Art 97 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XVII, Issue 1- 2018, pag. 97 - 102 STUDY ON TOTAL PHENOLIC CONTENT IN SOME ROMANIAN FOREST MUSHROOM SPECIES, BEFORE AND AFTER HEAT TREATMENT Marcel AVRAMIUC 1Faculty of Food Engineering, Stefan cel Mare University of Suceava, Romania avramiucm@fia.usv.ro *Corresponding author Received 17th November 2017, accepted 23th March 2018 Abstract: The purpose of this work was to study the phenolic compounds in ten forest mushroom species to see if and to what extent the thermal processing can modify the content of these compounds. The biological material consisted of ten forest mushroom species, colected from Suceava county area: Agaricus campestris, Morchella esculenta, Cantharellus cibarius, Armillaria mellea, Lactarius deliciosus, Boletus edulis, Pleurotus cornucopiae, Russula vesca, Sparassis crispa, Ramaria aurea. The experiment consisted in mushrooms thermal processing (boiling for 30 and 60 minutes, and roasting for 10 minutes at 160°C), followed by the analysis of Total Phenolic Content (TPC) which values were compared to the ones obtained from raw samples. As compared to raw material, the boiling process led to a significant increase of percent of TPC between 13.71% and 31.91% (after 30 min. of boiling) and between 12.33% and 61.36% (after 60 min. of boiling). As compared to raw material, the roasting of mushrooms for 10 minutes (at 160°C) led to significant increases of the phenolic compounds between 29.31% and 95.23% in all samples. High TPC values recorded by some species may be explained by the specific tissue structure of these species, which promotes a penetration and a more intense heat action on bound phenols. Keywords: phenolic content, mushroom, boiling, roasting 1. Introduction Widespread in plants and present in many species, phenolic compounds highlight various properties, from antioxidant, anti- microbial, anti-inflammatory, anti- allergenic to cardio-protective and vasodilatory ones [1- 6]. There is a relationship between the consumption of phenolic-rich food products and a low incidence of coronary heart disease, atherosclerosis, certain forms of cancer and stroke [7-10]. According to Huang et al., 1992 [11], due to their antioxidant properties, plant phenolic compounds have potential benefits for health. Plant species and varieties influence the content of phenols and of other metabolites [12- 16]. Lately, a number of scientific papers have revealed the presence of some mineral elements (Ca, Mg, P, Cd, Se), vitamins (B1, B2, B12, niacin, folates, C, D), flavonoids, lignans and phenolic acids in different species of cultivated mushrooms [17]. Also, have been reported some mushroom species used to prevent hypertension, hypercholesterolemia and even cancer [18, 19, cited by 20), due to the presence of chitin [21], and of beta glucans with β (1-3), β (1-4) and β (1-6) glycosidic linkages [22, 23 cited by 20]. The heat processing influences the concentration and quality of food nutrients http://www.fia.usv.ro/fiajournal mailto:avramiucm@fia.usv.ro Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 1 – 2018 Marcel AVRAMIUC, Study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, Food and Environment Safety, Volume XVII, Issue 1 – 2018, pag. 97 - 102 98 (carbohydrates, lipids, proteins), but there are less information, in this regard, on natural antioxidants and their activity. Cooking induces changes in physiological and chemical composition, influencing the concentration and bioavailability of bioactive compounds in food, the thermal treatments decreasing the total phenolics in squash, peas and leek [24]. Dewanto et al., 2002a [25] showed that in sweet corn, cooking led to an increase in the level of phenolic compounds. This paper studies the phenolic compounds in ten forest mushroom species in order to see if and to what extent the thermal processing can modify the content of these compounds. 