Evaluation Studies on Bioactivities and Medicinal Properties of Aqueous Extract of Pleurotus Ostreatus


Bachelor Thesis, 2020

54 Pages, Grade: A


Excerpt


1.0 Introduction

Mushrooms have been regarded as gourmet cuisine across the globe since antiquity for their unique taste and subtle flavor. They are considered as sources of important nutrients including dietary fiber, minerals, and vitamins, in particular, vitamin D (He, Perera Hemar, 2012). More than 2,000 species of mushrooms exist in nature, but only around 25 are widely accepted as food and few are commercially cultivated (Valverde et al., 2015). Recently, they have become increasingly attractive as functional foods due to their potential beneficial effects on human health. Hence, food industry is especially interested in both cultivated and wild edible mushrooms. The most extensively cultivated mushroom worldwide is Agaricus bisporus (J. E. Lange) Emil J. Imbach., followed by Lentinula edodes (Berk.) Pegler and Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. Mushrooms production is continuously increasing. The commercial production in 2012 hit 7,959,979 tonnes of mushrooms, with China accounting for most of the production (5,150,000 tonnes), while 1,869,091 tonnes were harvested in Europe (Grujic et al., 2015). Due to the increase in population and consumption, the world demand for mushrooms is projected to grow 15% a year (Kamarudzaman et al., 2015). The genus Pleurotus (Fries) Kummer (Basidiomycota, Agaricales) was defined by Paul Kummer in 1871. It is a cosmopolitan group of mushrooms with high nutritional value and therapeutic properties, besides a wide array of biotechnological and environmental applications (Knop, Yarden Hadar, 2015). Usually regarded as oyster mushrooms, these edible basidiomycetes are among the most popular worldwide, as much as they achieved the third position in the production of edible mushrooms, behind the species of the genus Agaricus and Lentinula (Fernandes et al., 2015). The most important Pleurotus species cultivated in large scale are P. ostreatus and P. pulmonarius ( Fr.) Quel. (Bazanella et al., 2013). P. pulmonarius has been often marketed by spawn manufacturers and cultivators under the incorrect name "Pleurotus sajor-caju". The real Pleurotus sajor-caju (Fr.) Singer is in fact a separate species of mushroom, which was returned to the genus Lentinus by Pegler (1975), and is correctly named Lentinus sajor-caju (Fr.) Fries (Buchanan, 1993).

Since the first report of hypotensive activity of the Pleurotus mushroom in a mouse model in 1986, many researchers have demonstrated their medicinal potentialities and classified them as 'mushroom nutraceuticals'; that were posteriorly added to the group of functional foods (Patel, Narain Singh, 2012). In the last decade, the number of patents and scientific articles regarding the genus Pleurotus has exponentially increased, with an increment of more than 2-fold in the total of scientific research/review articles in the last 5 years.

Extensive research on cultivation techniques (Gregori, Svagelj Pohleven, 2007; Carvalho, Sales-Campos Andrade, 2010), chemical composition and nutritional profile (Reis et al., 2012; Atri et al., 2013; Maftoun et al., 2015) has been done in the last ten years, along with a comprehensive account of the biotechnological capabilities of the genus Pleurotus including enzyme production (Inacio et al., 2015a; Knop, Yarden Hadar, Sephnowi 2015). More recently, the scientific reports referring to Pleurotus species have also focused on novel approaches for taxonomic issues (Menolli Jr., Breternitz Capelari, 2014; Maftoun et al. 2015), isolation and characterization of new functional compounds, besides the in depth-study of their medicinal properties (Khan Tania, 2012; Patel, Narain Singh, 2012; Yahaya, Rahman Abdulhah, 2014).

i. Biodiversity and Taxonomy

As of 2015 the Index Fungorum lists 202 species in the Pleurotus genus. It presents the most studied species in the past ten years, the main areas of publications regarding these mushrooms, as well as their geographical distribution worldwide.

