Oenococcus oeni

Oenococcus oeni è un batterio lattico di notevole importanza enologica per la sua capacità di condurre la fermentazione malolattica in vino. Isolato principalmente da uva fermentata e succhi di frutta, O. oeni è il batterio responsabile della fermentazione malo-lattica nel vino. Questo processo non è una vera e propria fermentazione, ma una degradazione dell’acido malico ad acido lattico necessario per la maturazione del vino.

O. oeni is a Gram-positive bacterial species in the genus of Oenococcus. It was the only species in the genus until 2006, when the species Oenococcus kitaharae was identified. As its name implies, O. oeni holds major importance in the field of oenology, where it is the primary bacterium involved in completing the malolactic fermentation.

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Esso presenta peculiari caratteristiche genetiche (mancanza del sistema mutS-mutL, codificanti per due enzimi chiave del Mismatch Repair Pathway, caso unico tra le Lactobacillales) e genomiche (genoma più piccola finora descritto per un batterio lattico, circa 1,8 Mb), ed è perfettamente adattato ad una nicchia specifica estremamente selettiva come il vino.

The genus Oenococcus has one main member involved in winemaking, O. oeni, once known as Leuconostoc oeni. Despite having the name Oenococcus, under the microscope, the bacterium has a bacillus (shape) rod shape. The bacteria is a Gram-positive, facultative anaerobe that can utilize some oxygen for aerobic respiration but usually produces cellular energy through fermentation. O. oeni is a heterofermenter that creates multiple end products from the use of glucose with D-lactic acid and carbon dioxide being produced in roughly equal amounts to either ethanol or acetate. In reductive conditions (such as near the end of alcoholic fermentation), the third end product is usually ethanol while in slightly oxidative (such as early in alcohol fermentation or in an untopped barrel), the bacteria are more likely to produce acetate.

O. oeni funge da stabilizzatore delle comunità microbiche del vino consumando le fonti alimentari e tenendo sotto controllo le altre specie microbiche, potenzialmente dannose. A seconda delle caratteristiche dell’uva, del clima e del tipo di vino, questo batterio ha un impatto significativo e determinante sulle qualità del vino. A livello industriale, vengono solitamente utilizzati ceppi selezionati e standardizzati di O. oeni, ma sono comunque estremamente importanti le condizioni ambientali di crescita per questo microrganismo. O. oeni comunemente domina la fermentazione secondaria per la sua estrema tolleranza a variazioni di pH e di livelli di etanolo. Grazie alle sue proprietà eterofermentanti, è considerato come un ingegnere dell’ecosistema e riesce a creare condizioni favorevoli per la crescita di altri batteri lattici.

Some O. oeni strains can use fructose to create mannitol (which can lead to wine fault known as mannitol taint), while many other strains can break down the amino acid arginine (which can be present in the wine that is resting on the lees after fermentation from the autolysis of dead yeast cells) into ammonia.

L’Oenococcus oeni è un batterio Gram-positivo appartenente alla categoria dei batteri lattici, più precisamente alla famiglia delle Leuconostocaceae. Le cellule di O. oeni hanno forma di bacillo: generalmente, sono disposte in coppia o a catenelle e sono immobili. Solitamente, non producono spore.

In addition to the hexose glucose and fructose sugars, most strains of O. oeni can use the residual pentose sugars left behind from yeast fermentation including L-arabinose and ribose. Only around 45% of O. oeni strains can ferment sucrose (the form of sugar usually added for chaptalization that gets converted by yeast into glucose and fructose).

O. oeni è un batterio anaerobio facoltativo: utilizza l’ossigeno per la respirazione cellulare, ma produce energia tramite fermentazione. A differenza degli altri lattobacilli, è in grado di sopravvivere ad ambienti particolarmente acidi, con pH inferiore a 3 e tollera livelli di etanolo superiori al 10%. Per queste sue caratteristiche è il principale responsabile della fermentazione malo-lattica, ovvero della conversione del glucosio e dell’acido malico ad acido lattico. Per questo pathway metabolico, O. oeni è classificato come eterofermentante: ovvero, è in grado di produrre diverse molecole dalla fermentazione degli zuccheri. Nello specifico, tra i prodotti principali ci sono l’anidride carbonica, l’etanolo, l’acetato e il diacetile, che è una molecola che compone il bouquet aromatico del vino.

