International Journal of Food Science and Technology 2020 1 Original article Pleurotus ostreatus and Agaricus subrufescens: investigation of chemical composition and antioxidant properties of these mushrooms cultivated with different handmade and commercial supplements Anna Beatriz Sabino Ferrari,1 George Azevedo de Oliveira,1 Helena Mannochio Russo,2 Luiza de Carvalho Bertozo,4 Vanderlan da Silva Bolzani,2 Diego Cunha Zied,3 Valdecir Farias Ximenes4 & Maria Luiza Zeraik1* 1 Laboratory of Phytochemistry and Biomolecules (LabFitoBio), Department of Chemistry, State University of Londrina (UEL), Rodovia Celso Garcia Cid, Pr 445 Km 380, Londrina PR, 86051-990, Brazil 2 NuBBE, Department of Organic Chemistry, Institute of Chemistry, Sao˜ Paulo State University (UNESP), Av. Prof. Francisco Degni, 55, Araraquara SP, 14800-060, Brazil 3 Faculty of Agrarian and Technological Sciences, Sao˜ Paulo State University (UNESP), Rod. Cmte Joao˜ Ribeiro de Barros, km 651, Dracena SP, 17900-000, Brazil 4 Departament of Chemistry, Faculty of Sciences, Sao˜ Paulo State University (UNESP), Av Eng° Luiz Edmundo Carrijo Coube, S/N, Bauru SP, 17033-360, Brazil (Received 2 April 2020; Accepted in revised form 27 May 2020) Abstract In this study, the chemical composition, total phenolic content and antioxidant activity (DPPH, ORAC and FRAP assays) of A. subrufescens and P. ostreatus, cultivated with handmade and commercials sup- plements, were compared. Additionally, the compounds ergosterol, saccharopine, and hexitol were identi- fied in A. subrufescens by HPLC-MS/MS. The antioxidant compound p-coumaric acid and dihexoses was found in both mushroom species. A. subrufescens presented higher total phenolic content (73.8 Æ 0.6 mg GAE 100 g−1) and antioxidant activity than P. ostreatus (16.6 Æ 0.5 mg GAE 100 g−1). The handmade supplement based on the waste of noble grains presented statistically similar phenolic content to the mushrooms cultivated with commercial ones Spawn Mate II SE (86.1 Æ 1.4 and 92.9 Æ 0.3 mg GAE 100 g−1, respectively). Therefore, the results support the use of handmade supplements based on agro- wastes as a viable alternative to the use of high-cost commercial ones. Keywords Agaricus subrufescens, functional food, HPLC-MS, mass spectrometry, MS, Pleurotus ostreatus, supplementation. considered powerful antioxidant sources. These sec- Introduction ondary metabolites are mainly phenolic compounds, Edible mushrooms have been reported as a source of tocopherols, carotenoids and phenolic acids (Sanchez,´ bioactive compounds with important pharmacological 2017). However, it is necessary to study different spe- properties and are considered as functional foods with cies of mushroom, since the kinds of bioactive com- high amounts of minerals, proteins and fibres (Tep- pounds produced might differ, also depending on the songkroh et al., 2020). Due to these properties, the sci- cultivation conditions of each species (Sanchez,´ 2010). entific community has studied the extracts from many The mushrooms from the Pleurotus genus are consid- edible mushrooms species, such as Agaricus bisporus, ered valuable for cultivating and feeding. Comparing to Hericium erinaceus, Lentinus edodes, Pleurotus ostrea- other edible fungi, they are mainly cultivated for nutri- tus, and various therapeutic activities have been tional and therapeutic values (Grabarczyk et al., 2019). reported, as anticarcinogenic, antioxidant, anti-inflam- The species Pleurotus ostreatus is also known as ‘oyster matory and immuno-suppressor (He et al., 2020). mushroom’ or ‘shimeji’ and has been intensely studied and These effects can be explained by the presence of a cultivated in many different parts of the world (Ruhl¨ et al., variety of secondary metabolites that can be 2008). Their fruiting bodies present numerous therapeutic properties, like anti-inflammatory, antioxidant, antibacte- *Correspondent: E-mail: [email protected] rial and immunomodulatory activity (Baeva et al., 2019). doi:10.1111/ijfs.14660 © 2020 Institute of Food Science and Technology The peer review history for this article is available at https://publons.com/publon/10.1111/ijfs.14660 2 Evaluation of supplemented mushrooms A. B. SABINO FERRARI et al. Mushrooms from the Agaricus genus are largely A. bisporus), besides four different handmade supple- studied, and due to their chemical composition, contri- ments based on agro-industrial wastes: peanut (20% bution to the diet and beneficial health effects, they grain or nut, 75% hulls of peanut waste and 0.5% of have been considered functional food (Yahia et al., calcium carbonate), acerola (obtained after the extrac- 2017). The genus comprises approximately 400 species tion of the acerola juice, with the addition of 0.5% of and can be found in different countries (Karunarathna calcium carbonate), noble grains (30% of cotton, corn, et al., 2016), particularly in the tropics (Callac & wheat, 9.5% bran of soybean, with the addition of Chen, 2018). The Agaricus subrufescens species, known 0.5% of calcium carbonate) and a mix of these three as ‘sun mushroom’, is a Brazilian mushroom that has handmade supplements (33.3% each). Additionally, a demonstrated antitumor activity, immunological substrate without supplementation was used as a con- enhancement, and can be used in AIDS, diabetes, trol. After harvest, each mushroom was dried at 40°C hypotension and hepatitis treatments (Al-Dbass et al., for 24 h and grounded. 