TITLE …………………… 398 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XIV, Issue 4- 2015, pag. 398 - 406 ESTABLISHING THE ANTIOXIDANT ACTIVITY BASED ON CHEMICAL COMPOSITION OF WILD EDIBLE MUSHROOMS * Ana LEAHU 1 , Cristina DAMIAN 1 , Mircea OROIAN 1 , Sorina ROPCIUC 1 1 Faculty of Food Engineering, Stefan cel Mare University of Suceava, Universitatiistr. 13,Suceava, Romania, analeahu@usv.ro *Corresponding Author ReceivedNovember10 th 2015, accepted December 15 th 2015 Abstract: The aim of this study is to determine the chemical compositions of fruiting bodies of widely used wild edible mushroom species (Agaricus albolutescens, Armillaria mellea, Russula virescens, Cantharellus cibarius and Boletus edulis), commonly consumed in the Bukovina region, from the Northeast of Romania. The content in water, crude protein, lipids, carbohydrates and ash, in fruiting bodies of mushroom samples was assessed. The antioxidant activities were performed on the 1,1´- diphenyl-2-picrylhydrazyl (DPPH) test. In addition to this, phenolic compounds were also analyzed. The carbohydrate contents were in the following order: Agaricus albolutescens>Boletus edulis>Armillaria mellea>Russula virescens and Cantharellus cibarius. The protein also varied and ranged from 10.12 to 15.15 % dry weight. The polyphenol content was the same in the two mushrooms, whereas the antiradical activity was higher in Russula virescens. The results suggested that consumption of Agaricus albolutescens, Armillaria mellea, Russula virescens,Cantharellus cibarius and Boletus edulis might be beneficial to the antioxidant protection system of the human body against oxidative damage and others complications. Keywords: mushrooms, carbohydrate, antioxidant, total phenolic content. 1. Introduction Mushrooms are an important dietary component for the modern consumer across the world, used for nutritional and medicinal purposes, due to the presence of metabolites with pharmacological potential. Wild mushrooms are becoming more and more important in our diet due to their nutritional characteristics [1-3]. Vegetables and mushrooms occupy an important position among functional food products due to their contents of many bioactive components that have a beneficial effect on human health and sense of wellbeing [4]. Dietary fibers are the compounds resistant to hydrolysis, which the human body cannot use, but which play a considerable role in the prevention of diseases. They are: cellulose, chitin, and glucans. The impact of fiber consumption is positive because it can influence the digestion rate and it was also associated with a reduced risk of type 2 diabetes. Recent clinical studies recommended mushrooms for inclusion in "functional foods" [5]. Functional foods are those foods enriched/modified and consumed as normal diet to provide health benefits [6]. Manzi et al., [7] evaluated the chemical composition of mushrooms, commercial samples, widely consumed in Italy (Boletus group, Agrocybe aegerita and Pleurotus eryngii). The results obtained in this study consider that the dried and re- hydrated Boletus samples showed higher levels of soluble and insoluble dietary fiber mailto:analeahu@usv.ro Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 399 than the other samples (Agrocybe aegerita and Pleurotus eryngii) [7]. Mushrooms have low energy content but remarkable protein content, from this point of view their consumption being advantageous, as compared to other vegetable products. Moreover, as compared to meat products or milk, fat content is negligible. These considerations justify the recommendation and preference to include mushrooms in a consumer’s usual diet. Wild mushrooms are rich sources of proteins and have low amounts of fat, making it an ideal snack material [1]. Dried Pleurotus ostreatus and Lentinula edodes mushrooms exhibited high contents of protein, total dietary fiber, K, Mg, Zn, Fe, Cu and low contents of fat, Na, Ca [8]. Carbohydrates constitute the most important component in terms of weight, the dry matter of mushrooms being the largest source of energy. Di Anibal et al. [9] tested the chemical composition and nutritional value of four edible mushroom varieties (Champignons, Portobellos, Girgolas and Shiitakes) commonly cultivated and consumed in Argentina. The study reveals that edible mushrooms are rich sources of carbohydrates and have low amounts of fats; also they are rich in potassium and phosphorus whereas they have a low content of sodium [9]. Mushrooms contain water-soluble compounds including reducing sugars and soluble polysaccharides. The dominant compound is mannitol, which in A. bisporus has the concentration of 3- 30g/100g dry matter [10]. Other simple sugars, such as: glucose, galactose, mannose and fructose have been also identified. Edible mushrooms have a discreet aroma, but