Species delimitation within the Pleurotus genus has been a complex issue for decades (Menolli Jr., Breternitz Capelari, 2014). Years ago, Kitamoto et al. (2004) pointed out the main causes of the taxonomic controversy involving Pleurotus species: initial misidentification, absence of type specimens, instability of morphological characters due to environmental changes, limited reports on physiological characteristics, and the lack of mating compatibility studies. Fortunately, in recent years the adoption of biochemical and molecular approaches has brought some clarifications for species delimitation in the genus, mainly when combined with morphology and sexual compatibility (Menolli Jr., Breternitz Capelari, 2014). The currently adopted methodologies of identification include isozyme electrophoresis, sequence analysis of ribosomal DNA, internal transcribed spacer region (ITS), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) and mating compatibility testing (Maftoun et al., 2015). Recently, molecular approaches made it possible to confirm the taxonomic status of some important Pleurotus varieties such as P. eryngii, P. ferulae, and P. elaeoselini. It also enabled reclassifications and the identification of new species (Zervakis et al., 2014).

In 2009, sequencing of the P. ostreatus genome was completed. Thanks to this accomplishment, a broad picture of the ligninolytic peroxidase gene family has been obtained. Besides, molecular techniques have also enabled progresses such as targeted gene replacement, RNAi-based gene silencing, and overexpression of genes in P. ostreatus. By this way, the recent information of the genomics of P. ostreatus secondary metabolism will allow an upgrade in the production of these compounds (Knop, Yarden Hada, 2015).

As a more affordable option to expensive molecular techniques, the diffuse reflectance infrared Fourier transform (DRIFT) has also been used for studying the molecular composition and for identifying biological samples. It consists in a fast, reagent-free, noninvasive and highly specific approach (Movasaghi et al., 2008). Zervakis et al. (2012) used the DRIFT spectroscopy to exam 16 taxa of the genus Pleurotus, concluding that it was a fast, reliable, and cost-efficient methodology for the classification of pure cultures from closely related mushroom species.

ii. Nutritional Aspects

Pleurotus spp are famous for owning all three properties expected from a food — nutrition, taste, and physiological functions —being thus appreciated for both their sensory characteristics and outstanding nutritional profile. The terpenes, lactones, amino acids, and carbohydrates of their composition determine a range of precious aromas and flavor characteristics to their fruiting body and mycelial biomass (Smiderle et al., 2012). P. ostreatus, the most popular species of the genus, is commonly used in the preparation of soups, in stir-fry recipes with soy sauce or eaten stuffed. P. eryngii (DC.) Quel. , another species with gastronomic prestige, is considered ideal for vegetarian dishes (consumed fresh), being served sauteed, grilled, braised, stewed, or boiled (Reis et al., 2012).

Concerning the amount of crude protein, mushrooms are ranked below animal meats, but well above most other foods, including milk, which is an animal product. Not to mention the fact that mushroom proteins contain all nine essential amino acids required by humans, enabling their use as a substitute for meat diet (Kakon, Karim Sah, 2012). However, their nutritional supremacy in relation to the vegetarian diet is also virtue of their chitin rich cell wall that acts as a source of dietary fiber, along with their vitamin content (including thiamine, riboflavin, ascorbic acid, ergosterine, and niacin), considerable contents of micro and macro-elements as phosphorus and iron, carbohydrates and very low fat tenor (Maftoun et al., 2015).

Fresh fruiting bodies of Pleurotus spp contain 85-90% moisture (Khan Tania, 2012), and the moisture percentage depends on the mushroom species besides other parameters related to harvest, growth, culinary and storage conditions (Reis et al., 2012). Atri et al. (2012) investigated the nutritional composition of P. floridanus Singer , P. pulmonarius, P. sapidus Quel. , P. cystidiosus O. K. Mill . and P. sajor-caju (Fr.) Sing and reported, on dry weight basis, contents of carbohydrates of 85.86-88.38%, proteins 0.98-2.17%, crude fat 0.62-0.84%, crude fibers 2.76-3.12% and ash 1.03-2.20%. In turn, Khan Tania (2012) found some diverse values in their review study on the nutritional value of P. ostreatus, P. sajor-caju, P. florida (Mont.) Singer, P. cystidiosus, P. geesteranus Singer , P. eryngii, P. tuber-regium (Fr) singer and P. flabellatus (Berk. Br.) Sacco . They found, on dry weight basis, contents of carbohydrates ranging from 36 to 60%, proteins from 11 to 42% and lipids from 0.2 to 8%.

