pleurotus, are widely studied and reports for cultivation methodologies are described for several strains (Table 1).
Pleurotus sajor-caju, grown in flasks, were studied for the optimization of the medium, with the different variables being a source of nitrogen (NH 4 ) 2 SO 4 , yeast extract and soy peptone. For this fungus, two concentrations of (NH 4 ) 2 SO 4 and yeast extract were tested in addition to the high concentration of soy peptone, which caused the EPS concentration to reach 0.60 g/L (Assis et al., 2013). The cultivation of this species served as a model for an optimization process based on the response surface methodology, taking into account three parameters, with a number of combinations of 2 3 . In 500 mL flasks, Erlenmeyer flasks containing 100 mL of medium were established that the highest concentrations of soy protein and yeast extract, combined with the lowest concentration of (NH 4 ) 2 SO 4 , were more efficient in biomass production (Confortin et al., 2008).
Also for Pleurotus djamour, whose optimization of the carbon source (glucose) and pH were tested as a function of biomass and EPS production, showed that the initial combination of 40 g/L of glucose and pH 3.0 decreased the consumption time of carbohydrate when compared with the other conditions (Borges et al., 2013). Maftoun et al. (2013) cultivated Pleurotus ostreatus of 250 and 50 mL of medium. 7 different means were tested in order to select the most suitable one. For the growth of biomass and generation of EPS, the medium containing glucose and corn steep liquor as a source of carbon and peptone and yeast extract as a source of nitrogen, favored the production of EPS over the other media. In this study, the kinetics of obtaining EPS showed a dependence on the carbon source, especially glucose. Another similar experiment conducted by Gern et al. (2008) studied the effect of different culture media on the production of biomass and polysaccharides in the same species. The media placed in 500 mL Erlenmeyer flasks containing 100 mL of the media showed different growth rates, affected by the interaction of the factors. Differently from what was previously reported, the growth rate is affected by the interaction between the factors, with organic nitrogen increasing the studied variables and the inorganic nitrogen having a negative effect, except in the global polysaccharide productivity.
In two other strains, P. ostreatus "Florida" and Pleurotus ostreatoroseus, Rosado et al. (2003) used a medium containing a high concentration of glucose as a carbon and peptone source, yeast extract, K 2 HPO 4 , MgSO 4 ⋅7H 2 O, (NH 4 ) 2 SO 4 in pH 6.0, in 500 mL flasks with 100 mL of medium. The results showed that for this medium, low concentrations of (NH 4 ) 2 SO 4 and high concentration of glucose favored both fungi with higher EPS production, 22.8 g/L for P. ostreatus "Florida" and 16.8 g/L for P. ostreatoroseus.
The cultivation of Agaricus brasiliensis was performed, aiming at the production of EPS, in which sucrose, yeast extract, pH at 6.1 and temperature of 30°C were the most effective in increasing EPS production (Fan et al., 2007). This type of behavior shows that different species have different cultivation behavior and, therefore, there is a need to adapt the culture medium.
Ganoderma lucidum, widely cultivated for its nutritional characteristics, is also among the most cultivated by the submerged environment. In flask cultivation, to develop effective and economical solid seeds suitable for use in submerged culture for the production of EPS, four supplements were examined as sources of nitrogen for the formulation of substrate with sawdust as the basal ingredient. The higher biomass and EPS yield obtained for the four solid test seeds on the liquid seed were attributed to the formation of a large number of growth points of hyphae in cultures, resulting from the dissociation of inoculated solid seeds into numerous tiny particles (Liu and Zhang, 2019).

Cultivation in biorreactors
Larger-scale crops use bioreactors, which are closed growing equipment, in which fermentation processes take place. The size of the equipment varies according to its use and the object of use, since they are adapted to the research and industrial scale. The term fermenters is used because primarily, bacteria and yeasts were cultivated and as a rule, almost all are anaerobic (Oosterhuis et al., 2013, Camelini et al., 2014. In the same way that flask culture media are optimized for the production of the metabolites of interest and for switching to production in a bioreactor, in the latter, there will also be a need for scaling in relation to thermodynamic aspects, fluid dynamics, transfers of gases and mass, need for aeration and agitation must be carried out (Schmidell, 2001).
