Research regarding the influence of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. on chemical composition of red wines

Romanian Biotechnological Letters Vol. 21, No. 2, 2016 Copyright 2016 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER Research regarding the influence of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. on chemical composition of red wines Received for publication, May 05, 2014 Accepted, August 27, 2014 LIVIU GIURGIULESCU 1*, IOANNIS VAGELAS 2, NIKOLAOS GOUGOULIAS 2 1 Technical University of Cluj Napoca, North University of Baia Mare Centrum, Department of Chemistry Biology, Victor Babes, 62/A, 430083, Baia Mare, România; 2 Technological Educational Institute of Thessaly, Department of Plant Production, 41110 Larissa, Greece; * Correspondence address: Giurgiulescu Liviu, Victor Babes, 62/A, 430083, Baia Mare, România, Tel.0740310674; Email: giurgiulescu@gmail.com Abstract The objective of this study was to determine the influence of Penicillium chrysogenum, Penicillium expansum, Phanerochaete spp. on the chemical composition of red wines produced in Romania. These three species of fungi produce metabolic compounds which modify the acidity, polyphenols structure, alcohol degree and total solids of red wines. The present study provides useful data for understanding the metabolism of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. in different types of red wines and can be used as effective tools in assessing the risk of fungal spoilage and predicting the evolution of wine composition. Keywords: molds, red wine, contamination, chemical composition, volatile acidity, total acidity 1. Introduction The growth of filamentous fungi in foods is an important quality problem and may lead to significant economic losses in the food industry [1]. Fungi can grow in moist environments of dairy plants, and develop themselves on ceilings, floors, walls and even in floor drains if these areas are not properly cleaned and sanitized [2]. Recent advances in predictive microbiology have allowed the development of effective validated models which can be applied to improve food safety and food quality. In the last decade an increased number of predictive models for fungal growth (Penicillium expansum, Aspergillus ochraceus, Penicillium verrucosum, Penicillium digitatum, Penicillium italicum, Geotrichum candidum, Fusarium verticilliodes, Aspergillus flavus, Aspergillus carbonarius) have been developed [3-15]. Penicillium expansum is a fungal species highly damageable during the postharvest preservation of numerous fruits. In vineyards, this fungus is sometimes collected from grape berries where its growing may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas [15]. The development of a specific test allowing an early identification of Penicillium expansum is of great interest not only in the fruit storage industries but also in the viticulture Penicillium expansum is one of the most common ascomycetous fungi involved in the postharvest decay of numerous fruits such as apples, peaches, pears, cherries and grapes, leading to important economic losses in the fruit industry [16, 17]. In 2011, Ajila and his collaborators studied the ability of white rot fungi, 11290 Romanian Biotechnological Letters, Vol. 21, No. 2, 2016

Research regarding the influence of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. on chemical composition of red wines Phanerochaete to release phenolic antioxidants from apple pomace by solid-state fermentation. This study focused the evolution of chemical composition of red wines contaminated with Penicillium chrysogenum, Penicillium expansum, and Phanerochaete spp. These molds produce in wines metabolic transformations which modify alcoholic degree, total and free SO2 concentration, level of free and total acidity, Cu, Zn, K contents. 2. Materials and methods Fungal species 2.1. Isolation of filamentous fungi from damaged corks Fungi were isolated from samples obtained from contaminated cork (corkwood) stoppers. These fungi were isolated from damaged cork samples. After 4 days of incubation at 25 0 C individual fungal colonies were subcultured. Further, pure fungi cultures were obtained from single spores isolates using standard microbiological techniques: Petri A, Penicillium chrysogenum (mycobank.org); Petri dish B, Penicillium expansum (mycobank.org); Petri dish C, Phanerochaete chrysosporium (mycobank.org). 