2. Experimental 2.1. Research material and samples preparation The biological material was represented by ten forest mushroom species, colected from Suceava county area: Agaricus campestris, Morchella esculenta, Cantharellus cibarius, Armillaria mellea, Lactarius deliciosus, Boletus edulis, Pleurotus cornucopiae, Russula vesca, Sparassis crispa, Ramaria aurea. All these species are edible and tasteful, being consumed with pleasure by people. From each species they have prepared control (raw material) and thermal processed samples. 2.2. Procedure and research methods The experiment consisted in thermal processing (boiling and roasting), whose results were compared to those ones obtained from raw samples. For boiling 30 minutes and 60 minutes, they have taken (for each procedure) 50 g mushrooms of each sample, which have been placed in a stainless steel vessel of two liters capacity. The boiling has done in one liter of tap water in the pot covered, for 30, respectively 60 minutes (timed from the moment when the water began to boil). For roasting, 50 g of each sample were heated for 10 minutes at 160°C, in an electric oven. In order to determine Total Phenolic Content (TPC), first an extract for each sample (raw or thermal processed) was obtained, weighing 10 g mushrooms, which were ground and subjected to extraction with a mixture methanol and water (80/20), by stirring, centrifuging and recovering the supernatant. The estimation of Total Phenolic Contents in extract was carried out through a colorimetric assay, by measuring its reducing capacity with Folin- Ciocalteu reagent [26, 27]. TPC was expressed as mg Gallic Acid Equivalent/100 g matter (mg GAE/100g). For this purpose, a standard curve was generated, representing the absorbance values of gallic acid standard solutions in relation to their concentrations [28]. Because during boiling, some of the cellular compounds can pass into the boiling water, TPC was dosed from both mushroom samples and boiling water, calculating the total amount for each sample. 2.3. Statistical analysis The data of experiments, coming from four replicates of each determination, were statistically processed, using SAS Version 8.02 [29]. To analyze the significance of differences among samples, generalized linear model analysis was carried out. For multiple comparisons Duncan’s multiple range test was used (P<0.05). 3. Results and discussion In the Table 1 are rendered the values of Total Phenolic Content (TPC), in ten forest mushroom species. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 1 – 2018 Marcel AVRAMIUC, Study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, Food and Environment Safety, Volume XVII, Issue 1 – 2018, pag. 97 - 102 99 Table 1 Comparative TPC mean values (±SD) in raw and thermal processed forest mushrooms Test TPC (mg GAE/100 g) Samples (Species) Raw material Boiled (30 min.)* Boiled (60 min.)* Roasted (10 min.) Agaricus campestris 23.25±1.82 C 27.70±0.78 BC 29.67±1.23 B 35.82±0.72 AB Morchella esculenta 18.43±1.07 CD 22.06±1.25 C** 26.03±1.84 BC 29.56±0.88 B Cantharellus cibarius 16.55±0.96 D 20.12±1.47 C** 22.85±0.39 C 27.43±1.09 BC Armillaria mellea 28.74±1.35 B 34.34±0.93 AB 37.23±0.96 AB 40.27±1.94 A Lactarius deliciosus 19.88±0.78 CD 23.53±0.74 C 23.66±1.02 C 28.09±0.48 B Boletus edulis 22.07±0.54 C 27.30±1.43 BC** 27.49±0.45 BC** 30.05±2.01 B Pleurotus cornucopiae 22.38±1.36 C 24.89±2.01 C 25.14±1.58 BC 28.94±1.65 B Russula vesca 24.66±0.97 C 28.04±1.44 BC 29.27±0.91B 33.67±1.03 AB Sparassis crispa 17.39±1.04 CD 22.94±1.09 C 28.06±1.35 BC 33.95±0.57 AB Ramaria aurea 20.12±0.89 C 26.15±0.53 BC 31.43±0.48 B 38.44±1.22 A SD=standard deviation; *Sum of boiled sample and its boiling water; **Means with the same letters within a row or a column are not statistically diferent (P<0.05) As seen from the table, in the raw material the values of TPC ranged between 16.55±0.96 (Cantharellus cibarius) and 28.74±1.35 mg GAE/100 g (Armillaria mellea). Beetween these extreme values, there are some close ones (without significant differences among them) in five species (Agaricus campestris, Boletus edulis, Pleurotus cornucopiae, Russula vesca and Ramaria aurea) on one hand, and in other three species (Morchella