Kántor A. et al./Scientific Papers: Animal Science and Biotechnologies, 2014, 47 (2)

Chemical and Microbiological Analysis of Red during Storage at Different Temperatures

Attila Kántor*, Jana Petrová, Miroslava Kačániová

Slovak University of Agriculture in Nitra, 949 76, Tr. A. Hlinku 2, Nitra

Abstract Overall, chemical and microbiological analyses are very important for the quality of during and after process. One of the most important factors during wine storage is the temperature of storage. The aim of our study we used two different wines, specifically and Blaufränkisch and chemically and microbiologically analysed these wines during storage at different temperatures. These wines were bottled in 2011 and 2013. We stored these samples at different temperatures. The first four samples were stored at 6-8°C in refrigerator, and the next four were stored at 20-25°C in room temperature. We had together eight wine samples. We had determined in all wine samples sequentially the free and total sulphur dioxide content, ethyl-alcohol content, extract, residual , total and volatile acids. Microbiological parameters were observed during wine storing after filtration through different micro filters. We determined the total count of bacteria (TCB), Acetobacter cells, Lactobacillus cells, yeast and moulds in wine samples with classic plate dilution method. The highest quality wines from microbiological properties were wines from 2013, which was filtered through micro filter and aseptically filled into the bottles, but wines from 2011 was filtered through cross-flow filter and samples directly collected from the storage tanks without microfiltration. Keywords: Chemical content, red wines, storage, temperature, total count of bacteria

1. Introduction dioxide [2]. MLF is a secondary fermentation that usually occurs during storage of young wines Wine fermentation is, as in many other food several weeks after the alcoholic fermentation. fermentations, characterized by complex chemical MLF normally occurs spontaneously and is a very and microbial interactions. Lactic acid bacteria slow and unpredictable process that can undergo and yeast are the first to develop after crushing of for weeks and even months, but not always give a grapes. Bacterial numbers increase to 103 or 104 satisfactory result [3]. From lactic acid bacteria CFU/ml, but decline to almost undetected levels (LAB) is Oenococcus oeni usually predominates during alcoholic fermentation [1]. At the end of in wines with low pH (<3.5), while Pediococcus alcoholic fermentation, growth commences and damnosus grows in wines with higher pH cell numbers increase to approximately 107 resulting in spoilage of wine [4, 5]. A number of cells/mL. The two main fermentation processes in acetic acid bacteria have been isolated from wine, winemaking are alcoholic fermentation conducted e.g. Acetobacter aceti, Acetobacter pasteurianus, by yeasts that transform sugars into and Gluconobacter oxydans [6], Gluconacetobacter , and liquefaciens and Gluconacetobacter hansenii [7]. (MLF) carried out by lactic acid bacteria that Yeast numbers at time of range between convert malic acid to lactic acid and carbon 104 and 106 CFU/mL. The most predominant genera from yeasts are Rhodotorula, Cryptococcus, Candida, Kloeckera and

* Corresponding author: Attila Kántor, Metschnikowia [8]. +421 37 641 5812, [email protected]