2012; Carvajal et al., 2012; Wisitrassameewong et al., 2012). Extraction procedure These mushroom species have been cultivated using different types of substrates, and several studies The extracts were obtained by adding 1 mL of etha- revealed improvements of their chemical, functional nol:water (80:20, v/v) to 5 mg of dried mushroom and sensorial characteristics (Zied et al., 2010), espe- samples, orbital shaking at 500 rpm for 3 h at 60°C cially because these fungi obtain their necessary nutri- and centrifuging at 800 g for 10 min. These extracts ents from the substrates, such as carbon, nitrogen and were subjected to the antioxidant and total phenolic vitamins (Maseko et al., 2013). According to Zied content assay. In order to tentatively identify the et al. (2018), supplements based on agricultural wastes compounds, the dried mushrooms (a total of 1.5 g are a viable alternative to the use of commercial sup- for each species, resulted from a mix of all the sam- plements, especially for A. subrufescens cultivation. ples) were sonicated sequentially with 30 mL of etha- Additionally, the use of supplements based on agro- nol:water (80:20), three times for 30 min, at room wastes represents an effective and economical method temperature. The supernatants were concentrated and contribute to decreasing environmental pollution under reduced pressure and subjected to chromato- (Zied et al., 2018). graphic analysis. Therefore, the aim of this study was to compare the chemical composition and antioxidant activity of the Determination of total phenolic contents edible mushrooms Pleurotus ostreatus and Agaricus subrufescens cultivated with different handmade and The total phenolic content was determined by the commercial supplements. The correlations between Folin–Ciocalteu method described by Swain & Hills antioxidant activities and total phenolic content were (1959), with some modifications. An aliquot of 100 µL also evaluated, as well as the identification of the com- of the supernatant separated from the extraction of pounds responsible for the antioxidant properties by the samples was added to 1500 µL of Milli-Q water HPLC-MS/MS analyses. and 100 µL of Folin and Ciocalteu’s phenol reagents. After 5 min, 300 µLofNa2CO3 was added, and these solution mixtures were incubated at 500 rpm with Materials and methods orbital shaker for 30 min at 37°C. A calibration curve was constructed using various concentrations of gallic Mushroom species acid concentrations as standard, and the results were The mushrooms P. ostreatus and A. subrufescens were expressed as gallic acid equivalents (GAE) per 100 g cultivated in the experimental area of Sao˜ Paulo State of dried extract. University (UNESP), campus of Dracena, Sao˜ Paulo, Brazil (21° 29’ longitude South and 51° 32’ latitude Antioxidant properties West; 396 m altitude), in July 2016. The authorisation for studying these mushrooms was conceded by the DPPH˙ scavenging activity National System for Management of Genetic Heritage The DPPH• assay is based on scavenging of the radi- and Associated Traditional Knowledge (SISGEN), n˚ cal 1,1-diphenyl-2-picrylhydrazyl (DPPH•) by the A934000. mushroom antioxidant compounds present in the The mushrooms were cultivated and harvested extracts, and the method performed as described by according to Zied et al. (2018), with six different sup- Brand-Williams et al. (1995), with some modifications. plements, two commercials: Spawn Mate II SE® (rec- An aliquot of 333.3 µL of the supernatant, separated ommended for the production of P. ostreatus) and Pro from the extraction of the samples, was added to Mycel Gold® (recommended to the production of 12.5 µL DPPH• solution (8 mmol L−1) with 654.2 µL International Journal of Food Science and Technology 2020 © 2020 Institute of Food Science and Technology Evaluation of supplemented mushrooms A. B. SABINO FERRARI et al. 3 of ethanol:water (80:20). These solution mixtures were ionisation modes, separately. The HPLC comprised a kept in the dark for 1 h, and the absorbance was mea- DGU 20-A3R online degassing unit, LC-20AD pump, sured at 517 nm. Gallic acid was used as