esculenta, Lactarius deliciosus and Sparassis crispa), on the other hand. As as result of boiling process, the TPC values showed modifications depending on boiling duration and species. Thus, after 30 minutes the highest TPC value was registered by Armillaria mellea, followed by Agaricus campestris, Boletus edulis, Russula vesca and Ramaria aurea, with close values (P<0.05). The other species registered the least values and close between them. After 60 minutes the highest TPC value was registered by Armillaria mellea, followed by Agaricus campestris, Russula vesca and Ramaria aurea, with close values, and by Morchella esculenta, Boletus edulis, Pleurotus cornucopiae and Sparassis crispa. Cantharellus cibarius and Lactarius deliciosus had the least values (P<0.05). The roasting process caused the highest modifications of TPC values in all mushroom species. The greatest TPC values was registered by Armillaria mellea and Ramaria aurea, followed by Agaricus campestris, Russula vesca and Sparassis crispa (with close values). The other four species registered the least values. Comparing the TPC of mushrooms thermal processed with raw materials, one can observe a raising of TPC values after heating in all analyzed samples (P<0.05). The table 2 highlights the mushrooms TPC increase percents after boiling and roasting. Thus, compared to raw materials, boiling for 30 minutes led to a significant raising percent of TPC (except Pleurotus cornucopiae) beetween 13.71% (Russula vesca) and 31.91% (Sparassis crispa). Compared to raw materials, 60 minutes of boiling determined significant raising percent of TPC in all samples, the great values being registered in Sparassis crispa and Ramaria aurea, and the least one in Pleurotus cornucopiae. From 30 to 60 minutes of boiling were four species whose TPC values have not singnificantly modified (Cantharellus cibarius, Armillaria mellea, Lactarius deliciosus and Boletus edulis. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 1 – 2018 Marcel AVRAMIUC, Study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, Food and Environment Safety, Volume XVII, Issue 1 – 2018, pag. 97 - 102 100 Table 2 TPC mean values raising percent in mushroom heated samples, compared to raw material Samples (Species) TPC raising percents Boiled (30 min.)* Boiled (60 min.)* Roasted (10 min.) Agaricus campestris 19.14% 27.60% 54.00% Morchella esculenta 19.70% 41.24% 60.40% Cantharellus cibarius 21.57% 38.10% 65.74% Armillaria mellea 19.48% 29.54% 40.12% Lactarius deliciosus 18.36% 19.00% 41.30% Boletus edulis 23.70% 24.56% 36.16% Pleurotus cornucopiae 11.22% 12.33% 29.31% Russula vesca 13.71% 18.69% 36.54% Sparassis crispa 31.91% 61.36% 95.23% Ramaria aurea 29.97% 56.21% 91.05% *Sum of boiled sample and its boiling water Investigating the effect of thermal treatment on corn, Xu and Chang, 2009 [30], found changes of phenols caused by liberating of free forms from bound phenols. The results of the thermal treatment (boiling) applied to samples of the ten mushroom species, analysed in this work, are consistent with data reported by Choi et al, 2006 [31], which searching the effect of heat treatment (at 100°C and for 15 or 30 min.) on Shitake (Lentinus edodes) mushroom extracts, found the polyphenolic contents and antioxidant activities increased as heating temperature and time increased. Although the increase in TPC mean values on heating could be attributed to the release of free phenolics from combinations, the differences between the studied species could be explained by the difference in structure between these species. For example, some species have their body composed by thin and wavy formations (Sparassis crispa) or by a number of vertical and cylindrical branches (Ramaria aurea), which increase the contact surface of the internal tissues with the heat agent, favoring a more intense heat action on cellular compounds, including bound phenols. As to mushrooms roasting, the highest raising percent was in Sparassis crispa, followed by Ramaria aurea, and the least one in Pleurotus cornucopiae. The thermal treatment causes