Within the universe of cultivation in bioreactors, there is a series of equipment that can be used for the cultivation of fungi. The materials from which they are manufactured  Tang et al., (2008) also vary from stainless steel to glass, which can withstand variations in pressure and temperature, especially at the time of sterilization. Another advantage of bioreactors over flask culture lies in the control factor. The bioreactors are equipped with temperature, pH and oxygen availability control systems by coupling sensors, which are also sterilizable. Unlike flask cultivation, bioreactors allow sampling and thus the process can be followed over the culture period (Oosterhuis et al., 2013;Xu et al., 2013). As the culture is established, the rheological parameters in the medium are modified by the increase in mycelial mass and by the production of metabolites secreted into the medium. This modification also alters the transference and in the case of filamentous fungi, this viscosity determines its growth mode in the free form or forming pellets. The form of pellets is widely observed in the cultivation of fungi and this morphology helps to avoid variations in the density of the medium. At the same time, this formation interferes with the rheological properties that can cause difficulty in transferring oxygen to the interior of the pellets, generating anaerobic conditions (Prosser and Tough, 1991;Rossi et al., 2002).
In the industry, two types of bioreactors are widely used, according to the characteristics of the cultivation and the microorganism used: those with agitated tanks and tires (Chisti, 1989;Kavanagh 2005). Currently, what limits the full use of the potential of bioreactors and the scheduling of processes for some types of microorganisms is the limited knowledge of bioprocess engineering (Elisashvili, 2012).

The cultivation of fungi in bioreactors
The most mentioned type of bioreactor used in fungus cultivation is the stirred tank reactor (STR), which is equipped with a system of blades or turbines to agitate and aerate the culture medium (Table 1). This model has a sophisticated construction that includes axles, supports and blades, which influences the cost. The aseptic mode of this model also makes it a choice, since the set of the vessel, sensors and culture medium can be sterilized in a single step and remains aseptic for a long time. A problem in relation to sterilization lies in the many moving parts that increase the points of contamination and the complexity of the mechanical seals of the equipment (Nienow, 2014).
Even though they are the most common, agitated tank bioreactors are not always the most suitable. The agitation generated by the equipment's blades necessary for aeration and mass transfer can cause damage to cells due to high shear (Duobin et al., 2013).
Mushrooms like P. sajor-caju showed good growth in an optimized medium with glucose, soy protein, yeast extract and ammonium sulfate, producing a good amount of biomass quickly even without pH control in agitated tank bioreactors (Confortin et al., 2008). In other strains of fungi of the genus Pleurotus such as P. ostreatus grown in an 18 L volume bioreactor with an 8 L working volume, two experiments with pH 5.5 and uncontrolled were studied in terms of biomass production and EPS.
This study showed the importance of pH control in cultures, where at 5.5 there was an increase in EPS in the order of 0.445 g/g (Maftoun et al., 2013), as well as influencing glucose consumption rates (Kurosumi et al., 2006). Changes in the pH of the culture are usually caused by the presence of organic acids and other metabolites excreted in the medium, produced by the fungi themselves (Xiao et al., 2006;Fang et al., 2002;Kim et al., 2005a).
Other variables are also important in cultivation in bioreactors. This is demonstrated for P. ostreatus grown in a stirring bioreactor, where the cultivation conditions varied in terms of agitation (120-180 rev min -1 ); the initial pH (pH, 4.5-5.5) and keeping the temperature fixed at 25°C. This type of test, allowed the variation of the constitution of the medium, when it is desired to study the optimization, separating them from the physical factors (Petre et al., 2010).
Bioreactors from agitated tanks of different volumes are reported in experiments in the cultivation of fungi of the genus Agaricus in which the optimization of exopolysaccharide production conditions was determined by response surface, in which starch was established as the best source of carbon and yeast extract as a source of nitrogen (Hamedi et al., 2007). Likewise, Agaricus brasiliensis showed a similar behavior when grown in the presence of glucose (Fan et al., 2007).
For other species of fungi, such as the Ganoderma genus, the optimization processes established in STR also show the importance of controlling the inoculum density in the growth process, biomass production and intra and extra-cellular polysaccharides. In the study conducted by Fan and Zhong (2002), the higher inoculum density promoted a stimulus in production. This species when cultivated at an initial pH 6.5 showed lower biomass and intra and extra-cellular polysaccharides compared to pH 3.5 (Fang and Zhong, 2002).
A less used type is represented by pneumatic bioreactors, which do not have mechanical agitation, as is the case of bubble column and airlifts bioreactors (AR). Pneumatic bioreactors are bioreactors with high height, usually above 10 times the diameter, in contrast to the STR that have a height of two to three times their diameter. This is necessary because the bubbles need time to contact the liquid to transfer oxygen and absorb carbon dioxide. In STR, forced agitation keeps the bubbles in contact longer with the liquid, in addition to being continuously broken, increasing the transfer area (Merchuk, 2003).
