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New World Wine Maker Blog - Technical Articles

The malolactic enzyme – parameters effecting expression

By Wineland Media

Lynn Engelbrecht, Senzo Mtshali, Bronwen Miller & Maret du Toit

The results obtained showed that pH, ethanol and malic acid play an important role in the expression of the malolactic enzyme gene.Malolactic fermentation is a very important process in winemaking, resulting in deacidification, microbial stability and aroma modifications. The direct conversion of l-malic acid to l-lactic acid during malolactic fermentation is catalysed by the malolactic enzyme. Here we report on the results of two studies investigating the effects of pH, ethanol and malic acid on the expression of the malolactic enzyme gene (mle) from Oenococcus oeni and Lactobacillus plantarum. The expression of the mle gene was enhanced at lower pH levels (pH 3.2 vs. 3.8), as well as in the presence of malic acid, while expression decreased in the presence of ethanol. A higher expression level of the mle gene encoding the malolactic enzyme may be linked to a faster and/or successful malolactic fermentation and a better understanding of which parameters and how they affect mlegene expression, could aid in managing a successful malolactic fermentation. Our results also support the use of co-inoculation as a malolactic fermentation inoculation strategy.

Introduction

Genes are part of a living organism’s genome and are responsible for specific traits and characteristics. Each gene contains a set of instructions on how to produce a functional product, for example an enzyme. The process by which the information contained within a gene is used to produce this functional product or enzyme is called gene expression. Not every gene product is needed all the time. The organism assess the environment and then reacts on internal and external signals which triggers the expression of certain genes necessary for the development and survival of the organism at that specific moment.

Malolactic fermentation is a very important step in the winemaking process. By the conversion of l-malic acid to l-lactic acid, it contributes to deacidification of the wine, microbial stability, as well as softening, while the aromatic profile is also being influenced. Oenococcus oeni is the lactic acid bacteria mainly associated with malolactic fermentation and is the most favourable species used in malolactic starter cultures. However, the species Lactobacillus plantarum, which is also frequently found in grape must and wine, and effective in completing malolactic fermentation successfully,1 has also now been used in commercial malolactic starter cultures either as a single strain or mixed with O. oeni.

Ideally, O. oeni prefers to grow at a pH of 4.8, in a medium with £10% (v/v) ethanol and at a temperature of 22°C,2 whereas in wine O. oeni is exposed to harsh environmental conditions, including high ethanol concentrations (>12%), low pH (<3.8), sulphur dioxide, low temperatures (<18°C) and limited nutrients. However, it is able to survive this multi-stress environment and therefore the best adapted wine lactic acid bacteria. In order to survive these conditions, O. oeni employs different stress response mechanisms to preserve energy and to defend and protect the cell envelope. The main mechanism of survival is the metabolism of l-malic acid which generates a proton motive force, resulting in the production of energy through ATP synthesis and deacidification of the intracellular pH3 and in the presence of ethanol for example, O. oeni has showed to respond by increasing the fatty acid content in its membrane to regulate membrane fluidity.4,5

The direct transformation of l-malic acid into l-lactic acid by wine lactic acid bacteria is the result of the malolactic enzyme. A better understanding of the when, the where and what conditions promotes or prevents the expression of the gene coding for the malolactic enzyme, provides valuable information on predicting the effectiveness of malolactic fermentation.

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When microbiology is a data problem: Putting science together to make better pictures of yeast

A Portuguese-based group is suggesting that winemakers could have more useful information about choosing a yeast strain if scientists did a better job of putting together data from different kinds of experiments.

Scientific research generates a lot of different shapes and sizes of data. How does anyone make it work together?

Contemporary scientific research has a lot of big challenges, but here are three: funding, replicability, and integration. Funding is a great big gory topic for another day.

Replicability has seen a lot of attention in recent science news: scientists across disciplines have been reporting difficulty duplicating their colleagues’ results when they try to repeat the same experiments. This is worrisome. (Most) science is supposed to be about making observations about the world that remain the same independent of who is making the observations. Two careful people should be able to do the same experiment in two different places and obtain the same results. Well-trained scientists, however, are finding themselves unable to replicate the results described in scientific papers, and the community isn’t sure what to do about it.