Winemakers tend to prefer O. oeni for several reasons. First, the species is compatible with the main wine yeast Saccharomyces cerevisiae, though in cases where both MLF and alcoholic fermentation are started together, the yeast most often outcompetes the bacterium for nutritional resources which may cause a delay in the onset of malolactic fermentation. Second, most strains of O. oeni are tolerant to the low pH levels of wine and can usually deal with the standard alcohol levels that most wines reach by the end of fermentation. Additionally, while sulfur dioxide levels above 0.8 molecular SO2 (pH dependent but roughly 35-50 ppm) will inhibit the bacteria, O. oeni is relatively resistant compared to other LAB. Finally, O. oeni tends to produce the least amount of biogenic amines (and most lactic acid[3]) among the lactic acid bacteria encountered in winemaking.

O. oeni non è un microrganismo patogeno per l’uomo o gli animali. Anzi, è un microrganismo in grado di contrastare i batteri patogeni che proliferano negli alimenti, come Salmonella enteridis e Listeria monocytogenes.

Il raggiungimento di elevati livelli qualitativi nei vini prodotti è spesso influenzato da un corretto compimento della “fermentazione” malolattica, trasformazione batterica utile e gradita nella tecnica enologica, non solo per equilibrare i vini dal punto di vista acidico, ma anche per l’ottenimento di prodotti sensorialmente più complessi e strutturati.

Malolactic fermentation (MLF) is the process by which bacteria convert malic acid into lactic acid and carbon dioxide. These lactic acid-producing bacteria can include Oenococcus oeni and other species of Pediococcus and Lactobacillus. Bacteria may be naturally present in the winemaking equipment (such as used oak barrels), or the winemaker may inoculate the wine with a specific malolactic culture, such as O. oeni. Malolactic conversion happens after or during yeast fermentation (primary fermentation), which is why it's sometimes called secondary fermentation.

La fermentazione malolattica, in realtà, non è tecnicamente una fermentazione dal punto di vista chimico, bensì si tratta di una conversione malo-lattica, ovvero una reazione di decarbossilazione dell’acido malico ad acido lattico catalizzata dall’enzima malolattico. Contemporaneamente avviene la conversione del gruppo acido (-COOH) in acqua e anidride carbonica.

Malolactic fermentation (MLF) is an integral part of winemaking, resulting from the metabolic activity of lactic acid bacteria (LAB). It gives the final wines better palatability by reducing the tart taste associated with malic acid, and provides additional improvements, like microbial stability and enhanced aroma complexity. Oenococcus oeni is the typical bacteria species isolated from spontaneous MLF, and it is used as a widespread starter culture for this process. Some Pediococcus strains have also been shown to be suitable to drive wine MLF. These are the main wine LAB involved in this process as well as the influence of some wine-related physicochemical factors on their growth and metabolism.

La reazione è mediata dai lattobacilli, batteri lattici appartenenti principalmente alle specie Oenococcus oeni e Lactococcus lacti, che sono naturalmente presenti nel mosto. I batteri lattici si trovano sulle bucce delle uve, ma anche negli ambienti di cantina come silenti colonizzatori. Possono dar luogo a fermentazioni spontanee attaccando gli zuccheri residui nei mosti dei vini. In questo caso danno vita a fermentazioni eterolattiche, generando anche dei sottoprodotti indesiderati come l’acido acetico.