individualized depending on the species, which contributes to their attractiveness to the consumer. Previous scientific reports have demonstrated the contribution of about 150 volatile compounds that outlines their aromatic profile[10]. According to Ashmore L. et al. [11], the characteristic volatile compounds found in fresh “raw” Agaricus bisporus mushroom sample are 4-methyl, 3-penten- 2-one, 1-octen-3-ol, 3-octanone, benzaldehyde, 3-octanol, and 2-octen-1-ol, and the dried sample had a mild aroma but upon rehydration, a stronger cooked aroma developed [11]. Among the sensory qualities, the most valued by the consumer are the visual ones: color, shape and size. Studies on the components which determine the color were focused on the carotenoids. The analysis of the extracts obtained from the seventeen fresh mushrooms, collected from different places in North-Eastern Portugal, allowed the identification of carotene only in C. cibarius [12]. Cultivated mushrooms (Agaricus bisporus/white, Agaricus bisporus/brown, Lentinus edodes, and Pleurotus ostreatus) were found to be good sources of vitamin B2, niacin, and folates, with contents varying in the ranges 1.8-5.1, 31-65, and 0.30-0.64 mg/100 g dry weight (dw), respectively [13]. It is considered that mushrooms are the only products that contain whole complex B. Mushrooms contain high levels of pro-vitamin D2 (ergosterol) [14]. Edible mushrooms also provide a nutritionally significant content of minerals, may constitute a good source of iron, zinc and copper. Various studies have been carried out on the minerals composition of the European edible wild mushrooms from several countries like Romania [15-16]; Greece [17]; Turkey [18]; Croatia [2]; Portugal [1]; Finland [13]. Mushrooms are known for their ability to accumulate heavy metals and radioactive elements, due to the texture of very good conducting properties in comparison to plants. That is why Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 400 mushrooms could be used as potential bio sorbents for heavy metals from aqueous solutions [19]. Mushrooms are considered bio indicators of environmental pollution. As mentioned previously, the antioxidant activity of mushrooms is positively correlated firstly to the amount of phenolic compounds, followed by the flavonoids, tocopherols, ascorbic acid and carotenoids [20]. Many studies have confirmed some large parts of traditional knowledge regarding the medicinal effects of mushrooms due to their antifungal, antibacterial, antioxidant and antiviral properties, besides being used as functional foods [21]. The main focus of this work was to determine the nutritional value and chemical composition, including the antioxidant activity in five types of wild mushrooms, Agaricus albolutescens, Armillaria mellea, Russula virescens, Cantharellus cibarius and Boletus edulis. 2. Materials and methods 2.1. Samples and storage conditions Mushrooms grow abundantly in the wild during the rainy season in every part of Dragomirna Forrest, Suceava County. The fruits of five mushrooms (Agaricus albolutescens, Armillaria mellea, Russula virescens, Cantharellus cibarius and Boletus edulis) species were harvested during early mature fruiting stage, in Dragomirna Forrest, Suceava County (Northeast of Romania) in autumn 2014. Sample preparation: the mushroom sample (600 g each) was cleaned, sliced and dried on paper towel prior to weighing. Mushrooms were air-dried in the oven at 40 0 C before making the analysis. 2.2. Chemical analysis The determination of moisture (according SR. ISO 1026:2008) in mushrooms samples was made by the drying process in a drying chamber at the temperature of 105 °C, until it reached a constant weight. Total ash composition was obtained by calcination of 5g of sample at 600 °C for 240 min (according SR ISO 763: 2008). The protein content (Nx4.38)of the samples was analyzed by the Kjeldahl method. The oil content was determined in a Soxhlet apparatus, extracting the fats from 10 g of mushrooms sample with petroleum ether, followed by extract evaporation to dryness and gravimetric determination. The carbohydrate content was estimated by difference of the other components using the following formula [8]: carbohydrate content = 100% - (% moisture + % protein + % fat + % ash) (1) (1). Energy was expressed as kilocalories/100g, using the factors mentioned in the Romanian Legislation: Energy (kcal) = 4.1x (g protein + g carbohydrate) + 9.3x (g lipid) (2) (2) The content of total polyphenolic compounds in dried mushroom methanol extracts diluted 1/10 was determined by Folin-Ciocalteu method. For the preparation of the calibration curve 0.5 mL aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 μM/mL aqueous gallic acid solution were mixed with 10.0 mL Folin-Ciocalteu reagent (diluted ten-fold) and 1.0 mL sodium carbonate (20.0%) and the volume made up to 10.0 mL with H2O. The absorbance was read after 2 h at 25°C, at 760 nm. All determinations were performed in triplicate. Total phenols were determined as gallic acid equivalents on a dry weight (mg GAE g -1 D.W.). 