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The secret to good winemaking is to manage the samples of wine were prepared in advance by process in such a way that interactions between sequential diluting based on decimal dilution yeast and lactic acid bacteria are controlled at all system application. For microorganism cultivation times. Very important yeast for alcoholic three types of cultivating mediums were used, to fermentation is Saccharomyces cerevisiae which segregate individual microorganism groups. For has a very active metabolism, tolerates oxygen identification of TCB we used Tryptic and relatively high levels of SO2. During alcoholic Yeast Agar (Biolife, Italy) and incubated the fermentation, some strains produce SO2, short samples at 30°C for 72h aerobically. Acetobacter chain fatty acids, peptides, proteins or cells cultivated on Glucose yeast extract CaCO3 glycoproteins, such as killer toxins, and lytic medium (GYC) (Conda, Spain) and incubated at enzymes that are used to inhibit the growth of 30°C for 48h aerobically. Lactobacillus species malolactic bacteria [9-11]. Saccharomyces cultivated on MRS agar (Conda, Spain), incubated cerevisiae is more tolerant to low oxygen levels at 37C for 72h in microaerophilic condition. compared to other yeast [12]. Finally yeast and molds cultivated on The main objectives of this study were the Chloramphenicol agar (YGC agar) (Conda, Spain) identification of Total Count of Bacteria (TCB), and incubated at 25°C for 5 days. After incubation Acetobacter, Lactobacillus cells, yeast and molds we collected the results and express in CFU/mL. in wine samples, which were storage at two different temperatures. The next objective was Chemical parameters collection of data from chemical parameters for three times in wine samples Blaufränkisch and In wine samples we have measured the ethanol Cabernet Sauvignon. content, residual sugars, total acids, volatile acids, total extract, free and total sulphur dioxide (SO2). Ethyl alcohol or ethanol, CH3CH2OH, is the most 2. Materials and methods important product in alcohol fermentation process. Species of yeasts metabolized the sugars in wine We used two different samples from and in reduced-oxygen content they produce 2011 and 2013 especially Blaufränkisch and ethanol and CO2. Ethanol is colorless liquid with a Cabernet Sauvignon dry wines. We collected the characteristic subtly spicy and aroma. We samples after final bottling. The samples from determined the alcohol content in wine samples 2013 filtered through micro filter, but at wine with wine alcohol meter (wine meter) at 20°C and samples from 2011 weren’t filtered through micro 6°C. The residual or reducing sugars in wine was filter. The bottles were storage at two different determinate by Schoorl’s titrate method. Reducing temperatures, first at 6-8°C in refrigerator and sugars are fermentable sugars present in must or second at 20-25°C at room temperature. We used wine namely, glucose and . Residual these temperatures, because in the refrigerator is sugars include all forms of in the wine, better maintain a steady temperature than in the including non-fermentable pentoses. Reducing room temperature. sugars was determined by reaction of a water soluble portion of the sample with an excess of Microbiological analysis standard copper sulphate in alkaline tartrate (Fehling's) solution under controlled conditions of For microbiological analysis the wine samples time, temperature, reagent concentration and were processed immediately after collection. The composition, so that the amount of copper reduced total counts of bacteria (TBC), number of is proportional to the amount of reducing sugars in Acetobacter cells (A), Lactobacillus cells (L) and the sample analysed [13]. The volatile acidity is yeast and molds (Y/M) were assessed. Plate derived from the acids of the acetic series present diluting method was applied for quantitative CFU in wine in the free and combined as salts. Carbon (Colony Forming Units) counts determination of dioxide was first removed from the wine. Volatile respective groups of microorganisms in 1 mL of acids are separated from the wine by steam wine. Petri dishes of gelatinous nutritive substrate distillation and titrated using standard sodium were inoculated with 1 mL of wine samples (TBC, hydroxide [13; 14]. A, L and Y/M) in three replications. Homogenized

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The sugar-free extract is the difference between Lactobacillus cells ranged from 0 to 28 CFU/mL the total dry extract and the total sugars. The in wine samples. The highest level of Acetobacter reduced extract is the difference between the total was found in the wine sample Cabernet Sauvignon dry extract and the total sugars in excess of 1 g/L 2011, and the highest level of Lactobacillus cells potassium sulphate in excess of 1 g/L, any was found in the same wine as Acetobacter. The mannitol present and any other chemical highest level of yeast and molds was found in substances which may have been added to the wine sample Cabernet Sauvignon 2011 and the wine. The total dry extract is calculated indirectly number of yeast and molds ranged from 0 to 84 from the specific gravity of the must and, for CFU/mL. In the study Kačániová et al. [15] the wine, from the specific gravity of the alcohol-free number of Lactobacillus cells ranged from 0 to 92 wine [13]. Sulphur dioxide is present in wines as CFU/mL in Cabernet Sauvignon wine. Yeasts and free SO2 and also bound to acetaldehyde. Free bacteria are widely distributed in wine sulphur dioxide is defined as the sulphur dioxide ecosystems, and they play an important role in present in the must or wine in the following forms: winemaking by contributing to the quality and - H2SO3, HSO3 , whose equilibrium as a function of desirable properties of the product. Nonetheless, + - pH and temperature is: H2SO3 H + HSO3 . Free their activity is not only beneficial, but also sulphur dioxide is determined by direct titration detrimental to wine quality [16]. with iodine. The combined ⇌sulphur dioxide is subsequently determined by iodometric titration Table 2. Number of microorganisms at 20-25°C after alkaline . When added to the free Type of wine 20-25°C TCB A. Lb. Y. / M. Blaufränkisch 14/10/13 160 5 9 25 sulphur dioxide, it gives the total sulphur dioxide. 2011 11/11/13 250 20 30 90