According to Khan Tania (2012) the carbohydrates in Pleurotus spp. are mainly in the form of polysaccharides or glycoproteins. The most abundant polysaccharides are chitin, a- and / -glucans, and other hemicelluloses (e.g., mannans, xylans, and galactans). The glucans present various types of glycosidic linkages, such as branched (1^3), (1 6)- / -glucans and linear (1 3)- a- glucans. The contents of these polysaccharides in the fruiting bodies range from 36 to 60 g/100 g dry weight. Total dietary fiber (mainly chitin) in Pleurotus mushrooms ranges from 10 to 31 g per 100 g dry weight, glucans being also components of soluble or insoluble dietary fibers.

Reis et al. (2012), in an inter-species comparative study on the most widely cultivated and appreciated mushrooms, found that P. ostreatus and P. eryngii had higher levels of monounsaturated fatty acids compared to Agaricus bisporus, Lentinula edodes and Flammulina velutipes (Curtis) Singer. Atri et al. (2013) reported that, among the fatty acids, the monounsaturated are present in a higher proportion (37.17-68.29%) than the saturated ones (26.07-47.77%) in Pleurotus spp. Maftoun et al. (2015), in their broad compilation data of the nutritional composition of Pleurotus mushrooms, reported that oleic acid (C18:1) was the major monounsaturated fatty acid while linoleic acid (C18:2n-6c) was the major polyunsaturated fatty acid in P. ostreatus. They also found that the most common monounsaturated fatty acid present in P. sajor caju, P. cystidiosus, P. pulmonarius, P. floridanus and P. sapidus was oleic acid. Among the saturated fatty acids (20.2%), the main contributers were palmitic acid (C16:0; 11.2%), followed by pentadecanoic acid (C15:0; 2.55%) and stearic acid (C18:0; 2.53%). Among the polyunsaturated fatty acids (69.1%), linoleic acid (68.1%) was the most common and abundant.

Atri et al (2012) detected three main sugars including sucrose (0.338-2.011 %), glucose (0.553-0.791%) and xylose (0.01%) when analyzing Pleurotus spp. They also found ascorbic acid content ranging from 0.46 to 0.49 mg/100 g, total phenolics ranging from 6.76 to 16.92 mg of gallic acid equivalents/100 g, P-carotene ranging from 0.134 to 0.221 |ig/100 g and lycopene from 0.055 to 0 .075 |ig/100 g.

For detailed information about essential amino acids, fatty acids, minerals, vitamins, soluble sugars and volatile compounds profiles of the most studied Pleurotus species, the recent review of Maftoun et al. (2015) might be consulted.

iii. Cultivation and Post-Harvest of Pleurotus spp.

Mushroom Production

The production of mushrooms with better flavor, appearance, texture, nutritional qualities, and medicinal properties at a sustainable cost constitutes a challenge for both industry and independent farmers, since many important operations are involved in this biotechnological process (Sanchez, 2004). This study summarizes the main cultivation techniques, postharvest treatments and industrial applications of Pleurotus spp. during the last decade.

Numerous articles have reported the viability of producing the Pleurotus spp. basidiome using a wide range of byproducts as substrates, e.g., elephant grass, coast-cross, cotton waste textile, rice straw, by-products of corn production, sawdust, husk of coffee, wheat straw, crushed sugarcane and stalks (banana tree, pea, peanut). Further, several kinds of materials were applied as supplementation, with high biological efficiency being obtained with wheat bran and rice bran (Carvalho, Sales-Campos Andrade, 2010). However, in the past years, a wide range of alternative, sustainable and green substrates were used for Pleurotus mushrooms production, such as casing material in a compost mixture (Mishra et al. 2013), handmade paper and cardboard industrial wastes (Kulshreshtha et al. 2013), agro-residues combined with biogas digester residues (Chanaky, Malayil Vijayalakshmi, 2015) and blank/printed paper (Fernandes et al., 2015).

