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How to Deal with Uninvited Guests in Wine: Copper and Copper-Containing Oxidases

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How to Deal with Uninvited Guests in Wine: Copper and Copper-Containing Oxidases fermentation Review How to Deal with Uninvited Guests in Wine: Copper and Copper-containing Oxidases Harald Claus Institute of Molecular Physiology, Microbiology & Wine Research, Johannes Gutenberg University of Mainz, Becherweg 15, D-55099 Mainz, Germany; [email protected] Received: 4 March 2020; Accepted: 15 March 2020; Published: 24 March 2020 Abstract: Copper is one of the most frequently occurring heavy metals in must and wine. It is introduced by pesticides, brass fittings, and as copper sulphate for treatment of reductive off-flavors. At higher concentrations, copper has harmful effects on the wine. It contributes to the oxidation of wine ingredients, browning reactions, cloudiness, inhibition of microorganisms, and wine fermentation. Last but not least, there is also a danger to the consumer. At present, some physicochemical methods exist to reduce the copper content in must and wine, but they all have their shortcomings. A possible solution is the biosorption of metals by yeasts or lactobacilli. Copper can also reach must and wine in the form of copper-containing phenol oxidases (grape tyrosinase, Botrytis cinerea laccases). Similar to free copper, they oxidize phenolic wine compounds, and thus lead to considerable changes in color and nutritional value, making the product ultimately unsaleable. All measurements for enzyme inactivation such as heat treatment, and addition of sulphites or bentonite are either problematic or not effective enough. The application of oenological tannins could offer a way out but needs further research. Keywords: wine browning; copper casse; phenoloxidases; bentonite; tannins; biosorption 1. Introduction Many wine constituents including phenols are susceptible to oxygen, and thus lead to the development of oxidation products during vinification [1,2]. Wine browning is one of the most common problems that occurs during winemaking and affects color and sensory characteristics, and also leads to loss of nutritional values of the wine [3]. Wine browning is caused by enzymatic and non-enzymatic oxidations. Non-enzymatic oxidation occurs in wine by direct reaction of phenols with air. Iron and copper play an important catalytic role. Enzymatic oxidation in must and wine takes place under the influence of copper-containing enzymes (tyrosinase and laccase) [4–7]. An excess of free copper cations also has negative effects on wine microorganisms, wine fermentation, and last but not least, on the health of the consumer. Therefore, there is an urgent need to minimize the concentration of copper and activities of copper-containing oxidases in must and wine. This review describes the effects of copper (oxidases) on wine quality and presents some possible countermeasures. 2. Copper Copper is an essential trace element involved in many natural metabolic processes of living organisms. It plays a major role in redox reactions and oxygen transport, contributes to the biological energy production of cells, and is an important component of enzymes [8]. The WHO guideline for drinking water was set at 2 mg/L, as a figure considered to be safe for chronic population exposure [9,10]. However, an elevated copper level leads to disorders of liver metabolism, increased formation of free radicals, and can also promote disorders of the central nervous system [9]. The physiological oxidation Fermentation 2020, 6, 38; doi:10.3390/fermentation6010038 www.mdpi.com/journal/fermentation Fermentation 2020, 6, 38 2 of 14 states of copper are Cu1+ and Cu2+, whereas Cu3+ is not a biologically relevant species because of the high redox potential of the Cu3+/Cu2+ couple [8]. 2.1. Occurrence and Effects of Cu in Must and Wine The advantages of copper as a widely applicable fungicide are countered by the problem of soil contamination. Vineyard areas that have been treated with copper preparations over several decades sometimes have high excessive copper contents that exceed the EU permissible limit value (140 mg/kg) several times [11]. Copper is also one of the most frequently occurring heavy metals in wine and can reach the wine via pesticides, brass fittings, and as copper sulphate for treatment of reductive off-flavors [12,13]. According to a study in which 72 wines were analyzed, the average copper content was 0.18 mg/L with a maximum of 0.55 mg/L[10]. The Organisation International de la Vigne et du Vin (OIV) recommends a maximum copper content of 1.0 mg/L for wines [14]. National regulations allow the presence of 2.0 mg/L Cu, in German wines [10]. Possible negative consequences of increased copper contents in must and wine, as well as corresponding limits are summarized in