2.2. Identification The identification of fungi (single spores isolates) was made on the basis of their macroscopic (cultural and morphological characters on agar medium) and microscopic structures (e.g. in Penicillium described; a) the forms of individual conidia, b) the chains of conidia, c) the structure and types of conidiophores and d) numbers of phialides). Identification of all isolates, used in this study was further confirmed using more criteria (morphological data) obtained from the Mycobank (mycobank.org) fungal web site. Wine Analysis 2.3. Experimental process In this study, four types of wines with a high volume alcohol and sugar were used. Wine types were Roșca (local wine obtained from hybrid variety) Merlot, Cabernet Sauvignon, Syrah (Table 1). Each type of wine was divided in four Erlenmeyer flasks and contaminated with different fungi which had previously isolated from contaminated corks as described above. In details, volumes of 150 ml of each wine were added into 250 ml conical flasks and further flasks were contaminated, under a sterile environment, with tree mycelia plugs, i.e. Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. (Table 1). Flasks with untreated wine and without any mycelia plugs, were kept as control (Table 1). After contamination all flasks were placed into an orbital shaker with 150 rpm and were incubated for 12 days at 25 0 C. After the incubation period all flask contents were analyzed following the standard parameters of wine analysis. Each experiment was repeated twice. 2.4. Parameters of wine analysis At the end of fungal incubation period wine samples were analyzed using the following methods of OIV [19]: (i) Total SO 2 was extracted with ΚΟΗ 1N and H2SO4 25% and realized titration of the remaining reagent with iodium solution 0,02Ν in presence of indicator starch solution; (ii) Free SO 2 was extracted with H2SO4 25% and realized titration of the remaining reagent with iodium solution 0,02Ν in presence of indicator starch solution; (iii) Alcohol-Volume (%) was determined by pycnometry method; (iv) Total acidity was titrated with NaOH 0.1N with bromothymol blue as indicator. Carbon dioxide is not included in the Romanian Biotechnological Letters, Vol. 21, No. 2, 2016 11291

LIVIU GIURGIULESCU, IOANNIS VAGELAS, NIKOLAOS GOUGOULIAS total acidity; (v) Volatile Acidity. Carbon dioxide was firstly removed from the wine and further volatile acids were separated from the wine by steam distillation and titrated using NaOH 0,1N and two drops of phenolphthalein solution. (vi) Residual Sugar was determined with reagents: Fehling A (5.464 g CuSO4.5H2O dissolved in 100 ml distilled H2O) and Fehling B (12.0 g KOH and 7.5 g Potassium Sodium tartar dissolved in 100 ml distilled H2O). (vii) Total solid and Ash. Total solid was estimated by the dry solid matter at 105 0 C. Ash was collected at 550 0 C after being previously dried at 105 0 C. (viii) Cu. A volume of 20 ml sample was placed in a 100 ml volumetric flask and brought up to 100 ml with doubledistilled water. The dilution was modified if necessary to obtain a response within the dynamic range of the detector. The absorbance was measured at 324.8 nm. The zero was set with double distilled water. Measure the absorbance of standard solutions and the sample prepared in and repeat each measurement with Cecil UV DietQuest CE3410 spectrophotometer. (ix) K. Pipette 2.5 ml of wine (previously diluted 1/10) into a 50 ml volumetric flask, add 1 ml of the cesium chloride solution and make up to the mark with distilled water. Measure the absorbance with Cecil UV DietQuest CE3410 spectrophotometer. Set the wavelength to 769.9 nm. Zero the absorbance scale using the zero standard solution. Aspirate the diluted wine directly into the spectrophotometer, followed in succession by the standard solutions. Record the absorbance for each solution and repeat. (x) Zn. Remove the alcohol from 100 ml of wine by reducing the volume of the sample to half its original value