Which Wines Undergo Malolactic Fermentation? The growth of MLB is generally encouraged where MLF is required to reduce the acidity of the wine. The reduction of acidity is beneficial to the quality of wines made in cool winegrowing regions, because the grapes naturally contain high levels of organic acids. Worldwide consumer preference currently favours fruit-driven wines with moderate acidity, resulting in acid reduction becoming a critical issue with wines produced in cool climates. This, coupled with positive flavour changes associated with growth of MLB in wine, has made MLF a desirable process for almost all red wines and for certain styles of white wines. Growth of LAB in wine must be controlled to ensure desirable MLB that produce no off-flavours. In most cases, MLF should complete rapidly to save processing time and achieve early stability of the product. In no instance should indigenous strains of LAB be relied on to conduct the MLF

A causa dell’elevata acidità dei vini e delle basse temperature stagionali, la trasformazione malolattica rischia di instaurarsi con difficoltà dopo un forzato periodo di attesa che può creare ripercussioni negative sulla qualità del vino. Da qui nasce l’esigenza di pianificare la trasformazione microbiologica attraverso l’impiego di colture starter costituite da miscele di batteri sicuri e selezionati.

After alcoholic fermentation, most red wines—like pinot noir—undergo the purposeful conversion of malic acid into lactic acid, and about one-fifth of white wines do as well. Certain white wine grapes, such as chardonnay and cabernet sauvignon, lend themselves better to MLF than others like riesling and gewürztraminer, which tend to be more sugary. The region and climate also impact the use of MLF. Malolactic fermenting is more likely to occur in colder regions, such as Burgundy and Champagne, where low temperatures can cause grapes to become more acidic. Red wines prior to malolactic fermentation tend to be harsh and astringent on the palate. After the malic acid is converted to lactic, the wine becomes noticeably softer and more approachable. Wines are perceivably heavier and rounder on the palate and will continue to gain weight through the aging process.

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La fermentazione malolattica è un evento caratteristico che porta il vino a maturazione e avviene successivamente alla fermentazione alcolica. Generalmente, inizia spontaneamente a fine inverno quando il rialzo termico (18-20 °C) favorisce la crescita dei batteri lattici. Affinché questo tipo di fermentazione abbia inizio, infatti, sono necessarie alcune condizioni: • Il pH del vino non deve essere eccessivamente basso; • Non deve esserci un eccesso di anidride solforosa; • L’alcol etilico non deve superare il 15%; • La temperatura del vino deve essere compresa tra i 18 e i 20°C.

Malolactic fermentation (MLF) is generally considered to be a simple bacterial breakdown of L-malic acid in red wines and some white wines, with the accompanying release of CO2, the formation of L-lactic acid and a reduction in the total acid content. The simple conversion of L-malic acid to L-lactic acid could be an oversimplification of the process, as MLF involves much more than that. The breakdown of L-malic acid to L-lactic acid imparts microbiological stability, while the formation of various metabolic end products influences sensory aspects of the wine. The overall reduction in acid, together with the accompanying increase in pH, results in better, “softer” and “rounder” wines with increased body

Durante la fermentazione malolattica, l’acido malico, che è più aspro, si trasforma in acido lattico, che è più debole e dona un gusto più delicato e meno acre al vino. Questo permette quindi di ottenere un vino più morbido ed equilibrato, ma allo stesso tempo corposo per via della concentrazione di polisaccaridi.

Malolactic fermentation is typically conducted in the same fermentor as alcoholic fermentation, prior to racking and maturation. Nevertheless, there have been many anecdotal reports that wine having undergone malolactic fermentation in oak cooperage is preferable to the same wine having undergone malolactic fermentation in stainless steel. This has been confirmed by Vivas et al. (1995). Their results indicate that relative color intensity and stability of red wine is increased following malolactic fermentation in oak cooperage. This is correlated with increased anthocyanin–tannin polymerization. In addition, astringency is apparently reduced if malolactic fermentation occurs in oak cooperage, generating a smoother, richer wine. The researchers also commented that oak and fruit flavors were considered more balanced and harmonious. These perceptions persisted for at least 3 years after bottling.

THE INFLUENCE OF MALOLACTIC FERMENTATION ON WINE COMPOSITION

MLF is not only the simple decarboxylation of L-malic acid to L-lactic acid and CO2. Wine components are consumed by wine LAB, which means they will produce metabolic end products from those components. This has an influence on grape-derived aroma compounds and on those arising from the AF, and confers biological stability on the final product.