2,2-Di (4-tert-octylphenyl)-1- pycrilhydrazyl (DPPH) scavenging capacity assay The method used to determine the antioxidant activity of mushrooms is based Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 401 on scavenging 2,2-Di (4-tert-octylphenyl)- 1-picrylhydrazyl (DPPH) radicals. The mushroom samples aliquot (0.5 mL) was added to freshly prepared DPPH reagent. After incubating for 5 min, the absorbance of the resulting solutions was measured at 517 nm using a spectrophotometer T70 UV-VIS PG Instruments Ltd. The control was conducted in the same manner, except that the distilled water was used instead of sample. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The capability to scavenge the DPPH radical was calculated using the following equation: IC50 (%) = [(A0-A1/A0) x 100] (3) where A0 was the absorbance of the control reaction and A1 the absorbance in the presence of the sample of mushroom species extract [22]. 2.3. Statistical analysis Three replications were used to obtain average values and standard deviations for proximate biochemical properties. Multiple linear regression (PLS), a method based on the criterion of covariance has been used, to analyze the relationship between the explanatory variables (chemical characteristics of mushrooms) or independent variables (different species of mushrooms) and between explanatory variables and independent. 3. Results and discussion Figure 1 presents the contents of macro components in five types of wild edible mushrooms: Agaricus albolutescens, Armillari amellea, Russula virescens, Cantharellus cibarius and Boletus edulis. Fig. 1. The contents of macrocomponents, g/100 g dry matter of dried Agaricus albolutescens, Armillaria mellea, Russula virescens, Cantharellus cibarius and Boletus edulis Mushrooms varied in their content of raw protein more than the other macroelements determined. All dried mushrooms were found to be good sources of proteins, with Moisture % Crude protein % Fat % Ash % 0 2 4 6 8 10 12 14 16 B. edulis C. cibarius R. virescens A. mellea A. albolutescens Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 402 contents varying in the ranges of 10.12– 15.15 g/100 g dry weight (dw), while the fat content was very low (0.93 –1.48 g/100 g dw). Minimum and maximum ash levels in the present study were 1.75 mg/100 g d.w. and 2.47 mg/100 g d.w. for Russula virescens and Boletus edulis, respectively. The distribution of minerals in the fruiting bodies of the mushrooms is not uniform, caps concentrating, generally, the largest amount. Barros et al. [1] reported similar ash content (0.81 ± 0.03 g/100 g), but higher moisture (93.05 ± 0.51g) and protein (2.12 ± 0.08 g/100 g of weight matter) levels in samples from Northeast of Portugal, while Ouzouni et al. [17] described similar moisture (87.6 g/100 g) and ash (6.25 ± 0.02 g/100 g) contents, and higher protein levels (27.17 ± 0.15 g/100 g weight matter) in Boletus edulis samples from forests in the West Macedonia and Epirus. Carbohydrates, calculated by difference, were also an abundant macronutrient and ranged from 70.85 g/100 g in Boletus edulis and 74.94 g/100 g in Agaricus albolutescens. The relative homogeneity in wild mushrooms suggests a uniform composition of the soil, which is the main reservoir of nutrients. The energy value differed significantly (p < 0.001) in samples tested dried products, while the higher energy value was found 367.64 Kcal/100g for dried Russula virescens (Figure 2). Fig. 2. The contents of energy value, Kcal/100 g dry matter, of dried Agaricus albolutescens, Armillaria mellea, Russula virescens, Cantharellus cibarius and Boletus edulis Generally talking, mushrooms are characterized by low energy value. The content of total phenolic compounds was determined in methanol extract of mushrooms samples and characterized using gallic acid equivalents (GAE) per 1 g of mushroom dry matter. Total polyphenol content reveals a significant difference between species, Boletus edulis proving to be the richest in such compounds and was found to be 18.38 mg GAE/g weight matter (figure 3). Jeena et al. [23] determined total poliphenol content of three mushrooms types Pleurotus (P. sajorcaju, P. ostreatus et P.sapidus) and the study revealed that P. 352 354 356 358 360 362 364 366 368 370 A. albolutescens A. mellea R. virescens C. cibarius B. edulis Energy value Kcal/100g Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 403 sajorcaju 1.53 mg/g>P. ostreatus 1.32 mg/g> P. sapidus 1.10 mg/g [23]. The results obtained by Gençcelep and Hüseyin [24] indicated that total phenolics in the methanolic