The first is made in a single part, with an internal column that receives the injection of air, whereas the airlift bioreactor is composed of a column, which is divided into the region that contains the upward flow of liquid gas (the riser) and the region containing thedownward flow of liquid gas (the downcomer) and in this model, the difference in liquid density between these regions causes the fluid to circulate in the bioreactor by an air-lift action (Znad et al., 2004;Merchuk and Yunger, 1990;Chisti, 1989). The circulation of liquid within the bioreactor is essential for its proper performance, as it is related to the most important variables of the process, such as the turbulence that affects the size of the bubbles and the mixing time.
The cultivation of fungi in this type of bioreactor has an advantage when studying the shear force imposed on the pellets or in the cell. These biological processes, another advantage of airlift over the bubble column and agitated tank reactors is related to the shear force imposed by the turbulent field in cells or "pellets". Numerous studies have been conducted investigating the effects of shearing on microorganisms and cells in an effort to quantify the level of shear variation that microorganisms can tolerate (Merchuk and Yunger, 1990). Thus, in biological processes the shear force generated by the turbulence in the pellets generated during the growth of the fungi is less than in the agitation tanks, causing less damage.
Many fungi of medicinal and food interest were grown in bioreactors (Table 1). According to work by Xu et al., (2013), the cultivation of fungi of the genus Trametes demonstrated a similar behavior, regardless of the design of the bioreactor or the agitation rate, since the yield of products was similar. Higher EPS yield was achieved in STR than AR, and higher EPS production was related to advantageous morphological characteristics of the fungus defined by lower shear forces in AR. Recently, the cultivation of T. ochracea was optimized according to the different carbon sources, in a bioreactor of the stirred tank type, where sucrose proved to be the best sugar for the development of the fungus and the production of IPS and EPS (Bai et al., 2020). Other species, such as Phellinus vaninii grown in a stirred tank bioreactor, using lactose as a carbon source, proved to be efficient, producing EPS at a rate of 3.75 g/L (Li et al., 2019).
Pneumatic systems based on airfilt-type bioreactors showed important effects on Agaricus subrufascens crops when compared with agitated tank bioreactors, even when pH parameters were not controlled (Camelini et al., 2013). Likewise, different formulations of culture media for Pleurotus flabellatus, grown in an airlift bioreactor, showed the highest biomass productivity (0.180 g/L/day), with potato extract and a β-glucan yield of 7.70±1.11 g/100 g (Mohamad et al., 2015).
In some cases, as in Trametes trogii cultivation, the pneumatic airlift system did not promote biomass and EPS production in 6 days of cultivation, when compared with STR cultivated for 5 days. In this case, agitation can be beneficial to the growth of fungi and performance, improving the characteristics of mass transfer in relation to substrates, products by-products and oxygen (Xu et al., 2013).
One application of pneumatic bioreactors lies in the production of mycelium for use as an inoculant for growing mushrooms. Another great advantage is that due to the simplicity of these bioreactors, they can be manufactured at a lower cost and applying only basic techniques of turning and welding in stainless steel. The reduced diameters of these equipment also imply the need for less thick walls than the STR with consequent Campestrini and Salles-Campos 105 material savings. For these simple reasons, you can easily build a cultivation system that can be used by mushroom producers or companies that work with this type of inoculant.

CONCLUSION AND PERSPECTIVES
The experimental results described for submerged fermentation methods are important to direct and plan equipment configurations, improve the culture media according to the species studied or that have not yet been cultivated using this methodology. When aiming at greater productivity in terms of biomass and polysaccharide production, the definition of the type and quantity of the culture media and the inoculum are particularly important. The submerged culture systems described in this review include fungi that have a medicinal or edible use and are cultivated in that system. In the works described, the system proved to be quite efficient when compared with what is described in solid state fermentation, regarding the control of parameters. Certainly, the results described demonstrate that much can still be improved even if other elements are inserted in the system, such as extracts, and broths. The biotechnological processes that involve the cultivation of fungi have a potential yet to be explored. Tropical forests are a repository of fungi that can be explored for the metabolites of interest, especially polysaccharides. For these species, the described methods can be a guide to direct the cultivation of these species in order to take advantage of their maximum potential. The polysaccharides obtained from these species represent a promising and attractive field and can be used in future research projects, where they will be used against a wide range of diseases.

CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.