Integration – how to fit together large amounts of lots of different kinds of data – looks like a separate kind of problem. Scientists (microbiologists, biochemists, systems biologists, geneticists, physicists…) study a thing – yeast, say – in many, many different ways. They generate data in many different shapes and sizes, using all manner of different kinds of instruments to make numbers that don’t just tidily line up with each other. But, at least in theory, all of those data are about the same thing – the same yeast – and so finding ways to integrate data from different kinds of experiments should massively improve our understanding of how yeast works as a whole ….

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Sauvignon blanc – role of phenotypic plasticity in cultivar typicity

Grapevine cultivars are remarkably adaptable to their environments and responsive to production manipulations. This adaptability is (scientifically) described as phenotypic- or metabolic plasticity. You might not have heard these terms before, but they underpin the observation that under certain conditions the same cultivar can produce very different styles of wines, or in other words, display plasticity. To understand the plasticity of a cultivar, it is necessary to study the underlying physiology and metabolism. To do that, grapevine cultivars need to be studied in interaction with their environment (natural and manipulated). It sounds relatively easy, but it is no simple task. Considering the multitude and complexity of the individual factors potentially affecting field grown grapes, how can one reliably predict the outcome of a viticultural treatment? From a scientific perspective it comes down to the need to establish “cause-and effect” (causality) type vineyard studies. A causal relationship exists when the results/trends of an experiment are proven to be caused by the manipulation, or a specific factor. Such a study of a leaf removal treatment in a Sauvignon blanc vineyard could explain why wine style/typicity can be shifted by increased bunch exposure and provide proof of this cultivar’s metabolic plasticity.

Introduction

Producers and viticulturists are confronted with a multitude of compounding factors to contend with to produce quality grapes. Some viticultural decisions are long term, and are decided during the initial establishment phase of the vineyard, and include: site selection (e.g. climate, altitude, aspect/inclination and soil), cultivar/clone selection, scion/rootstock combination, row orientation, vine/row spacing and trellising system. Needless to say, these decisions influence the ultimate quality of the grapes and are costly to change once a vineyard has been established.

Other decisions are seasonal, and can include the choice of cover crop(s), the implementation of canopy manipulations (e.g. shoot thinning, shoot trimming and leaf removal), bunch manipulations (e.g. cluster thinning), and timing of winter pruning. The grape yield and/or quality is then further influenced by the prevailing seasonal conditions (vintage) which can be considered as the sum total of all factors that the grapes are exposed to in any given season and will include wind, water (rain and/or irrigation), light, temperature, humidity and disease load (pathogens and pests). These factors do not occur in isolation, and for each of these factors both the timing and intensity is relevant. The challenge is to link these factors to outcomes in causal relationships to ultimately understand their impacts on grape/wine quality.1

We used an early leaf removal treatment in Sauvignon blanc in the moderate (cool night) region of Elgin to study the impact of increased bunch exposure on grape composition throughout berry developmental stages (i.e. green pea size through till the ripe/harvest stage).

Leaf removal is used for diverse purposes, usually with a predetermined viticultural and/or oenological outcome …

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The Relationship Between Sensory Characteristics and Emotion in Consumer Wine Preferences

Consumer wine preference is an oft-studied topic, as understanding wine preference is paramount in determining how to market and sell any given wine.  It can also help wine marketers not only observe what consumers like, but also how these preferences can change over time and between different segments.

Often, wine preference is determined via the hedonistic scale, or how much a consumer says they like a particular style of wine. However, research in food and other industries have found that the role of emotions may provide an extra level of understanding in regard to consumer preferences and that this type of analysis may be very useful in wine as well. For example, studies have found many associations between certain flavor types and emotions in various foodstuffs: in dark chocolate, studies have linked “powerful” and “energetic” with cocoa flavor; and in beer, studies have linked herbal flavors with “sadness” and citrus flavors with “disappointment.