phytochemical degradation, oxidation, and Maillard reactions resulting in changes in antioxidant property [32]. By Lin et all [33], Maillard reaction products may protect phytochemicals from oxidation, and can maintain or even enhances the overall antioxidant properties of food products [34]. Heat treatment at 150°C for 40 min. liberated bound phenolics in citrus peels having as result a significant increasing of TPC after treatment [35]. Heating at 121°C for 30 min Shitake mushroom extracts, Choi et al, 2006 [31] found the free polyphenolic content increased by 1.9-fold compared to that in the extract from the raw sample. 4. Conclusions The thermal processing of mushrooms, belonging to ten species colected from Suceava county area, significantly influenced their Total Phenolic Content (TPC). Thus, as compared to raw material, the boiling process led to a significant increase in percent of TPC between 13.71% and 31.91% (after 30 min. of boiling) and Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 1 – 2018 Marcel AVRAMIUC, Study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, Food and Environment Safety, Volume XVII, Issue 1 – 2018, pag. 97 - 102 101 between 12.33% and 61.36% (after 60 min. of boiling). Compared to raw material, the roasting of mushrooms for 10 minutes (at 160°C) evidenced in all samples, significant increases of the phenolic compounds between 29.31% and 95.23%. It seems that heating at this temperature, for 10 minutes, led to an increase in the free phenols content by releasing bound phenols. High TPC values recorded by some species may be explained by the specific tissue structure of these species, which promotes a penetration and a more intense heat action on bound phenols. 5. References [1]. BENAVENTE-GARCIA O., CASTILLO J.,MARIN F.R., ORTUNO A., DEL RIO J.A. - Uses and properties of citrus flavonoids. Journal of Agricultural and Food Chemistry, 45, 4505-4515 (1997) [2]. SAMMAN S., LYONS WALL P.M., COOK N.C. - Flavonoids and coronary heart disease: Dietary perspectives. In C.A. Rice-Evans & L. Packer (Eds.), Flavonoids in health and disease (pp. 469-482), New York: Marcel Dekker (1998) [3]. MIDDLETON E., KANDASWAMI C., THEOHARIDES T.C. - The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacological Reviews, 52, 673-751 (2000) [4]. PUUPPONEN-PIMIÄ R., NOHYNEK L., MEIER C., KÄHKÖNEN M., HEINONEN M., HOPIA A., et al. -Antimicrobial properties of phenolic compounds fom berries. Journal of Applied Microbiology, 90, 494-507 (2001) [5]. MANACH C., WILLIAMSON G., MORAND C., SCALBERT A., RÉMÉSY C. - Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 81 (suppl), 230S – 242S (2005) [6]. BALASUNDRAM N., SUNDRAM K., SAMMAN S. - Phenolic compounds in plants and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food Chemistry 99, 191-199 (2006) [7]. HERTOG M.G.L., FESRENS E.J.M., HOLLMANN P.C.H., KATAN M.B., KROMBOUT D- Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen elderly study. Lancet 342, 1007-1011. (1993) [8]. DIAZ M.N., FREI B., VITA J.A, KEANEY J.F. - Antioxidants and atherosclerotic heart disease. New Engl. J. Med., 337, 408-416 (1997) [9]. ITO N., HIROSE M. - Antioxidants- carcinogenic and chemo-preventive properties. Adv. Cancer Res. 53, 247-302 (1989 [10]. NESS A.R., POWLES J.W. - Fruit and vegetables and cardiovascular disease: a review. Int. J. Epidemiol. 26, 1-13 (1997) [11]. HUANG M.T., HO C.T., LEE C.Y. - Phenolic compounds in food and their effects on helth II: antioxidants and cancer prevention. American Chemical Society Symposium Series 507. Washington, DC: American Chemical Society, pp 2-7 (1992) [12]. ABDEL-AAL E.-S.M., HUCL P. - A rapid method for quantifying total anthocyanins in blue aleurone and purple pericarp wheats. Cereal Chem., 76, 350–354 (1999) [13]. ZHAO Z.H., EGASHIRA Y., SANADA H. - Phenolic antioxidants richly contained in corn bran are slightly bioavailable in rats. J.Agric.Food Chem., 53, 5030–5035 (2005) [14]. PEDRESCHI R., LUIS C.