Table1. Copper casse (haze) can develop after wine bottling in the absence of oxygen, presence of sulfur dioxide and copper contents > 0.5 mg/L. It is composed of cupric and cuprous sulfides which form a reddish haze or deposit especially in white wine and less in rosé wines [12,13]. Table 1. Copper concentrations in must and wine with corresponding effects [15]. Limits and Normal Values/Effects Cu Concentration (mg/L) Normal value in wine 0.7–0.8 Maximum content allowed in wine 2.0 Concentration range in must 0.09–0.99 Copper catalyzed oxidations (browning reaction) in traces Copper casse > 0.5 Influencing fermentation of sparkling wine (very rare) > 20 Sluggish must fermentation 25–50 Inhibition of Oenococcus oeni Microenos B1 5*; 10** Inhibition of Saccharomyces cerevisiae SN9 / SN41 / CCT0472 32/320/75 Inhibition of Lactobacillus fermentum CCT1400 / CCT0559 75/300 Inhibition of Lactobacillus mesenteroides CCT0582 / CCT0367 75/150 *Copper as CuSO4 x 5H2O; **Copper as CuCl2 x 2H2O. 2.2. Measurements to Reduce Cu Concentrations in Must and Wine 2.2.1. Chemical and Physical Methods If the remaining copper content exceeds the permitted limits, metal removal procedures must be applied. In the so-called "blue fining" process, potassium ferrocyanide II is added to the wine, which forms an insoluble complex with copper ions that precipitates the copper. The remaining cyan compounds, then, react with the iron already present and precipitate [12,13]. In the case of iron deficiency, an excess amount of potassium hexacyanoferrate II remains in the wine, making it usuitable for consumption. A further disadvantage of blue fining is the resulting toxic hazardous waste, which must be disposed of separately and at great expense. Another procedure is the use of the natural plant polysaccharide gum arabicum which presents the most applied protective colloid in winemaking authorized within the European Union. It is used at a dosage of 10 to 15 g/hL to prevent copper casse, as long copper levels do not exceed 1.0 mg/L[16]. The colloid prevents metal precipitation but does not eliminate the copper. Because clarification of a wine treated with gum arabicum is difficult, it is generally mixed into the wine just before bottling [16]. Fermentation 2020, 6, 38 3 of 14 Apart from plant resins, artificial exchange resins have been developed to overcome the disadvantageous use of traditional bluefining. These include polyvinylimidazole-polyvinylpyrrolidone copolymers (PVI/PVP) to combine the benefits of PVPP with selective binding of metals such as copper or iron [17,18]. In that case, special care must be taken to avoid possible migration of unpleasant monomers or degradation products into the wine [17,18]. With the aim of preventing turbidity, bentonite is often used to remove proteins (see Section 3.3.4) and also copper from the wine. However, this process can exert adverse effects on color and the taste of the wine [19]. 2.2.2. Biosorption An alternative method for metal removal is biosorption (Table2). This is a passive, non-metabolic process in which, for example, heavy metals are bound to biomass (bacteria, fungi, and algae) [20–26]. For a suitable application in winemaking, preference is given to microorganisms for which wine represents a natural habitat and which are not harmful to consumers, for example, wine yeasts [20,21]. Other candidates in this respect are lactobacilli [22] which are regularly found on grapes, as well as in must and wine [27]. Table 2. Compilation of biosorbent materials and their maximum specific copper binding capacity (qh) [20–26]. Biomass Added q Biosorbent pH Optimum h (mg/mL) (µg Cu2+ /mg Cells) Bacteria Arthrobacter sp. 3.5–6.0 0.4 148.0 Bacillus sp. (ATS-1) 5.0 2.0 16.3 Bacillus subtilis IAM 1026 5.0 0.5 20.8 Enterobacter sp. J1 5.0 1.0 32.5 Lactobacillus brevis ID9262 5.0 0.5 26.5 Lactobacillus buchneri DSM 20057 5.0 1.0 10.5/9.9 (living / dead) Lactobacillus hilgardii 5.0 1.0 10.0/8.8 DSM 20176 (living/dead) Lactobacillus plantarum 5.0 1.0 9.7/8.6 DSM 20174 (living /dead) Lactobacillus plantarum ID9263 5.0 0.5 15.5 Lactobacillus vini DSM 20605 5.0 1.0 12.3/9.7 (living /dead) Leuconostoc mesenteroides ID9261 5.0 0.5 26.3 Micrococcus luteus 5.0 0.5 33.5 Pseudomonas aeruginosa PU21 5.0 1.0 23.1 Pseudomonas cepacia 7.0 n. d 65.3 Pseudomonas putida 6.6 n. a. 6.6 Pseudomonas putida CZ1 4.5 1.0 15.8 Pseudomonas stutzeri IAM 12097 5.0 1.0 22.9 Pseudomonas stutzeri KCCM 34719 5.0 1.0 36.2 Pseudomonas syringae n.d. 0.28 25.4 Sphaerotilus natans 5.5 n.d. 5.4 Streptomyces noursei 5.5 3.5 9.0 Synechocystis sp. 4.5 1.0 23.4 Zoogloea ramigera 5.5 0.83 270.0 Zoogloea ramigera 4.0 n. d 29 Yeasts Cryptococcus terreus 5.5 1.0 71.8 Pichia guilliermondii 5.5 1.0 34.0 Saccharomyces cerevisiae1) 5.5 1.0 43.3 Saccharomyces cerevisiae2) (living/dead) 4.0 2.0 0.8/0.4 n.d., not determined; (1), [21]; (2), [20].
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