According to the authors of a new study, available online in late December 2017 and to be published in print in June 2018 in the journal Food Quality and Preference, there have been no studies linking specific wine sensory characteristics with emotional responses, nor is there a dedicated lexicon for such relationships in wine products like there are with food (i.e. the EsSense Profile). In this new study, the researchers aimed to analyze the associations between sensory characteristics of wine and elicited emotional responses of consumers, further subcategorized by gender and age.

Brief Methods

This study had two parts:  a sensory evaluation of the wines by a trained panel (11 total: 5 women, 6 men; faculty and researchers from the School of Agricultural, Food and Biosystems Engineering at the Universidad Politécnica de Madrid in Spain), and a consumer evaluation of the wines with an additional emotional response analysis.

6 commercially-available wines were used in the study: 2 whites, 1 rosé, and 3 reds.

For the sensory evaluation by the trained panel, each wine was scored for various aromatic and sensory attributes using an unstructured 15-cm line scale that had labels “low” and “high” on the ends (with variation throughout the line that could be translated to a specific intensity level of any given attribute).  Wines were presented in random order.

For the consumer evaluation, participants were first asked to complete questionnaires on demographics and wine consumption habits. Next, they participated in a “warm-up” or “practice” tasting session with 7 wines presented [blind] at the same time.  Finally, after the warm-up, they were presented with the sample of 6 test wines briefly mentioned above.

After tasting the wines (which were presented in random order), participants were asked to rate their liking of each wine (using a 9-point hedonic scale), and what emotions were elicited by each wine (using the EsSense 25 software). Emotions were rated using a 10-cm line scale with the labels “very low” and “very high” at the ends (and everything in between).

Participants were recruited from the School of Agricultural, Food and Biosystems Engineering at the Universidad Politécnica de Madrid and were required to consume wine at least once per month. A total of 208 people participated in the study (48.5% male, 51.5% female).  Participants were categorized by three age groups (for studying potential age effects): young adults (18-35 years old; 44.9% of the total); middle-aged adults (36-55 years old; 29.3% of the total); and older adults (55 years old and older; 25.9% of the total) …

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Can yeasts be used to prevent protein haze?

Winemakers add bentonite to prevent protein haze in white wines. Although this treatment reaches its goal, it also leads to volume losses and sometimes a decrease in wine quality. The question is: are there alternatives available?

Protein haze – some background information

The removal of proteins is a key step during the production of white and rosé wines to avoid the possible appearance of a harmless, but unsightly haze. Haze formation is an aesthetic problem that consumers usually regard as a fault (e g microbial spoilage) leading to potential economic losses. Proteins that are responsible for haze formation in wine have been identified as pathogenesis-related proteins of grape origin. The most abundant class of haze-forming proteins are chitinases and thaumatin-like proteins and are continuously produced in the grape berry and even more so in response to pathogen attack. Because of their physical structure and properties, these proteins are very resilient and are not or poorly degraded during the course of fermentation. Over time and upon exposure to warm/hot temperatures during storage for instance, these proteins denature and aggregate into light dispersing particles resulting in what is referred to as ‘haze’.

The mechanisms of haze formation has received much attention from researchers over the last decade. It is complex by nature and depends on several factors, one of the most important being the presence of sulphate. The removal of these proteins is usually achieved via bentonite fining, but several issues including volume loss, aroma stripping and sustainability have been identified with the use of this clay. Several strategies have therefore been investigated over the past few years. One of the most attractive alternatives would consist in degrading these haze-forming proteins with enzymes. This is particularly appealing since enzymatic degradation of proteins (protease activity) would not lead to any of the issues mentioned for bentonite and could have the additional benefit of releasing yeast assimilable nitrogen.

Where does one find enzymes capable of degrading haze-forming proteins? …

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TAKE A BIG WHIFF!