using a rotary evaporator (50 to 60 0 C). Make up to the original volume of 100 ml, with double distilled water. Use the set the absorbance wavelength to 213.9 nm. Zero the absorbance scale using double distilled water. Aspirate the wine directly into the burner of the spectrophotometer, followed in succession by the four standard solutions. Record the absorbance and repeat each measurement. Use for read each absorbance spectrophotometer UV Cecil UV DietQuest CE3410. Each experiment was repeated twice and had a completely randomized design. Fisher s protected least significant difference (LSD) procedures were used to detect and separate the mean treatment differences at P=0.05. The program MiniTab ver 13 was used to conduct the analysis of variance. 3. Results and discussion In this research we used the following molds: Penicillium chrysogenum (figure 1A), Penicillium expansum (figure 1B) and Phanerochete spp. (figure 1C). A. B. C. Figure 1. Fungal cultures on PDA. Penicillium chrysogenum (A); Penicillium expansum (B); and Phanerochaete spp. (C). 3.1. Total SO 2 The experiment performed on Roșca hybrid wine, showed that Penicillium chrysogenum and Penicillium expansum did not influence the level of total sulfur dioxide, while Phanerochaete spp. decreased the level with 14.28 percent. For Merlot wine, Penicillium 11292 Romanian Biotechnological Letters, Vol. 21, No. 2, 2016

Research regarding the influence of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. on chemical composition of red wines chrysogenum decreased the level of total SO2, Penicillium expansum did not influence the level and Phanerochaete spp. increased the level of total SO2 with 21.42 percent. This increase is in concordance with Popa A. [20] stating that molds produce in wines high quantities of compounds with ketone group (ketone acids; diacetyl acid; 2.5 fructose and xylose) which block sulfur dioxide in chemical compounds. Cabernet Sauvignon wine recorded in all of contaminated samples a decrease of total SO2. Syrah wine showed a decreased level of total SO2 for samples contaminated with Penicillium chrysogenum and Penicillium expansum with 60.71 % in both cases, Frank et al., [21]. Phanerochaete spp. increased the level of total SO2 and blocked the turnover of free SO2 in ketone groups (Table 1). Table 1. Wine analyses contaminated with different fungal species, total SO 2, free SO 2, alcohol, total acidity, volatile acidity and reducing carbohydrates. Wine treatment* Total SO2 (mg.l -1 ) Free SO2 (mg.l -1 ) Alcohol/ Volume (%) Total acidity (H2SO4 g.l -1 ) Volatile acidity (CH3COOH g.l -1 ) Reducing Carbohydrates (g.l -1 ) Rosca Control 22.4±0.12 a** 16±0.17 a 11.8±0.01 b 2.74±0.02 c 1.15±0.02 b 32±2.08 b 2011 Rosca-A 22.4±0.01 a 12.8±0.01 b 11.8±0.01 b 2.55±0.01 a 0.89±0.02 a 32±1.33 b Rosca-B 22.5±0.03 a 12.8±0.12 b 11.8±0.06 b 2.60±0.01 b 0.89±0.01 a 34±3.06 b Rosca-C 19.2±0.10 b 12.8±0.01 b 11.0±0.01 a 2.60±0.01 b 1.58±0.02 c 21±2.01 a P value >0.001 >0.001 >0.001 >0.001 >0.001 >0.013 Merlot Control 2009 44.8±0.27 b 38.4±0.18 c 13.5±0.01 3.14±0.01 a 0.71±0.03 c 6.6±0.12 ab Merlot-A 41.6±0.03 a 32±1.15 a 13.5±0.01 3.28±0.02 b 0.59±0.02 b 6.8±0.09 b Merlot-B 44.8±0.20 b 35.2±0.12 b 13.5±0.01 3.33±0.01 b 0.22±0.01 a 6.9±0.12 b Merlot-C 54.4±0.012 c 44.8±0.31 d 13.5±0.06 3.43±0.02 c 0.94±0.02 d 6.2±0.13 a P value >0.001 >0.001 ns *** >0.001 >0.001 >0.007 Cabernet Control 73.6±0.32 d 48±0.01 c 12.5±0.06 3.82±0.02 c 1.05±0.01 c 2.8±0.01 ab 2007 Cabernet-A 57.6±0.03 c 48±0.23 c 12.5±0.01 3.43±0.01 a 0.74±0.01 a 2.9±0.01 b Cabernet-B 51.20±0.15 b 44.8±0.12 b 12.5±0.01 3.53±0.01 b 0.99±0.02 b 2.8±0.09 ab Cabernet-C 48.0±0.01 a 41.6±0.21 a 12.5±0.06 3.53±0.01 b 1.16±0.02 d 2.4±0.20 a P value >0.001 >0.001 ns >0.001 >0.001 >0.052 Syrah Control 89.6±0.10 c 57.6±0.27 d 12.5±0.06 b 3.87±0.01 a 1.32±0.01 b 6.2±0.1 b 2009 Syrah-A 35.2±0.25 a 38.4±0.01 c 12.5±0.06 b 3.92±0.01 b 1.32±0.01 b 6.2±0.31 b Syrah-B 35.2±0.01 a 35.2±0.27 b 12.5±0.01 b 4.07±0.01 c 0.85±0.01 a 6.4±0.12 b Syrah-C 51.2±0.25 b 32±1.17 a 11.0±0.06 a 3.92±0.01 b 2.59±0.3 c 3.4±0.07 a P value >0.001 >0.001 >0.001 >0.001 >0.001 >0.001 *where: Control untreated wine; A- wine contaminated with Penicillium