WELL-KNOWN FACTORS THAT AFFECT MALOLACTIC FERMENTATION

The best understood factors that govern successful MLF are SO2, pH, alcohol and temperature. For MLF to be successful, the values of these chemical parameters must correspond to those that allow the bacterial cultures to function successfully. A favourable level of any one of these components may compensate for an unfavourable level of one or several of the others. It is important to remember these factors function synergistically, i.e., their actions together have a greater total effect than the sum of their individual actions

INTERACTION BETWEEN YEAST AMD BACTERIA

Wine is not only a product made out of grapes and modified by technical means. Wine is a product of a complex microbial fermentation of the grape juice involving the sequential development of various species of yeasts and lactic acid bacteria. In wine fermentation, the perhaps earliest use of biotechnology, the final quality of the wine can be modified in two major ways, (1) by the quality of the grapes and juice preparation, and (2) by the selection of most suitable yeasts and bacteria and control of fermentation condition. The task of the winemaker is to ensure that the desired fermentative yeasts and malolactic bacteria strains predominate in the juice and wine and carry out the fermentations. Today, most major producers of wine inoculate the juice with selected strains of yeast and bacteria. Very often, starter culture failures are due to improper preparation and inoculation procedures. In some cases, starter culture failure may be due to antagonistic interactions between yeast and bacteria

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utilizzo

Acid reduction

La fermentazione malolattica origninariamente utilizzata nei vini rossi, è ormai stablmente introdotta ed utilizzarta anche nei vini bianchi più importanti, che grazie ad essa risultano dotati di grande morbidezza.

Malolactic fermentation decreases acidity, since malic acid is more acidic than softer lactic acid. MLF can be desired in such wines to confer a degree of biological stability and/or to impart flavour complexity, necessitating the use of acidulants to adjust wine acidity and pH to acceptable levels after MLF, the reduction of total acidity can lead to spoilage, so winemakers sometimes have to re-acidify wines by adding tartaric acid. The increase in wine pH accompanying MLF can also influence wine colour.

3 Ways Malolactic Fermentation Affects the Taste of Wine Malolactic fermentation can add flavor and a rounder, creamier mouthfeel to some wines, while reducing aroma in others. There are three primary reasons for this:

1. Diacetyl: Diacetyl is a byproduct of malolactic conversion that has a nutty, toasted flavor at low concentrations and an overwhelming buttery flavor at higher concentrations. Diacetyl is responsible for the buttery flavor of certain Chardonnays. The amount of diacetyl present in a wine depends on levels of citric acid, sulfur dioxide, temperature, oxygen, and pH during malolactic fermentation. 2. Malic acid: Malolactic fermentation reduces malic acid, which has a tart, green apple flavor. Depending on the wine style, winemakers may choose to avoid MLF or have only a portion of the wine undergo MLF to preserve the tart flavor of malic acid. 3. Acetic acid: Acetic acid may be another byproduct of malolactic fermentation. Too much acetic acid can make a wine taste vinegary.

Effetti organolettici della fermentazione malolattica

Flavor

I batteri lattici tendono a metabolizzare non solo l’acido malico, ma anche gli zuccheri residui, se sono ancora presenti nel mosto dopo la fermentazione alcolica. Inoltre, la maggior parte dei batteri lattici sono eterofermentativi: generano cioè non solo acido lattico e i suoi sali, ma anche metaboliti secondari (acetoino, diacetile, acido acetico). Questi possono alterare il profilo aromatico del vino sia positivamente che negativamente. Ad ogni modo, l’effetto principale della fermentazione malolattica è quello di ridurre l’acidità del vino: nello specifico, il consumo di 1 g/L di acido malico fa diminuire l’acidità totale di 0,4 g/LH2SO4. Nello specifico durante la fermentazione malolattica aumenta l’acidità volatile, ma diminuisce quella totale.

