GARCÍA-RUIZ ET AL., POLYPHENOLS AS A NATURAL ALTERNATIVE TO THE USE OF SULPHITES IN WINEMAKING, PAG. 1 POLYPHENOLS AS A NATURAL ALTERNATIVE TO THE USE OF SULPHITES IN WINEMAKING Almudena GARCÍA-RUIZ, M.Victoria MORENO-ARRIBAS, Pedro J. MARTÍN-ÁLVAREZ, Begoña BARTOLOMÉ* Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM). c/ Nicolás Cabrera, 9. Campus de Cantoblanco. Universidad Autónoma de Madrid. 28049 Madrid (Spain). E-mail: bartolome@ifi.csic.es Sulphurous anhydride or sulphur dioxide (SO 2 ) has numerous properties as a preservative in winemaking; these include its antioxidant and selective antimicrobial effects, especially against lactic acid bacteria (LAB). During winemaking, it is important to control the growth of these bacteria because could otherwise occur alterations wine quality and safety as the formation of biogenic amines. Although sulphiting is an essential technology procedure in the elaboration and conservation of wines, in recent years, there is a growing tendency to reduce the maximum content of SO 2 in musts and wines, mainly due to its possible effects harmful to human health and environmental protection reasons (Santos et al., 2012). This, coupled with the growing concern of consumers for the use of chemical compounds as food preservatives, has promoted a growing interest in seeking of total or partial alternatives, harmless to health, to the traditional use of SO 2 in winemaking. The use of natural products, among which are phenolic compounds or polyphenols is shown as one of the most promising possibilities because this extensive group of compounds are characterized by showing a dual effect as antimicrobials and antioxidants (García-Ruiz et al., 2008). This paper summarizes the results of a systematic study on the effect of polyphenols on the growth and metabolism of wine LAB, and its mechanism of action, also evaluated the possible use of natural phenolic extracts as antimicrobial additives during winemaking. In a first stage, it was determined the antimicrobial activity of phenolic compounds representative of the different classes of polyphenols present in grapes and wine (n= 18), against LAB strains of the species Lactobacillus hilgardii and Pediococcus pentosaceus -both generally considered as wine spoilage species-, and Oenococcus oeni -main species responsible for malolactic fermentation (MLF) in wine. This activity was evaluated by determination of inactivation and inhibition parameters: MIC and MBC, and IC 50, respectively. There was a certain relationship between the chemical structure-antimicrobial activity of the polyphenols, which in turn depend on the concentration of the compound as well as the intrinsic characteristics of each strain (Garcia-Ruiz et al., 2009, 2011). Among families of polyphenols studied, flavonols highlighted as being the most active (lowest values of MIC, MBC and IC 50 ) whereas, by contrast, flavanols did not show antimicrobial activity. O. oeni was the species most susceptible to the antimicrobial effect of wine/grape polyphenols. It was also determined IC 50 values of potassium metabisulfite and lysozyme -whose use is also authorized in oenology. The results showed, first, that polyphenols had higher antimicrobial activity than lysozyme and, secondly, that flavonols were characterized by exhibiting greater inhibition capacity than metabisulphite against strains of L. hilgardii and P. pentosaceus. Regarding the mechanism of action of polyphenols, the micrographs obtained by fluorescence and transmission electron microscopy revealed loss in bacterial viability and damage to the integrity of the membrane, respectively (Figures 1, 2). This result suggested that in the mechanism of
GARCÍA-RUIZ ET AL., POLYPHENOLS AS A NATURAL ALTERNATIVE TO THE USE OF SULPHITES IN WINEMAKING, PAG. 2 inhibition of polyphenols on BAL may be involved hydrophobic interactions between the phenolic compounds and the lipid fraction of the bacterial membrane, leading to the loss of its integrity and the subsequent cell death (Ibrahim et al., 1996). A B C D Figura 1. Epifluorescence micrographs (40x) of Pediococcus pentosaceus CIAL 85 non-incubated and incubated with antimicrobial agents for 3 h: (a) control, (b) incubation with potassium metabisulfite (600 mg/l), (c) incubation with kaempferol (100 mg/l) and (d) incubation with trans-resveratrol (300 mg/l). (Figure taken from García-Ruiz et al., 2009). A B
GARCÍA-RUIZ ET AL., POLYPHENOLS AS A NATURAL ALTERNATIVE TO THE USE OF SULPHITES IN WINEMAKING, PAG. 3 C D Figura 2. Electron micrographs of ultrathin sections of Pediococcus pentosaceus CIAL-85 non-incubated and incubated with antimicrobial agents. (a) control, (b) incubation with potassium metabisulfite (600 mg/l), (c) incubation with kaempferol (100 mg/l) and (d) incubation with kaempferol (100 mg/l). Bars = 1 m (a c), 0.2 m (d). (Figure taken from García-Ruiz et al., 2009). Overall, these results demonstrate the potential use of the polyphenols as an alternative to the traditional use of SO 2 in winemaking. However, practical level, it is unfeasible to think that the addition of individual compounds (obtained by organic synthesis) wine for controlling the growth of LAB, and therefore of MLF. The possible technological application of polyphenols with antimicrobial properties would necessarily pass through the use of phenolic extracts obtained by technical processes