Vinification

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Introduction

The process of making wine, also known as vinification, is very complex and involves intricate interactions between yeast and bacteria (1). In order to create the rich flavours that wine is known for, multiple steps of fermentation need to be carried out in a timed manner using various strains of yeast and bacteria in successive order (1). The first fermentation step involves the use of yeast to perform alcohol fermentation. The products of alcohol fermentation combine with grape juice to produce a pleasant aroma and flavour (8). Once alcohol fermentation is completed, lactic acid bacteria (LAB) perform malolactic fermentation (MLF) to form additional compounds which modify the existing grape juice and yeast compounds to further enhance the flavour (8). Traditional methods of vinification depended on naturally occurring LAB to spontaneously induce MLF. However, with increased knowledge about the growth of microbial species in wine, MLF can now be induced at the winemaker’s discretion using starter culture technologies (7, 9). Oenococcus oeni, the main contributor of MLF, can be inoculated in wine to control the timing and rate of MLF to reduce the chance of spoilage by other bacterial species (3). The activity of LAB can lead to improvement in wine quality due to the deacidification and stabilization of the wine (6).

Physical environment

Wine is a poor source of nutrients and makes MLF very difficult (1). The low pH, high ethanol content, presence of sulphur dioxide, and low nutrition all contribute to the inhibition of yeast and bacterial growth in wine (9). Because wine is an anaerobic environment, yeasts ferment sugars present in the grape juice (3). Following alcohol fermentation, LAB ferment the smaller organic compounds produced by the yeast. However, SO2 mainly exists as HSO3- at low pH and is able to react with carbonyl compounds (3). If the carbonyl products of the primary fermentation react with this HSO3-, the secondary fermenters are unable to perform their fermentation. Bacterial diversity is limited because of the extreme conditions present in wine.

Biological interactions

Because wine is a complex system of microbial activity, there are many interactions among various yeasts and bacteria. Yeasts produce many inhibitory compounds such as SO2, ethanol, medium chain fatty acids, and antimicrobial peptides, which act against bacterial growth (1, 3). It’s important that yeasts complete alcohol fermentation prior to the induction of MLF because 4% [v/v] ethanol is enough to inhibit growth of most LAB (9). High levels of medium chain fatty acids and a very low pH can inhibit the ATPase activity of O. oeni, which can reduce the rate of MLF (9). In addition, the completion of alcohol fermentation by yeasts yields the production of missing substances, like vitamins and amino acids, which allow LAB such as Lactobacillus to thrive (1). One example of a bacterial interaction that can have negative consequences on vinification is the production of Brevicin. Brevicin is a bacteriocin produced by Lactobacillus brevis that inhibits the growth of O. oeni (1). Even though few species of yeasts and bacteria grow in wine, the interactions between them are extremely important.

Microbial processes

Alcohol fermentation is the primary fermentation of wine in which sugars such as glucose are converted to ethanol and carbon dioxide. MLF is the secondary fermentation of wine which is the enzymatic [1] decarboxylation of L-malic acid to L-lactic acid (3). MLF leads to the deacidifcation of wine and results in an increase in growth rate of LAB as well as an enhancement of the wine’s flavour and aroma (9). The wine’s flavour is enhanced during MLF because LAB hydrolyze glycosylated aroma compounds in the grape juice (11).

Fermentation of the citric acid that is also present in the grape juice by LAB results in the formation of diacetyl, a compound that gives wine a buttery or nutty flavour (3). Acetic acid and ethyl acetate are fermentation products that need to be avoided because they give wine an undesirable flavour (5). The metabolism of each microorganism is a large determinant in the final taste of wine.

Key Microorganisms

Because vinification requires two types of fermentation, both fungi and bacteria are required to complete the process. Saccharomyces is a genus of unicellular fungi that can grow in both aerobic and anaerobic environments in the presence of a fermentable sugar. Saccharomyces cervisiae is the main contributor of alcohol fermentation because of its tolerance to high concentrations of ethanol (10). Pina et al. (2004) determined that S. cervisiae are able to modify the lipid composition of their plasma membrane according to the increasing stress applied by the increasing ethanol concentration (10).

Lactobacillus is a genus of Gram-positive microaerophilic bacteria that require rich media with a fermentable sugar to grow. These organisms are involved in the secondary fermentation of wine and contribute to MLF (1).

