Sources of acetic and other fatty acids and their role in sluggish and stuck red wine fermentations

Du Toit, Wessel J. (Wessel Johannes) (2000-04)

Thesis (MScAgric)--University of Stellenbosch, 2000.

Thesis

ENGLISH ABSTRACT: The quality of wine is influenced by numerous factors. These factors include the quality of the grapes, winemaking techniques and quality control throughout the winemaking process. It is thus very important that any process leading to the lowering of the quality of the wine be prevented. Evidence in the wine industry shows that bacterial spoilage is still very much a common problem in many wineries. The spoilage of wine by bacteria can lead to amongst other problems, elevated volatile acidity levels, of which only a certain concentration limit in wine is permitted. Usually more than 90% of the volatile acidity of wine consists of acetic acid. Different yeast strains, heterofermentative lactic acid bacteria and acetic acid bacteria (which can all be spoilage microorganisms) can produce acetic acid in high concentrations. It is thus important to be able to prevent the formation of this acid by controling the unwanted growth of these spoilage microorganisms. Acetic acid and other medium chain fatty acids, octanoic- and decanoic acid, can also lead to stuck or sluggish fermentations. A stuck or sluggish fermentation can also lead to wine spoilage, due to sugar remaining in the fermentation which can be utilized by spoilage microorganisms. Acetic- and other fatty acids enter the yeast cell by passive diffussion and releases its proton in the cytoplasm, thereby acidifying the cytoplasm and inhibiting some enzymes. These acids can also work synergistically with ethanol and its inhibitory effect is also dependent on the temperature. Yeast strains can also differ in their resistance to acetic and other medium chain fatty acids and these acids can also influence the growth of lactic acid bacteria. How acetic acid bacteria influence the winemaking process and the used measures to keep these bacteria from spoiling wine have been the subject of very little attention in the past. This was due to the belief that the anaerobic conditions prevailing in wine and the use of sulfur dioxide are enough to control these bacteria, since acetic acid bacteria were always described as being strictly aerobic microorganisms. Recently, some evidence showed that acetic acid bacteria can survive and even overcome the limits that the winemaking process places on its growth. These bacteria are also known to inhibit the yeasts growth and fermentation ability due to the production of acetic acid and other factors. A research programme on the origin of volatile acidity in South African wines had been initiated at the Department of Viticulture and Enology and at the Institute for Wine Biotechnology at the University of Stellenbosch after increases in volatile acidity in different South African wines had been reported. This spurred us to investigate the occurrence of acetic acid bacteria in South African red wine fermentations, which forms part of this study, and to identify the dominant acetic acid bacterial strains. The sulfur dioxide resistance of five representative strains were also determined, as well as the effect of metabolites which were produced by these bacteria on yeast growth and fermentation ability. Our results indicate that acetic acid bacteria can occur in high concentrations in the fresh must and during alcoholic fermentation. In the 1998 harvesting season acetic acid bacteria occurred at 106-107 cfu per ml in the fresh must. In 1999 these numbers were 104-105 cfu/ml. Acetic acid bacteria numbers decreased in 1998 to 102-103 cfulml during fermentation. The survival of these bacteria in 1999 correlated with the pH of the must, as well as sulfur dioxide dosages in the must. In must with a low pH and higher sulfur dioxide the number of acetic acid bacterial numbers decreased more drastically than in the high pH, low sulfur dioxide musts. This was also true for acetic acid bacterial counts during cold soaking of musts, with the number of acetic acid bacteria increasing during the cold soaking period in musts with a high pH. In musts with a low pH and higher S02 dosages acetic acid bacterial counts did not, however, increase during cold soaking. Gluconobacter oxydans dominated in the fresh must with Acetobacter liquefaciens and especially Acetobacter pasteurianus dominating during the fermentation. Different biochemical and physiological tests revealed that 52% of the 115 isolates tested belong to A. pasteurianus. The high occurrence of A. liquefaciens with A. pasteurianus during fermentation showed that the dominant acetic acid bacterial species in South Africa differed from reports from other wine producing countries. The sulfur dioxide resistance of the acetic acid bacteria tested also differed in white grape juice, with a molecular sulfur dioxide concentration of 0.64 mg/I being necessary to eliminate all the acetic acid bacterial strains tested. The A. hansenii strain was found to be the most resistant to sulfur dioxide and G. oxydans the least resistant. The latter strain was eliminated by only 0.05 mg/I molecular sulfur dioxide, while A. hansenii was only eliminated by 0.64 mg/I molecular sulfur dioxide. The A. pasteurianus, A. liquefaciens and A. aceti strains tested displayed varying degrees of resistance to sulfur dioxide. The volatile acidity produced by these bacteria profoundly influenced the growth and fermentation ability of yeast, which led to slow/stuck fermentation. The A. hansenii and A. pasteurianus strains produced the most volatile acidity in grape juice, with up to 4.02 g/I for A. hansenii within 4 days, which led to a stuck alcoholic fermentation. This was, however, prevented by inhibiting or eliminating the acetic acid bacteria with sufficient sulfur dioxide additions prior to yeast inoculation. Compounds produced by acetic acid bacteria can also influence wine quality. Certain organic acids were produced and metabolized by acetic acid bacteria, as well as acetoin. We could not, however, detect any other fatty acids that are inhibitory to yeast (produced by these bacteria). This study clearly showed that acetic acid bacteria could occur during fermentation and that certain winemaking techniques, like the maintenance of a low pH in the must and sulfur dioxide additions can influence the growth and survival of acetic acid bacteria. Acetic acid bacteria also influence both the winemaking process by inhibiting yeast as well as the quality of the wine by producing acetic acid and/or other compounds. This study also shed some light on the occurrence of acetic acid bacterial species in the South African context and could be important in assisting the winemaker, as well as the scientific reseacher, in finding ways to inhibit acetic acid bacteria in the ongoing battle against these spoilage microorganisms of wine.