and economically viable. If in addition the extracts come from vegetable, that is, have the status of natural products, would be adding a double attraction to the procedure. Therefore it was proceeded to test the antimicrobial effect of vegetable phenolic extracts (n= 54) against LAB (L. hilgardii CIAL-49, L. casei CIAL-52, L. plantarum CIAL-92, P. pentosaceus CIAL- 85, O. oeni CIAL-91 and CIAL-96) and acetic acid bacteria (Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107) oenological, whose presence in wine is always associated with alteration processes. The extracts tested, all food grade, were from vegetable with different origins (including vines), and had different composition and phenolic content. The results showed that phenolic extracts have a wide antimicrobial spectrum against microorganisms present in the wine, showing, as in the case of polyphenols, certain selectivity against bacteria tested. In particular, eucalyptus and almond skin extracts were more active against BAL non-o. oeni while pomegranate #1, grape seed, cinnamon, grape skin and grape pomace #2 extracts were only active against O. oeni, being the quebracho tannins extract the most active against both acetic acid bacteria. Overall, these results confirmed that the inhibitory effect of phenolic extracts depended on their composition and phenolic content (Baydar et al., 2004; Özkan et al., 2004). Finally, to confirm the potential use of phenolic extracts as an alternative to SO 2 was necessary to demonstrate its antimicrobial capacity during winemaking. For this, it was developed a vinification process which comprises the addition of antimicrobial phenolic extracts of vegetable origin after alcoholic fermentation (Bartolomé et al., 2011). In particular, this procedure was applied in experiments of MLF in red wines (var. Merlot) at laboratory-scale and during ageing in wooden barrels of white wines (var. Verdejo) at winery-scale. According the results obtained in the culture medium for these experiments was selected the eucalyptus leaves extract. MLF experiments, both inoculated and spontaneous, performed at laboratory-scale in an industrially obtained red wine, showed that the addition of eucalyptus leaves extract (2 g/l) significantly delayed MLF, both spontaneous and inoculated, but lesser extent than the addition of potassium metabisulphite (30 mg/l) (Table 1).
GARCÍA-RUIZ ET AL., POLYPHENOLS AS A NATURAL ALTERNATIVE TO THE USE OF SULPHITES IN WINEMAKING, PAG. 4 Table 1. Percentage of disappearance of residual malic acid during MLF assays in red wines tested RESIDUAL MALIC ACID (%) INOCULATED MLF SPONTANEOUS MLF After 14 days After 19 days After 24 days After 14 days After 19 days Control <0.03 n.d. n.d. 40 <0.03 + Eucalyptus extract 10 <0.03 n.d. 55 <0.03 + SO 2 89 35 <0.03 n.d.: not determined Furthermore, and with the aim to evaluate the organoleptic impact of the addition of phenolic extracts on wine, was proceeded to characterize the volatile and phenolic fraction of wine studied (García-Ruiz et al., 2013). In comparison with the control wine, the wines added with eucalyptus leaves extract were characterized by a lower volatile content (except volatile phenols) and a high content of gallic acid, trans-resveratrol and flavonols and anthocyanin concentration similar. This latter indicated that the addition of phenolic extracts does not induce changes in the characteristics of the colour of wine, since anthocyanins are primarily responsible for the colour of red wine (Monagas et al., 2005). It is also important to mention that, in general, changes were more evident in the MLF experiments using inoculated bacteria than those carried out by spontaneous microbiota, which indicates different susceptibility of LAB to the antimicrobial properties of phenolic extracts. Overall, these results suggested that the addition of phenolic extracts during the elaboration of red wine would not lead to higher organoleptic changes than those produced during MLF per-se. Finally, winery-scale trial during white wine ageing in wood (6 months of storage) showed a lower population of LAB in wines treated with eucalyptus leaves extract (0.1 g/l) and SO 2 (80 mg/l) than in the control wine (SO 2 = 160 mg / L) (Table 2). This result confirmed that the use of phenolic extracts during wine ageing ensured microbiological stability and allow reduction of sulphite content. On the other hand, the analysis of the volatile and phenolic composition of these wines again showed a higher content of volatile phenols in wines added with eucalyptus leaves extract than in the control wine, as well as higher flavonol content. In turn, the results also showed that the storage period in the barrel, as expected, has a minor effect on the phenolic fraction than on the volatile fraction, as well as that the changes observed in the volatile composition of wines studied were mainly due to the effect of the wood of the barrel and not to the addition of antimicrobial phenolic extracts. Finally and in order to evaluate possible differences in the sensory profile of wines treated or not with phenolic extract, it was realized a discriminatory sensory analysis (triangle test), in which was detected minimum significant differences between wines aged in barrels in absence of extracts and the wines treated with phenolic extract.
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