chrysogenum; B- wine contaminated with Penicillium expansum, C-wine contaminated with Phanerochaete spp.; ** Values followed by the same letter were not significantly different (p < 0.05) *** (ns) not significant 3.2. Free SO 2 The free SO2 is in correlation with total SO2, determining the redox potential of wines. In most part of the contaminated samples, the free SO2 concentration decreased. Rosca hybrid wine has registered the lowest content in free SO2 present the same mitigation with 20 percents, Du Toit et al., [22] At Merlot wine Penicillium chrysogenum and Penicillium expansum decreased the level of free SO2, Phanerochaete spp. increased the level by metabolic process, Ribéreau-Gayon et al., [23]. This fungus increased free SO2 with 16.66 % than control. All of the samples, in rest, recorded normal values for wines contamination with molds. These results are in accordance with Popa A. an important percent of SO2 remained blocked in ketone compounds (Table 1). 3.3. Alcohol concentration It wasn t record a significant variation of alcohol concentration in all of samples taken in to consideration. Wine Rosca obtained from hybrid variety recorded a decrease with 0.8 alcohol degree at sample contaminated with Phanerochaete spp and is in accordance with Romanian Biotechnological Letters, Vol. 21, No. 2, 2016 11293

11294 LIVIU GIURGIULESCU, IOANNIS VAGELAS, NIKOLAOS GOUGOULIAS A.M. Amerine. The same results were registered at sample Syrah contaminated with Phanerochaete spp. 0.5% of alcohol degree was transformed in volatile acidity (Table 1). 3.4. Total acidity This parameter gives information about the stability and quality of red wines. At samples taken in study the results where different, determinate by the type of wine. For Rosca hybrid wine and Cabernet Sauvignon total acidity decreased for all contaminated samples, with significant results at Penicillium chrysogenum. The molds transform in metabolic process tartaric and malic acids, Popa and Teodorescu [26]. For Merlot and Syrah wines, total acidity increased in all of contaminated samples, important results being obtained at Penicillium expansum and Phanerochaete spp. on Merlot wine. Results confirm capacity of Penicillium and Phanerochaete to produce gluconic acide and oxo acids in higly contaminated red wines, Fleet [27] (Table 1). 3.5. Volatile acidity Phanerochaete spp. increased in all wines contaminated volatile acidity. Significant results recorded at Rosca hybrid and Syrah wines. Volatile acidity was produced by transformation of alcohol and reducing carbohydrates in volatile acids. Sulphur dioxide from wines blocked NAD compose and reduced the rapport between NADH/NAD. This reaction determines an increase of acetic aldehyde. Acetic aldehyde generate in wines high level of volatile acidity, Lopez de Lermaa et al., [28]. Penicillium molds reduce or maintain constant volatile acidity in all samples. The significant reducing was recorded at Penicillium expansum with 35.60% at Syrah, 69% at Merlot, 22.60% for Rosca and 5.71% for Cabernet Sauvignon. Penicillium transforms malic and tartaric acids inducing the decrease of volatile acidity (Table 1). Table 2. Wine analyses contaminated with different fungal species, total solids, ashes, total Cu, total Zn, total K Wine treatment* Total Solids (g.l -1 ) (105 0 C) Ashes (g.l -1 ) ( 550 0 C) Total Cu ( mg.l -1 ) Total Zn (mg.l -1 ) Total K (mg.l -1 ) Rosca Control 15±0,06 c** 5±0,05 c <0.5 0.62±0,01 a 1213.2±15,0 c Rosca-A 13±0,01 a 3±0,01 a <0.5 0.91±0,01 b 1116.6±10,0 b Rosca-B 13±0,12 a 3±0,02 a <0.5 1.11±0,02 c 1060.8±0,0 a Rosca-C 14±0,12 b 4±0,02 b <0.5 0.57±0,02 a 1060.8±15,0 a P value >0,001 >0,001 ns *** >0,001 >0,001 Merlot Control 23±0,03 a 8±0,05 c <0.5 0.87±0,01 d 1264.0±17,0 c Merlot-A 24±0,09 b 1±0,01 a <0.5 0.45±0,02 c 1060.8±15,0 a Merlot-B 24±0,06 b 2±0,03 b <0.5 0.34±0,01 b 1213.2±0,0 b Merlot-C 24±0,01 b 1±0,01 a <0.5 0.18±0,01 a 1213.2±0,0 b P value >0,001 >0,001 ns >0,001 >0,001 Cabernet Control 30±0,06 a 6±0,02 c <0.5 0.99±0,02 a 1213.2±10,0 a Cabernet-A 32±0,01 c 3±0,02 b <0.5 1.24±0,03 b 1264.0±0,0 b Cabernet-B 31±0,44 ab 2±0,01 a <0.5 0.97±0,01 a 1416.4±8,7 c Cabernet-C 31±0,07 b 2±0,02 a <0.5 1.04±0,02 a 1264.0±0,0 b P