Several studies on the role of the culture medium on mushrooms growth yield and nutritional quality have been done, but Ryu et al. (2014), in an innovative study, have investigated media combinations and components responsible for producing fruiting bodies with a long shelf life. They developed a cultivation medium for extending the shelf life and improving yield of P. eryngii mushrooms, increasing the viability of the export procedures. This medium contained 4.5% of crude protein and 15% of nitrogen free extracts.

The cultivation of Pleurotus spp. at high temperatures has been studied for a number of mushroom producers and scientists. Considering that some Pleurotus species are unable to develop mushrooms at temperatures of more than 28°C, the most important step in the cultivation of these mushrooms under high-temperature conditions (usual condition in tropical countries) is the cold stimulation of the mature mycelium (Chen, 2007). Yingyue et al. (2014) evaluated the effect of cold in the production of P. pulmonarius mushrooms. They found that time and the interaction of temperature versus time of the cold stimulation treatment were the two major factors influencing density of pinheads, yield per bag and number of mushrooms per bag. Meanwhile, temperature was the major factor influencing the yield per bag and stability. The best performance was recorded following a 12 h cold stimulation at 5°C, suggesting that an appropriate cold stimulation may enhance the performance of the primordial initiation and yield of P. pulmonarius cultivation during the summer season.

Dulay, Ray Hou (2015) investigated the optimal liquid culture conditions for producing P. cystidiosus with reference to the nutritional and physical growth factors, as well as with respect to lipid composition. They reported Sabouraud dextrose broth (SDB) as the most suitable culture medium, with maximum mycelial biomass favorably produced in SDB at pH 7.6 when incubated at 28°C. Agitation did not improve mycelial growth of mushrooms.

In the production of Pleurotus mushrooms, every ton of mushroom produced generates about five tons of dry spent residual material. This spent mushroom substrate (SMS) has been under-exploited in the past decades, sometimes being used for land filling and crop production only. However, as the correct disposal of SMS is one of the main environmental issues for the mushroom industry, new alternatives for the biotechnological application of this abundant by-product have been explored. Newly, the Pleurotus SMS was identified as a low-cost biosorbent for heavy metals removal, and as an effective degradation agent of organochlorine pesticides (Juarez et al., 2011; Kamarudzaman et al., 2015).

4.2.Submerged cultivation

Ten years ago about 80% to 85% of all edible-medicinal mushroom products were derived from the fruiting bodies and only 15% proceeded from mycelia extracts (Lindequist, Niedermeyer Julich, 2005). However, the process of producing fruiting bodies or basidiomata is effortful and time-consuming, as it demands large volumes of substrate, space, and qualified labor, factors that hinder research in the laboratory. Cultivations that are performed in the vegetative phase are much more interesting and functional for research considering that they can be kept in the laboratory, performed on a small and medium scale, and important parameters such as temperature, humidity, pH and aeration can be easily controlled (Inacio et al, 2015a). Thus, submerged cultivation is a promising and still under-explored alternative for the extraction of bioactive molecules in short time, which also allows the mycelia storage for a long period without genetic alterations, beneficiating the conservation of biodiversity (Zilly et al., 2011). However, for using the mycelial biomasses, it is necessary to prove that they are similar to fruiting bodies (Soares et al., 2013). Submerged fermentation is also proper for enzyme production and waste bioconversion. With respect to submerged liquid fermentation with Pleurotus spp., recent studies reported the use of potato dextrose broth, amino acids, liquor maiz, reducing sugars (mainly glucose and xylose), casein hydrolyzate, soybean cake, yeast extract and peptone as the main carbon and nitrogen sources. The culture conditions reported refer to temperatures of 25-30 °C and culture pH of 4-6, in addition to the use of static culture or agitation ranging from 100 to 160 rpm (Arango Nieto, 2013). Most recent publications aimed substrate optimization for maximal production of hydrolytic and oxidative ligninolytic extracellular enzymes.