Results were expressed as SO2 in mg/l [13]. 9/12/13 348 38 42 138 Blaufränkisch 14/10/13 4 0 2 9 2013 11/11/13 35 6 20 43 9/12/13 147 22 18 89 3. Results and discussion Cabernet Sauvignon 14/10/13 121 0 20 28 2011 11/11/13 190 11 51 56 9/12/13 327 50 86 112 The results collected after plate dilution method Cabernet Sauvignon 14/10/13 5 0 0 11 and incubation time from three measuring for each 2013 11/11/13 60 5 12 37 wine samples (Blaufränkisch 2011, 2013 and 9/12/13 120 17 33 64 Cabernet Sauvignon 2011, 2013). The results were TCB - total count of bacteria, A. - Acetobacter cells, Lb. - Lactobacillus cells expressed in CFU/mL. Table 1 show the results Y. / M. - Yeast and Molds. Unit - CFU/mL from plate dilution method for wine samples storage at 6-8°C in refrigerator. The 14/10/13; Acetic acid bacteria and yeast species, such as 11/11/13 and 9/12/13 represent date of measuring. Acetobacter aceti, Acetobacter oeni, Acetobacter pasteurianus, Dekkera (Brettanomyces) Table 1. Number of microorganisms at 6-8°C bruxellensis, Hanseniaspora (Kloeckera) uvarum, Type of wine 6-8°C TCB A. Lb. Y. / M. Saccharomyces cerevisiae, Zygosaccharomyces Blaufränkisch 14/10/13 156 5 10 20 bailii and Zygosaccharomyces rouxii are among 2011 11/11/13 218 5 12 48 9/12/13 320 14 12 60 the most concerning microbial contaminants and Blaufränkisch 14/10/13 1 0 0 0 are well known for their potential to deteriorate 2013 11/11/13 20 0 6 28 wine [17; 18]. In the Table 2 we can see the 9/12/13 61 4 10 70 Cabernet Sauvignon 14/10/13 128 1 13 30 results from plate dilution method at wine samples 2011 11/11/13 169 10 28 38 which storage in room temperature at 20-25°C. 9/12/13 233 28 20 84 The results were following: number of total count Cabernet Sauvignon 14/10/13 3 0 0 1 2013 11/11/13 12 0 0 17 of bacteria ranged from 4 to 348 CFU/mL. The 9/12/13 32 0 2 21 highest level of total count of bacteria was found TCB - total count of bacteria, A. - Acetobacter cells, Lb. - Lactobacillus cells in the wine sample Blaufränkisch 2011. The Y. / M. - Yeast and Molds. Unit - CFU/mL number of Acetobacter cells ranged from 0 to 50 CFU/mL and the highest level of these bacteria The number of total count of bacteria ranged from was found in Cabernet Sauvignon 2011 wine 1 to 320 CFU/mL. The highest level of total count sample. The number of Lactobacillus cells ranged of bacteria was found in the wine sample from 0 to 86 CFU/mL and the highest level of Blaufränkisch 2011. The Acetobacter and lactobacilli was found in Cabernet Sauvignon

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2011 wine sample. The number of yeast and we can see the results from measuring some molds ranged from 9 to 138 CFU/mL in wine chemical parameters of wine sample samples, and the highest level was in the Blaufränkisch for three month storage at different Blaufränkisch 2011 wine sample. In Table 3 and 4 temperatures.