AFRIKAANSE OPSOMMING: Wynkwaliteit word deur verskillende faktore beinvloed. Dit sluit die druifkwaliteit, wynmaak tegnieke en kwaliteitsbeheer deur die wynmaakproses in. Enige prosesse en faktore wat tot die verlaging in wynkwaliteit kan lei moet dus ten alle koste voorkom word. Die bederf van wyn deur bakterieë kan en is 'n algemene probleem in enige kelder. Bakteriese bederf kan, onder andere, lei tot verhoogte vlugtige suurheid, waarvan 'n sekere konsentrasie limiet in wyn toegelaat word. Asynsuur maak gewoonlik 90% van die vlugtige suurheid uit. Asynsuur kan deur verskillende gisrasse, heterofermantatiewe melksuurbakterieë en asynsuurbakterieë (wat almal wyn kan bederf) gevorm word. Die vorming van asynsuur in wyn kan dus voorkom word deur die ongewenste groei van dié organismes te voorkom. Asynsuur en ander medium ketting vetsure, soos oktanoë- en dekanoësuur, kan ook tot slepende of gestaakte gistings lei. Suiker wat in die wyn agterbly wat In slepende/gestaakte fermentasie ondergaan kan deur bederf bakterieë gebruik word om die wyn te bederf. Ongedissosieerde asynsuur en ander vetsure dring die gissel binne deur passiewe diffussie en stel 'n proton vry in die sitoplasma wat sitoplasma versuur en sekere ensieme inhibeer. Hierdie sure werk ook sinergisties met etanol en hul inhiberede effek is ook temperatuur afhanklik. Gisrasse verskil in hul weerstandbiedendheid teen asynsuur- en ander mediumketting vetsure en dié vetsure kan ook melksuurbakterieë se groei beïnvloed. Hoe asynsuurbakterieë wyn bederf en die aksies wat geneem kan word om dit te verhoed is in die verlede nie baie ondrsoek nie. Dit is hoofsaaklik daaraan toe te skryf dat geglo is dat die anaerobiese kondisies in wyn en die gebruik van swaweidioksied die groei van asynsuurbakterieë, wat altyd beskryf is as streng aerobe mikroorganismes, kan beheer. Daar is onlangs aangetoon dat asynsuurbakterieë kan oorleef in wyn en selfs die ongunstige kondisies daarin kan oorkom. Hierdie bakterieë is ook in staat om gisgroei en fermentasie vermoë te inhibeer deur die produksie van asynsuur en ander faktore. In Navorsingsprogram om die oorsprong van verhoogde vlugtige suurheid in Suid-Afrikaanse wyne te bepaal is deur die Departement Wingerd- en Wynkunde en die Instituut vir Wynbiotegnologie van die Universiteit van Stellenbosch geinisieer. Dit het ons aangemoedig om die voorkoms van asynsuurbakterieë in Suid-Afrikaanse rooiwyngistings, wat deel vorm van hierdie ondersoek, en ook die dominante asynsuurbakterie rasse te identifiseer. Die swaweidioksied bestandheid van vyf verteenwoordegende rasse en die effek wat metaboliete wat deur dié bakterieë geproduseer is op gisgroei en gisitingsvermoë is bepaal. Ons resultate bewys dat asynsuurbakterieë teen hoë getalle in vars mos en gedurende alkoholiese gisting kan voorkom. Asynsuurbakterieë het gedurende die 1998 seisoen teen 106-107 kve/ml en in 1999 teen 104-105 kve/ml in die vars mos voorgekom. Gedurende fermentasie het hierdie getalle in die 1998 seisoen gedaal na 102-103 kve/ml. Die oorlewing van hierdie bakterieë het gedurende die 1999 seisoen