As members of the white-rot fungi (WRF), Pleurotus spp. present the ability to grow on a variety of lignocellulosic biomass substrates and degrade both natural and anthropogenic aromatic compounds. This occurs by virtue of the presence of non­specific oxidative enzymatic systems, which consist mainly in laccases, manganese peroxidases (MnPs) and versatile peroxidases (VPs) (Hofrichter et al., 2010), besides the newly explored dye decolorizing peroxidases (DyPs) and heme-thiolate peroxidases (HTPs). A lot of information has been accumulated in the past decade concerning the biochemistry, structure and function of the Pleurotus ligninolytic peroxidases (Knop, Yarden Hadar, 2015).

Recently, the possibility of extending the liquid culture technology for the production of mycelia to the mushroom spawn industry has been studied. Generally the edible mushroom cultivation industry utilizes grain spawn for this purpose. However, it is already known that preparation of grain spawn requires a longer growth period and poses higher risk of contamination compared to liquid spawn (Confortin et al., 2008). Abdulla et al. (2013) investigated the alternative of producing liquid spawn of P. pulmonarius by submerged fermentation in a 2-L stirred-tank bioreactor under controlled conditions and assessed its ability to colonise rubber wood sawdust substrate for sporophore production. The ideal liquid spawn cultivation medium contained 20 g L-[1] of brown sugar, 4 g L-[1] of rice bran, 4 g L-[1] of malt extract, and 4 g L-[1] of yeast extract (BRMY) with an initial pH of 5.5 and was incubated at 28 ◦C with agitation speed of 250 rpm and oxygen partial pressure of 30-40%. The maximal dry biomass production of 11.72 ± 5.26 g L-[1] was observed after 3 days of fermentation. The authors concluded that liquid spawn has the ability to colonise sterile rubber wood-sawdust as fruiting substrates in a shortened time and to produce a higher yield of sporophores in comparison with the regularly used grain spawn.

4.3. Post Harvested Treatment

The commercial value of mushrooms falls due to quality loss during postharvest storage because the storage conditions are quite different from the growing conditions. This provokes changes in the physiological and molecular mechanisms that lead to deterioration (Li et al., 2013). In the past years, diverse post-harvest treatments have been investigated in an effort to discover new alternatives for extending the mushroom shelf life: cold storage (Dama et al., 2010), modified atmosphere packaging (MAP) (Guillaume et al., 2010), gamma and electron beam irradiation (Xiong et al., 2009; Fernandes et al., 2012), and coating (Jiang, Feng, Li, 2012) treatments.

Li et al. (2013) investigated the high carbon dioxide and low oxygen treatment on the sensory characteristics, MDA (malondialdehyde) content, O2- production rate, and enzyme activities of SOD (superoxide dismutase), POD (peroxidase), CAT (catalase), and CCO (cytochrome C oxidase) in P. eryngii. They reported that 2% O2 + 30% CO2 treatment could maintain sensory characteristics of the mushroom and significantly prolong its shelf life.

In turn, Zhang et al. (2015) investigated the activity and molecular mechanisms of serine proteinase (Spr) during storage of P. eryngii. The activity of Spr in 2% O2 + 30% CO2-treated mushrooms was notably lower than in the controls. The spatio-temporal expression of PeSpr1 in the ambient air and 2% O2 + 30% CO2 storages correlated with the Spr activity. Thus, the authors concluded that PeSpr1 plays an important role in post harvested P. eryngii, information that is valuable for post harvest investigation.

Newly, Huang, Lin Tsai (2015) studied the effect of ultraviolet-B (UV-B) light irradiation on the vitamin D2 content of edible fruiting bodies and mycelia of P. eryngii, P. citrinopileatus Singer, P. ferulae Lanzi., P. ostreatus and P. salmoneostramineus L. Vass., and their antioxidant properties. The vitamin D2 content of irradiated fruiting bodies significantly increased from 0-3.93 to 15.06-208.65 mg/g, Vitamin D2 content in irradiated mycelia of P. citrinopileatus, P. ostreatus and P. salmoneostramineus mushrooms increased from 0.28-5.93 to 66.03- 81.71 mg/g, respectively. The three irradiated mycelium polysaccharide contents decreased from 1.3% to 24.6%. Despite the fact that UV-B irradiation affects the content of ergothioneine, flavonoids and total phenols, the irradiated samples still contained a sufficient amount of these antioxidant components.