value >0,002 >0,001 ns >0,001 >0,001 Syrah Control 28±0,23 c 11±0,24 c 0.92 1.37±0,03 c 1365.6±15,3 c Syrah-A 27±0,12 b 3±0,06 b <0.5 1.22±0,02 b 1314.8±2,9 b Syrah-B 27±0,01 b 1±0,02 a <0.5 0.68±0,02 a 1213.2±0,0 a Syrah-C 26±0,19 a 1±0,01 a <0.5 0.60±0,01 a 1314.8±8,7 b P value >0,001 >0,001 ns >0,001 >0,001 *where: Control untreated wine; A- wine contaminated with Penicillium chrysogenum; B- wine contaminated with Penicillium expansum, C-wine contaminated with Phanerochaete spp. ** Values followed by the same letter were not significantly different (p < 0.05) *** (ns) not significant 3.6. Reducing carbohydrates Merlot, Cabernet Sauvignon and Syrah are dry wines with low content in residual sugars. Rosca wine obtained from hybrids variety had 32 g L -1 residual sugar. Penicillium expansum Romanian Biotechnological Letters, Vol. 21, No. 2, 2016

Research regarding the influence of Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. on chemical composition of red wines and Penicillium chrysogenum maintained constant the concentration of reducing carbohydrates or increased a few by dextran production in contaminated wines, Fucelsang and Edwards [29]. The other mold Phanerochaete spp. metabolized part of reducing carbohydrates, glucose in special, and transformed in volatile acids, Vidal et al., [30]. Special remark can be done Syrah wine contaminated with Phanerochaete spp. where transformation of reducing carbohydrates was 45.16% (Table 1). 3.7. Dry substances The evolution of dry substances content was different depending on the contaminated wine. For Rosca and Cabernet Sauvignon dry substances decreased. Molds block free SO2 and transform total acidity, residual sugar in volatile compounds. For Merlot and Cabernet Sauvignon dry substances increased than control, in these wines was accumulated dextran and other compounds resulted by metabolic process (Table 2), Popa A. [31]. 3.8. Ash In all samples taken into consideration ashes content decreased in comparison with control. Special remark can be done for Merlot contaminated with Penicillium chrysogenum decreased with 87.5%, Penicillium expansum decreased with 75% and Phanerochaete spp. decreased with 87.5%; Syrah contaminated with Penicillium chrysogenum decreased with 72.72 % and Phanerochaete spp. decreased with 91% than control (Table 2). 3.9. Cooper, zinc and potassium Level of copper was under limit of detection at control and wines contaminated with molds. The concentration of Zn was different depending on the type of wine and contamination. In most samples taken into consideration concentration of Zn decreased except Rosca wine contaminated with Penicillium and Cabernet Sauvignon contaminated with Penicillium chrysogenum and Phanerochaete spp. Level of potassium recorded a decreasing at Rosca wine hybride, Merlot and Syrah and an increase at Cabernet Sauvignon contaminated with Penicillium chrysogenum, Penicillium expansum and Phanerochaete spp. Level of potassium in wine determines the absorption of sugars into the mold cell. 4. Conclusions Total SO2 and free SO2 recorded different evolution in contaminated wines by type of mold. All of molds decreased or maintained constant SO2 content, this fact being explained by reaction against antiseptic effect of SO2. Phanerochaete spp. Had a different behavior for Merlot contaminated wine? At this samples mold increases the content of total and free SO2 by metabolic process. The molds had different reactions for total acidity. For Rosca hybrid wine and Cabernet Sauvignon total acidity decreased and for Merlot and Syrah total acidity increased. The molds consume a part of acids from red wines causing reducing of acidity or produce volatile acids causing accumulation of total acidity in red wines. Penicillium chrysogenum and Penicillium expansum decreased volatile acidity in all of red wines contaminated. Phanerochaete spp. increased volatile acidity in all samples. This mold transforms a part of alcohol and reducing sugars in volatile compounds. At Syrah wine Phanerochaete spp. increased the content of volatile acidity with 96.21%. Romanian Biotechnological Letters, Vol. 21, No. 2, 2016 11295

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