Table 3. Chemical parameters of Blaufränkisch 2011 wine Wine Temperature (6-8°C) Temperature (20-25°C) Blaufränkisch 2011 14/10/13 11/11/13 9/12/13 14/10/13 11/11/13 9/12/13 Units Ethanol 12.24 12.24 12.07 12.24 11.90 10.73 % Residual sugars 2.4 2.4 2 2.4 2.4 2.2 % Total acids 5.7 5.7 5.7 5.7 5.7 5.9 % Volatile acids 0.468 0.468 0.482 0.456 0.468 0.816 g/L Total extract 26.9 26.9 27.4 27.4 27.4 28 g/L

Free SO2 15 8 3 8 5 3 mg/L Total SO 2 54 23 18 21 23 15 mg/L

Table 4. Chemical parameters of Blaufränkisch 2013 wine Wine Temperature (6-8°C) Temperature (20-25°C) Blaufränkisch 2013 14/10/13 11/11/13 9/12/13 14/10/13 11/11/13 9/12/13 Units Ethanol 12.31 12.04 11.72 12.31 10.98 9.94 % Residual sugars 1.3 1.3 1.3 1.3 1.3 1.2 % Total acids 5.7 5.7 5.8 5.7 5.6 5.6 % Volatile acids 0.428 0.531 0.683 0.428 0.783 1.341 g/L Total extract 26.6 25.6 25.6 25.6 25.4 25.3 g/L

Free SO2 23 18 12 20 13 11 mg/L Total SO 2 31 30 28 31 29 28 mg/L

Ethanol content in Blaufränkisch 2011 wine at 6- [19; 20]. The major volatile acid in wine is acetic 8°C was more stable than at 20-25°C after three acid (> 90%) [21]. months. Residual sugars were very stable for two Acetic acid has a threshold value from 0.7 to 1.1 months, but in third month was recorded a small g/L depending on the style of wine and above reduction of residual sugars. Total acids content these values it becomes objectionable [22]. The was changes only in third month at 20-25°C high levels of volatile acids produce by yeasts, sample. Volatile acids increased wild yeasts, lactic acid and acetic acid bacteria in step by step; in the second month the value was wine [21]. In wine sample Blaufränkisch 2013 the same 0.468 g/L at different temperatures. Total storage at 20-25°C was the volatile acids extract was reduced after month in small content, increased from 0.428 to 1.341 g/L and in wine but in free and total SO2 were recorded very high Cabernet Sauvignon 2013 at 20-25C was the differences between monthly measuring. Yeasts volatile acids increased from 0.324 to 1.256 g/L. are very important in winemaking process, but Probably the higher temperature of wine storage there are exists some species with negative impact was the starter to bacterial spoilage on these wine on wine quality. One of these yeasts is osmophilic samples. The microfiltration is useful for bottled Zygosaccharomyces, with the ability to grow at wines, but when the wine is opened and storage at high sugar concentrations and to ferment grape higher temperature; the bacteria species produce juice to dryness. Z. bailii is highly resistant to more final products than at 6-8°C. The volatile preservatives (SO2, sorbic and benzoic acid) used acids increase by production of acetic acid by in grape juice and wine, and possesses a high acetic acid bacteria or by yeast. ethanol tolerance (> 15 %) and a low pH < 2.0 tolerance, which makes it a difficult spoilage yeast

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Table 5. Chemical parameters of Cabernet Sauvignon 2011 wine Wine Temperature (6-8°C) Temperature (20-25°C) Cabernet Sauvignon 2011 14/10/13 11/11/13 9/12/13 14/10/13 11/11/13 9/12/13 Units Ethanol 12.14 12.05 11.90 12.05 11.90 10.48 % Residual sugars 2.3 2.3 2 2.4 2.4 2.3 % Total acids 5.6 5.6 5.7 5.6 5.6 5.9 % Volatile acids 0.444 0.456 0.556 0.456 0.468 0.486 g/L Total extract 25.6 26.7 26.9 27.2 27.5 27.7 g/L