Although there has been conjecture over the contribution of MLF to the sensory properties of wine, more recent research has provided greater insight into specific sensory changes associated with the growth and metabolic activity of malolactic bacteria in wine. It is clear that different strains of malolactic bacteria may increase or decrease the intensity of certain wine aroma and flavour attributes, and such changes are strain dependent. In addition to deacidification, the flavour attributes imparted by MLF can be described as buttery, toasty, fruity, spicy, lactic, nutty, yeasty, oaky, sweaty and earthy. Some common terms used to describe the positive effects of MLF on wine flavour are nutty,. These descriptors can be attributed to the production of flavour-active compounds, or to the modification of existing flavour components. In addition, the body of the wine can be affected, making it softer and richer. MLF may also impact fruity and vegetative aromas, as well as the mouthfeel of wine, MLF can add a buttery, creamy complexity to wine by mellowing out tart fruity flavors. It also can make for softer wines with a full, smooth mouthfeel.

INTERACTION BETWEEN YEAST AMD BACTERIA

Wine is not only a product made out of grapes and modified by technical means. Wine is a product of a complex microbial fermentation of the grape juice involving the sequential development of various species of yeasts and lactic acid bacteria. In wine fermentation, the perhaps earliest use of biotechnology, the final quality of the wine can be modified in two major ways, (1) by the quality of the grapes and juice preparation, and (2) by the selection of most suitable yeasts and bacteria and control of fermentation condition. The task of the winemaker is to ensure that the desired fermentative yeasts and malolactic bacteria strains predominate in the juice and wine and carry out the fermentations. Today, most major producers of wine inoculate the juice with selected strains of yeast and bacteria. Very often, starter culture failures are due to improper preparation and inoculation procedures. In some cases, starter culture failure may be due to antagonistic interactions between yeast and bacteria

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EFFETTI NEGATIVI

SPOILAGE BY LACTIC ACID BACTERIA

I batteri lattici eterofermentativi selvaggi possono avere potenzialmente un effetto negativo sulle proprietà organolettiche del vino. Specialmente quando la microflora è di dubbia natura, c’è il rischio che si producano sottoprodotti indesiderati. Per questo, soprattutto se si desidera produrre vini di eccellente qualità senza difetti, nelle grandi e piccole produzioni si consiglia l’utilizzo di batteri lattici specifici come starter per supportare la fermentazione malolattica ed allo stesso tempo condizionare positivamente la microflora generale e contrastare possibili colonizzazioni e preponderanze di microflore avverse selvagge.

Wines that have completed a controlled MLF are described with such positive sensory descriptors as buttery, nutty, yeasty, honey, vanilla, spicy, earthy, toasty and fruity. Texturally, these wines are described as having more body and roundness, greater length on the palate and silky tannins. Conversely, wines that have undergone uncontrolled MLF, especially at pH levels greater than 3.5, are described with such negative descriptors as intense lactic aroma, acid yogurt, rancid, sweaty, acetic, intense bitterness and animal notes MLF is not always beneficial and can be responsible for undesirable changes to the sensory properties of wine. As previously mentioned, several species of LAB may conduct MLF. MLF that occurs at a pH below 3.5 and is induced by catalogued, commercial strains is generally conducted by O. oeni, which is less likely to generate off-odours than indigenous MLB strains. Excessive amounts of acetic acid, as well as buttery, cheesy, milky, metallic or earthy odours, are usually present when indigenous strains of Pediococcus sp. and/or Lactobacillus sp. catalyze MLF at pH levels in excess of 3.5.

ORGANOLEPTIC DEFECTS ASSOCIATED WITH UNCONTROLLED MALOLACTIC FERMENTATION

Ropiness (oily wine) Ropy wine is characterized by an atypically high viscosity and an oily or slimy appearance. Ropiness is caused by production of the polysaccharides glucan or dextran from glucose. Synthesis of polysaccharides by LAB is a widespread characteristic, with dextran production by Leuconostoc mesenteroides being the best-known example. Some Oenococcus oeni strains have a “slimy” layer around their cells. Pediococcus damnosus , as well as Pediococcus sp., Streptococcus mucilaginous and Lactobacillus pasturianus also have this ability. In wine, 50 to 100 mg/L of glucose may be sufficient to allow the formation of polysaccharides. Ropiness may occur in tanks, barrels or the bottled product. It usually occurs in white wines and is very rare in reds because the causative organisms do not grow well in the presence of tannin. Volatile Phenols The negative impact of volatile phenols produced by Brettanomyces bruxellensis growth in wines is a major threat to wine quality. This yeast will grow in wine during maceration or while waiting to undergo MLF, even under conditions of high alcohol, high SO2 and limited nutrient availability, and will produce the undesired compounds 4-ethylphenol, 4-ethylgaiacol and 4-ethylcatechol. These volatile phenols are characterized by animal-like odours described as horse and barnyard, pharmaceutical odours characterized as band-aid and medicinal, as well as aromas associated with ink. It is interesting to note that Brettanomyces is not the only micro-organism capable of producing volatile phenols. L. brevis, L. collinoides, L. plantarum, L. mali, L. sake, P. damnosus and P. pentosaceus are also able to produce volatile phenols