-Z - Antimutagenic and antioxidant properties of phenolic fractions from Andean purple corn (Zea mays L.). J. Agric. Food Chem., 54, 4557–4567 (2006) [15]. LI W., WEI C.V., WHITE P.J., BETA T. - High-amylose corn exhibits better antioxidant activity than typical and waxy genotypes. J. Agric. Food Chem., 55, 291–298 (2007) [16]. LOPEZ-MARTINEZ L.X., OLIART-ROS R.M., VALERIO-ALFARO G., LEE C.-H., PARKIN K.L., GARCIA H.S. - Antioxidant activity, phenolic compounds and anthocyanins content of eighteen strains of Mexican maize. LWT- Food Sci. Technol. 42, 1187–1192 (2009) [17]. MATTILLA P., KÖNKÖ K., EUROLA M., PIHLAVA J.M., ASTOLA J., VAHTERISTO L., HIETANIEMI V., KUMPULAINEN J., VALTONEN M., PIIRONEN V. - Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. Agric Food Chem. 2001, May, 49(5): 2343-8 (2001) [18]. BOBEK P., GALBAVY S. - Hypocholesterolemic and antiatherogenic effect of oyster mushroom (Pleurotus ostreatus) in rabbit. Nahrung, 43(5), 339-342 (1999) [19]. BOBEK P., OZDYN L., KUNIAK L. - The effect of oyster mushroom (Pleurotus ostreatus), its ethanolic extract and extraction residues on cholesterol levels in serum lipoproteins and liver of rat. Nahrung, 39, 98-99 (1995) Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVII, Issue 1 – 2018 Marcel AVRAMIUC, Study on total phenolic content in some romanian forest mushroom species, before and after heat treatment, Food and Environment Safety, Volume XVII, Issue 1 – 2018, pag. 97 - 102 102 [20]. MANZI P., AGUZZI A., PIZZOFERRATO L. - Nutritional value of mushrooms widely consumed in Italy. Food Chemistry 73, 321-325 (2001) [21]. MANZI P., AGUZZI A., VIVANTI V., PACI M., PIZZOFERRATO L. - Mushrooms as a source of functional ingredients. Euro Food Chem X European Conference on: Functional foods. A new challenge for the food chemist. 22-24 September, Budapest, Hungary, Vol. I, 86-93 (1999) [22]. MANZI P., PIZZOFERRATO L - Beta glucans in edible mushrooms. Food Chemistry, 68, 315-318 (2000) [23]. MULLINS J.T. - Regulatory mechanism of β- glucan synthetases in bacteria, fungi and plants. Physiological Plantaurm, 78, 309-314 (1990) [24]. TURKMEN N., SARI F., VELIOGLU Y.S. - The effect of cooking methods on the total phenolics and antioxidant activity of selected green vegetables. Food Chem., 93, 713-718 (2005) [25]. DEWANTO V., WU X., LIU R.H. - Processed sweet corn has higher antioxidant activity. Journal of Agricultural and Food Chemistry, 50, 4959-4964 . (2002a) [26]. ADOM, K.K., LIU R.H- Antioxidant activity of grains. J. Agric. Food Chem. 50, 6182–6187 (2002) [27]. DEWANTO V., WU X., ADOM K.K., LIU R.H. - Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50, 3010-3014 (2002b) [28]. MOORE JEFFREY AND YU LIANGLI - Methods for antioxidant capacity estimation of wheat and wheat-based food products in: Wheat antioxidants, Edited by Liangli Yu, Published by John Wiley & Sons, Inc., Hoboken, New Jersey, 147-150 (2008) [29]. SAS INSTITUTE - SAS User’s Guide. Statistical Analysis System Institute, Cary, NC (2005) [30]. XU B. and CHANG S.K.C. - Total phenolic, phenolic acid, anthocyanin, flavan-3-ol, and flavonol profiles and antioxidant properties of pinto and black beans (Phaseolus vulgaris L.) as affected by thermal processing. Journal of Agricultural and Food Chemistry, 57, 4754–4764 (2009) [31]. CHOI, Y., LEE, S.M., CHUN, J., LEE H.B., LEE J. - Influence of heat treatment on the antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom. Food Chemistry, Vol. 99, Issue 2, 381-387 (2006) [32]. CHENG Z., SU L., MOORE J., ZHOU K., LUTHER M., YIN J.J., YU L. - J. Agric. Food Chem., 53, 2433–2440 (2006) [33]. LIN C.J., GUO G., MENNEL D.L. - Effects of postharvest treatments, food formulation, and processing conditions on wheat antioxidant properties in: Wheat Antioxidants, Edited by Liangli Yu, Published by John Wiley & Sons, Inc., Hoboken, New Jersey, 78-79 (2008) [34]. SLAVIN J.L., JACOBS D., MARQUART L. - Crit. Rev. Food Sci. Nutr., 40 (4), 309-327 (2000) [35]. JEONG S.-M., KIM S.-Y., KIM D.-R., JO S.- C., NAM K.C., AHN D.U., LEE S.-C. - Effect of heat treatment on the antioxidant activity of extracts from citrus peels. Journal of Agricultural and food Chemistry, 52, 3389-3393 (2004)