Free SO2 8 5 3 6 5 1 mg/L

Total SO2 21 18 16 18 18 15 mg/L

In the Table 5 and 6 we can see the results from reduced step by step in every measuring. The measuring some chemical parameters of wine concentration of molecular SO2 needed to prevent sample Cabernet Sauvignon for three month growth of microorganisms varies with wine or storage at different temperatures. The ethanol juice pH, temperature, population density and content reduced because the bacteria change the diversity, stage of growth, alcohol level, and other ethanol to acetic acid and then the volatile acids factors [23]. are increased. The total and free SO2 were rapidly

Table 6. Chemical parameters of Cabernet Sauvignon 2013 wine Wine Temperature (6-8°C) Temperature (20-25°C) Cabernet Sauvignon 2013 14/10/13 11/11/13 9/12/13 14/10/13 11/11/13 9/12/13 Units Ethanol 11.98 11.85 11.54 11.98 11.40 10.08 % Residual sugars 1.8 1.8 1.8 1.8 1.8 1.3 % Total acids 5.9 5.6 5.7 5.9 5.9 5.9 % Volatile acids 0.324 0.556 0.756 0.324 0.721 1.256 g/L Total extract 25.7 26.7 26.9 25.7 26.5 27.1 g/L

Free SO2 24 15 13 24 11 11 mg/L Total SO 2 42 31 18 42 31 28 mg/L

For inhibition of some species of yeasts: Pichia, However, caution should be exerted when using Saccharomycodes, Schizosaccharomyces, and membranes with pore sizes >0.45 µm because of Zygosaccharomyces require at least 2 mg/L variation in sizes of microorganisms to be molecular SO2 in wine. Molecular SO2 depends removed. For instance, older and larger yeast cells highly on pH. When the SO2 content is high the may be removed by a 1 µm filter whereas younger pH is high too (60 mg/L → 3.7pH), but when the and smaller cells may not be, thereby maintaining SO2 is decrease the pH is decrease too. (13 mg/L the risk of refermentation in the bottled wine. → 3pH) [24]. Sulphur dioxide (SO2) more inhibits Furthermore, use of >0.45 µm membranes will not the bacteria species in wines than the yeasts. Very remove bacteria. Millet and Lonvaud-Funel [25] important for final product is filtration. Sterile noted that the size of Acetobacter cells decrease filtration requires the use of membranes of during lengthy residency in wine, allowing the sufficient porosity that microorganisms are bacterium to pass through 0.45 µm membranes. physically excluded. To remove bacteria, Cross-flow or tangential filtration is finding membranes with maximum pore sizes of 0.45 µm increased interest and application in winemaking are recommended. Although attempts have been and juice concentrate production. Although small made to use membranes with pore sizes of <0.45 particulates can be removed, tangential filtration µm (e.g., 0.2 µm), flow rates are commonly too should not be considered a substitute for sterile slow to be economically feasible. Where spoilage filtration. In fact, a population of >104 CFU/mL of yeasts are believed to represent a threat, some Zygosaccharomyces was found in a “permeate” winemakers have relied on membranes with larger obtained from a commercial cross-flow filtration pores (0.65–1.0 µm).

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system, indicating the importance of using sterile step by step at all wine samples, especially the perpendicular-flow membranes at bottling [20]. ethanol content is decrease, the volatile acids were However, topic of food safety and protection of increase, total and free SO2 were rapidly decrease, consumers interest is always current and of for residual sugars were determinate small increasing concern to the general public. reduction, and for total extract and total acids were Consumer confidence is an essential outcome of a determinate small increase at third measurement. successful food policy and control. EU food safety and its legislation has evolved years, reflecting a blend of scientific, social, political and economic Acknowledgements factors [26]. This work was supported by the project: The research leading to these results has received funding grant of 4. Conclusions KEGA 013SPU-4/2012.

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