gekorrelleer met die pH en swaweidioksied konsentrasies van die mos. In die lae pH, hoë swaweidioksied moste het asynsuurbakterie getalle vinniger en meer dramaties gedaal as in die hoë pH, lae swaweidioksied moste. Asynsuurbakterie getalle het dieselfde tendens getoon in moste gedurende dopkontak by lae temperature. In moste met 'n hoë pH het asynsuurbakterie getalle toegeneem gedurende koue dopkontak, terwyl dit nie gebeur het nie in moste met 'n lae pH en hoë swaweidioksied konsentrasies. In die vars mos het Gluconobacter oxydans en gedurende die fermentasie Acetobacter liquefaciens en veral Acetobacter pasteurianus oorheers. Verskillende biochemiese en fisiologiese toetse het bepaal dat 52% van die 115 isolate A. pasteurianus is. Die hoë voorkoms van A. liquefaciens saam met A. pasteurianus gedurende gisting bewys dat die voorkoms en dominansie van asynsuurbakterieë in Suid- Afrika verskil van ander wynproduserende lande. Die swaweidioksied weerstandbiedendheid van die asynsuurbakterieë wat getoets het, het ook verskil, met 0.64 mg/I molekulêre swaweIdioksied nodig om hul almal te elimineer in wit druiwesap. Die A. hansenii en G. oxydans rasse was die mees weerstandbiedend en sensitiefste onderskeidelik ten opsigte van swaweidioksied. Slegs 0.05 mg/I molekulêre swaweidioksied was voldoende om G. oxydans te elimineer, terwyl A. hansenii deur 0.64 mg/I molekulêre swaweidioksied geëlimineer is. Die A. pasteurianus, A. liquefaciens en A. aceti rasse het verskillende swaweidioksied weerstandbiedendheid getoon. Die vlugtige suurheid wat deur dié bakterieë geproduseer is het die groei en gistingvermoë van gis drasties beinvloed, wat tot slepende/gestaakte fermentasies gelei het. Die A. hansenii en A. pasteurianus rasse het die meeste vlugtige suurheid geproduseer, met tot 4.02 g/I geproduseer deur A. hansenii na vier dae se groei, wat tot 'n gestaakte fermentasie gelei het. Dit is egter voorkom deur die asynsuurbakterieë te elimineer deur genoegsame swaweidioksied toevoegings tot die mos voor gisinokulasie te doen. Verbindings wat deur asynsuurbakterieë geproduseer word kan ook wynkwaliteit beinvloed. Sekere anorganiese sure is deur hierdie bakterieë geproduseer, terwyl ander gemetaboliseer is. Asetoïen is geproduseer deur die getoetse asynsuurbakterieë. Ons kon egter nie ander vetsure wat gis inhibeer opspoor nie. (Geproduseer deur die bakterieë). Hierdie studie bewys dat asynsuurbakterieë gedurende alkoholiese fermentasie kan voorkom en dat sekere wynmaaktegnieke, soos die verkryging van moste met 'n lae pH en genoegsame swaweidioksied toevoegings die groei en oorlewing van asynsuurbakterieë kan beivloed. Asynsuurbakterieë kan ook beide die wynmaakproses, deur giste te inhibeer, en die wynkwaliteit beivloed deur die produksie van asynsuur en/of ander verbindings. Hierdie studie het ook kennis oor die voorkoms van asynsuurbakterieë in Suid-Afrikaanse moste verbeter en kan ook as 'n nuttige hulpmiddel dien vir die wynmaker en navorser in die stryd om hierdie bederf organismes van wyn te elimineer.

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