extracts were the highest in Boletus edulis. Reis et al. [25] analyzed the total polyphenol content of different species of mushrooms, thus, in Agaricus bisporus (brown) was 37.33 mg GAE/g extract, while in Pleurotus eryngiiit has a lower value: 7.14 mg GAE/g extract. Fig. 3.Total phenolicsmg GAE/ g of analyzed samples Concerning mushroom sample extracts, the Russula virescens extract was the one that had the strongest antiradical activity (IC50 of 15.154), followed by Armillaria mellea and Cantharellus cibarius extracts, with very similar activities (IC50 of 20.12 and 21.55, respectively). Alam N. et al. [26] reported that the antioxidant activities of the fruiting bodies of Pleurotus ferulae was lower than the synthetic antioxidant, BHT and TOC, respectively at 0.5 mg/ml and at 0.5–20.0 mg/ml concentration. Their antioxidant activities of the acetonic, methanolic, and hot water extracts of P. ferulae ranged from 52.71% to 95.63%, 69.51% to 96.52%, and 34.88% to 93.22%, respectively [26]. Fig. 4. Free Radical Scavenging Activities % of analyzed samples 0 2 4 6 8 10 12 14 16 18 20 A. albolutescens A. mellea R. virescens C. cibarius B. edulis T o ta l p h e n o li cs ( m g G A E /g ) 0 5 10 15 20 25 30 35 A. albolutescens A. mellea R. virescens C. cibarius B. edulis IC 5 0 ( % ) Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 404 In a research conducted by Ferreira, I. C., et al.[27] it was reported that the extracts obtained from the mushroom cap proved to be a better source of antioxidants than extracts from mushroom stem [27]. Similar studies reported that the antioxidant activity of ethanolic extracts from mushrooms is due to phenolic compounds content [28]. Statistical analysis using the PLS (Partial Least Squares Regression) application dependent variables (content in carbohydrate, fat content and the quantity of minerals-ash %) for mushroom samples analyzed indicates that the energy is determined by lipid content (Fig. 5). In B. edulis samples it can be observed the high amount of protein and minerals content. The analyses conducted on Armillaria mellea and Russula virescens revealed a high energetic value and fats concentration, respectively. Samples with high humidity belong to the species of fungus Cantharellus cibarius. The high carbohydrate content occurs in Agaricus albolutescens mushrooms. Fig. 5.Correlation between carbohydrates % and the energy value Kcal/100g for analyzed samples Table 1. Pearson correlative bond of chemicals characteristics of mushroom samples Variables Energy, Kcal/100g Carbohydrate, % Moisture % Protein, % Fat % Ash % Energy value, Kcal/100g 1.000 -0.140 -0.958 0.663 0.830 -0.176 Carb. content (CHO) % -0.140 1.000 0.397 -0.933 -0.064 -0.687 Moisture % -0.958 0.397 1.000 0.472 -0.798 -0.073 Protein, % 0.663 -0.933 0.472 1.000 0.265 0.469 Fat % 0.830 -0.064 -0.798 0.265 1.000 0.148 Ash % -0.176 -0.687 -0.073 0.469 0.148 1.000 Energy value, Kcal/100g Carbohydrate content (CHO) % Moisture % Crude protein, % Fat % Ash % A. albolutescens A. mellea R. virescens C. cibarius B. edulis -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 -1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1 t2 t1 Correlations on axes t1 and t2 Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XIV, Issue 4 – 2015 Ana LEAHU, Cristina DAMIAN, Mircea OROIAN, Sorina ROPCIUC, Establishing the antioxidant activity based on chemical composition of wild edible mushrooms, Food and Environment Safety, Volume XIV, Issue 4 – 2015, pag.398 – 406 405 The correlation coefficients (Table 1) between chemicals’ characteristics analysis reveal that the protein gives to fungus a medium energy values (r=0.663) and once the humidity increases, the energy values decrease significantly.The protein content representstheonlysource of minerals in mushrooms. Pearson coefficient shows a significant correlation between the percentage of ash and protein content. 4. Conclusion Nutritional content represents the rational motivation for mushroom consumption. Due to their low fat content, the risk of cholesterol and triglyceride accumulation is eliminated. The main nutritional characteristics are presented in the current study (high water and protein content, but low fat content) along with the content of macro- and micro-nutrients. The fruiting bodies of widely used wild edible mushroom have all attracted considerable attention as sources of phenolic compounds. All of these recommend them as food with health benefits. The results of the research suggest that dried wild mushrooms can be used as additives in food products. It was also noted a high degree of biodiversity resulting from the specificities of the species, substrate composition, and harvesting conditions. 5. References [1]. BARROS, L., BAPTISTA, P., CORREIA, D. 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