5. Isolated Compounds and Bioactivity

Demand is growing in the food industry for new functional ingredients or bioactive compounds from natural sources, as they are widely applied in the formulation of functional foods. This has promoted, especially in the past years, an increasing interest in extracting ingredients from foods such as mushrooms and in developing functional foods (Li Shah, 2015).

Numerous bioactive compounds, namely polysaccharides, peptides, glycoproteins, phenolics, lipids and hydrolytic and oxidative enzymes have been extracted from crude extracts, mycelia, and basidioma of Pleurotus spp. for investigation purposes. Two of the most interesting bioactive compounds produced by Pleurotus mushrooms are the immune stimulant polysaccharides and the natural statins. The latter are hypocholesterolemic and with higher activity than the synthetic ones due to their milder side effects (Inacio et al., 2015a). Patel, Naraian Singh (2012) published a comprehensive account on the medicinal properties of extracts of both fruiting bodies and mycelium of Pleurotus mushrooms. Their list includes antihypercholesterolemic, antihypertensive, antidiabetic, antiobesity, antiaging, antimicrobial, and antioxidant activities in addition to a hepatoprotective action. Also, different types of extracts from Pleurotus mushrooms have been reported as potential anticancer agents in several tumor cell lines, acting through distinct mechanisms. Clear clinical evidence of anticancer activities of Pleurotus mushrooms, however, is still not available (Khan Tania et al. 2012).

Table 3 presents a compilation of the last decade most important studies on Pleurotus spp. mushroom fractions and isolated/identified compounds, including high (e.g. polysaccharides, small peptides and proteins) and low (e.g. terpenes, fatty acid esters and polyphenols) molecular weight compounds, as well as their corresponding bioactivities.

5.1. High molecular weight compounds

Several polysaccharides have been isolated from the fruiting bodies, cultured mycelia and culture filtrates of various mushrooms (Ren, Pereira Hemar, 2012). Those polysaccharides showing antitumor activity have a great variety of chemical composition and structure, with different types of glycosidic linkages, such as (1,3)-, (1,6)- P-glucans and (1,3)- a-glucans (Figure 3A). In what refers to the polysaccharides from Pleurotus sp, Facchini et al. (2014) reported the efficacy of a polysaccharide fraction obtained from the mycelium of P. ostreatus with NH4-oxalate at 100 ◦C in inhibiting the development of Ehrlich Tumor (ET) and Sarcoma 180 (S-180). Also, Llaurado et al. (2015) examined the in vitro antimicrobial and the complement/macrophage stimulating effects of a hot water extract from the mycelium of Pleurotus sp. The extract activated the microbial autolytic system of both bacterial and yeast strains, acting also on innate immunity by triggering the complement system via the alternative pathway (and presumably the classical pathway of adaptive immunity) and by enhancing macrophage functions. The authors suggested the polysaccharide-rich extract as an accessible and innovative antimicrobial food ingredient. Recently, Li Shah (2015) added a polysaccharide extracted from P. eryngii (PEPS) to milk before its fermentation process. They found that the addition of PEPS had a considerable effect on bacterial growth, texture properties, and proteolytic and ACE inhibitory activities of fermented milk during refrigerated storage and proposed its use as a nutritional and functional additive.

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Title
Evaluation Studies on Bioactivities and Medicinal Properties of Aqueous Extract of Pleurotus Ostreatus
Course
Biotehcnology
Grade
A
Author
Year
2020
Pages
54
Catalog Number
V1156356
ISBN (eBook)
9783346583802
ISBN (Book)
9783346583819
Language
English
Keywords
Pleurotus ostreatus, Medicinal, Pharmacology, Evaluation
Quote paper
Dr Praveen Kumar Vemuri (Author), 2020, Evaluation Studies on Bioactivities and Medicinal Properties of Aqueous Extract of Pleurotus Ostreatus, Munich, GRIN Verlag, https://www.grin.com/document/1156356

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