Oenococcus is a genus of Gram-positive acidophilic facultative anaerobes that can grow at a pH of 4.8 at 18-30 degrees Celsius and require rich media with tomato or grape juice (1).

These organisms are the main contributors of MLF because they are not inhibited by the high level of ethanol produced after alcohol fermentation is complete (1, 3). Additionally, Oenococcus do not produce any secondary compounds, like acetic acid, that can impair wine quality (1, 2). O. oeni and other LAB rely on ATPase activity to maintain their intracellular pH while growing in acidic environments (6).

Current Research

After analysis of LAB in vinification, it was discovered that LAB are able to inhibit the growth of pathogenic bacteria through their ability to outcompete pathogens for nutrients, survive the low pH, and secrete antimicrobial compounds (4). It was previously thought that by inoculating O. oeni into raw food, food-borne pathogens including Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enteridis can be controlled (4). Chiang et al. (2012) tested the ability of O. oeni to inhibit the growth of these common pathogens and found that of the 24 strains of O. oeni tested, 17 strains were able to inhibit E. coli O157:H7, L. monocytogenes, and S. enteridis (4). The application of O. oeni in pathogen control will have major economical, ecological, and health implications, but the safety of such a method needs to be confirmed by further testing.

References

(1) Costantini, A., Garcia-Moruno, E., and Moreno-Arribas, M.V. “Biochemical Transformations Produced by Malolactic Fermentation.” Wine Chemistry and Biochemistry, 2009, doi: 10.1007/978-0-387-74118-5 2

(2) Borneman, A.R., McCarthy, J.M., Chambers, P.J., and Bartowsky, E.J. “ Comparative analysis of the Oenococcus oeni pan genome reveals genetic diversity in industrially-relevant pathways.” BMC Genomics, 2012, doi: 10.1186/1471-2164-13-373

(3) Nielsen, J.C., and Richelieu, M. “Control of Flavor Development in Wine during and after Malolactic Fermentation by Oenococcus oeni.” Applied and Environmental Microbiology, 1999, 0099-2240/99

(4) Chiang, I., Worobo, R.W., Churey, J.J., and Henick-Kling, T. “Growth Inhibition of Foodborne Pathogens by Oenococcus oeni.” Journal of Food Science, 2011, doi: 10.1111/j.1750-3841.2011.02446.x

(5) Campos, F.M., Couto, J.A., and Hogg, T.A. “Influence of phenolic acids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii.” Journal of Applied Microbiology, 2003.

(6) Carrete, R., Vidal, M.T., Bordons, A., and Constanti, M. “Inhibitory effect of sulfur dioxide and other stress compounds in wine on the ATPase activity of Oenococcus oeni.” FEMS Microbiology Letters, 2002, doi: 0378-1097 / 02

(7) Mangani, S. Guerrini, S., Granchi, L., and Vincenzini M. “Putrescine Accumulation in Wine: Role of Oenococcus oeni.” Current Microbiology, 2005, doi: 10.1007/s00284-004-4425-1

(8) D’Incecco, N., Bartowsky, E., Kassara, S., Lante, A., Spettoli, P., and Henschke, P. “Release of glycosidically bound flavour compounds of Chardonnay by Oenococcus oeni during malolactic fermentation.” Food Microbiology, 2004, doi: 10.1016/j.fm.2003.09.003

(9) Alexandre, H., Costello, P.J., Remize, F., Guzzo, J., and Guilloux-Benatier, M. “Saccharomyces cerevisiae–Oenococcus oeni interactions in wine: current knowledge and perspectives.” International Journal of Food Microbiology, 2004, doi: 10.1016/j.ijfoodmicro.2003.10.013

(10) Pina, C., Santos, C., Couto, J.A., and Hogg, T. “Ethanol tolerance of five non Saccharomyces wine yeasts in comparison with a strain of Saccharomyces cerevisiae—influence of different culture conditions.” Food Microbiology, 2004, doi: 10.1016/j.fm.2003.10.009

(11) Ugliano, M., Genovese, A., and Moio, L. “Hydrolysis of wine aroma precursors during malolactic fermentation with four commercial starter cultures of Oenococcus oeni.” Journal of Agricultural and Food Chemistry, 2003, doi: 10.1021/jf0342019