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Colture Starter

STARTER COLTURES IN MLF

L’uso dei batteri lattici selezionati è una pratica indispensabile nella conduzione della fermentazione malolattica in un moderno protocollo di produzione riducendo il rischio di fermentazioni deviate che possono compromettere la qualità organolettica e la salubrità del vino. Lo sviluppo spontaneo porta con alta probabilità alla proliferazione di specie batteriche sconosciute, con il pericolo della produzione di metaboliti pericolosi, tra i quali ad esempio le ammine biogene, istamina, putrescina e cadaverina.
Le colture starter selzionate, oltre a non produrre metaboliti secondari indesiderabili, assicurano una fermentazione malolattica rapida, regolare e completa, con una rapida degradazione malica; sono consigliate su vini rossi strutturati e vini bianchi di qualità, nel pieno rispetto delle caratteristiche olfattive del vino e preservando le note fruttate originali.
Le colture starter di Oenococcus oeni sono coltura batterica ad alta concentrazione cellulare che hanno subito un ciclo di preadattamento nel processo finale di produzione. Le membrane cellulari sono state stimolate alla produzione di proteine da stress, adatte alla resistenza alle condizioni avverse del vino, elevata tolleranza all alcol e all’acidità, il ché garantisce anche elevate velocità di degradazione malica.

The use of selected strains of wine lactic acid bacteria (LAB) allows for better control of Malolactic fermentation (MLF) AND the timeframe of L-malic acid degradation occurs in wine as the result of the metabolic activity of wine lactic acid bacteria (LAB). MLF reduces wine acidity and modifies wine flavour, both of which are considered to be beneficial to wine quality Sensory studies show that flavour compounds produced by wine LAB impart recognizable changes to the flavour characteristics of wine and show that different strains of wine LAB will have different sensory effects in wines. The timing of the bacterial addition and the number of cells in the wine after inoculation influence the sensory profile.

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La Vinificazione

La vinificazione è il processo che riguarda la trasformazione del mosto in vino.

Dopo aver raccolto l’uva (vendemmia), nelle cantine inizia il processo di vinificazione propriamente detta, che comprende diverse fasi:
• Diraspatura e pigiatura: il processo meccanico mediante il quale gli acini d’uva vengono separati dal raspo e successivamente spremuti per separare la parte liquida da quelle solide (vinacce e vinaccioli). Queste ultime, dopo la macerazione assieme al succo, vengono allontanate dal mosto;
• Fermentazione alcolica: la fermentazione alcolica è la fase centrale della vinificazione. E’ il processo mediante il quale gli zuccheri naturalmente presenti nei mosti si trasformano in alcool grazie all’azione dei lieviti (principalmente Saccharomyces cerevisiae, ma anche altre specie);
• Affinamento e maturazione del vino: è la fase successiva alla fermentazione in cui il vino viene sottoposto a travasi e filtrazione. Segue poi un periodo di assestamento all’interno di recipienti inerti per un periodo variabile (da qualche settimana a qualche mese). E’ durante questa fase che si sviluppano gli aromi secondari del vino e tutti quegli elementi che contribuiscono a determinarne il profumo e le caratteristiche organolettiche. E tutto questo avviene, nuovamente, grazie all’azione di altri microrganismi, i batteri lattici, che operano la cosiddetta fermentazione malolattica e altre reazioni chimiche.

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