One of the most important parameters for the measurement of quality is the aroma/flavour profile of a wine (1). Up till now, more than 1000 compounds have been identified in grapes and wine. To add to the complexity of the wine matrix, the individual concentrations of these compounds may vary considerably (2). The aroma profile will also be influenced by production processes, be it in the vineyard or cellar and with an infinite number of variations possible in the production process, the final aroma profile of a wine is a complex matter to say the least (3).

There are various influencing factors that play a role in determining wine aroma composition. These include, amongst many more, climatic conditions (e.g. altitude above sea level), viticultural practices (e.g. canopy management) and enological practices, e.g. fermentation conditions, on which this article will focus (1).

Even though some aroma impact compounds exist for some varieties, seldom can the sensory perception of wine aroma be attributed to a single compound (1). The aroma attributes of a specific compound depends not only on its concentration or the specific odour threshold value (lowest concentration at which it can be detected), but also its interaction with other aroma compounds, be it the enhancement (even compounds present below their odour threshold) or suppression of another compound (1). Because of the complexity of the wine matrix it is almost impossible to predict the interaction between aroma compounds, but certain actions, like the selection of a specific yeast strain, could aid in driving the aroma profile to a certain extent (2). This is an important tool as it has been shown that a difference in flavour profile solely as a result of the choice of yeast strain, can be detected not only by trained panels and wine professionals, but more importantly, wine consumers (6). This implies that besides choice of viticultural practices and grape selection, selecting a specific yeast strain (usually Saccharomyces cerevisiae) for fermentation, as well as a bacteria strain for MLF, can greatly modify the aroma, flavour, mouthfeel, colour and chemical complexity of a wine, making this a tool to create a specific flavour profile according to market specifications (3).

The compounds that play a role in wine appearance, aroma, flavour and mouthfeel can be derived from three sources: the grapes, microbial modification during fermentation and then maturation, be it bottle ageing or wood maturation (3).

Grape-derived compounds do not only provide the basic wine structure, but also results in distinct varietal characteristics (3). The main grape-derived aroma compounds belong to the groups of monoterpenes, norisoprenoids and methoxypyrazines. Some examples of these include rose-like geraniol in Chardonnay, spicy eugenol and guaiacol in Gewürztraminer and floral, fruity and berry-like β-damascenone and violet-like β-ionone in Cabernet Sauvignon, Shiraz and Pinotage (6). While monoterpenes and norisoprenoids are very important in Muscat and aroma-rich varieties, fermentation-derived aroma compounds play a larger role in ‘neutral’ cultivars. The following section will focus on yeast-derived fermentation aroma compounds, although MLF also makes a significant contribution towards the final wine aroma profile.

While the main purpose of yeast is to metabolise sugar in order to produce ethanol and carbon dioxide, this microbial culture also produces a myriad other metabolites that, despite being present in small amounts, significantly alters the wine aroma profile and have a significant sensorial impact (3). Yeast strains are able to modify the wine aroma via three mechanisms (3):

1) via the extraction of compounds from solids in the grape must;
2) modification of grape-derived aroma compounds and
3) producing flavour-active metabolites.

The biosynthetic pathways responsible for aroma production via these mechanisms are influenced by various factors, to name a few (3):

  1. a) viticultural factors;
  2. b) composition and pH of grape must;
  3. c) nature and prevailing temperature of grape must and
  4. d) technological aspects and vinification methods.

As previously mentioned, the yeast can modify grape-derived aroma compounds for e.g. esters, higher alcohols and lactones in Chenin blanc contributes to varietal aroma; mercapto components formed during fermentation in Sauvignon blanc adds to passion fruit, guava and other tropical aromas and iso-amyl acetate adds to banana aromas in Pinotage (6). The table below also lists some of the most important yeast-derived aroma compounds important in determining the final wine aroma profile that serves as an important quality parameter (5).

Major aroma impact compounds produced and modified by yeast during fermentation

Volatile Acids
  • produce 0.2-0.7 g/L acetic acid during fermentation
Alcohols
  • ethanol: influence volatility of other aroma compounds
  • higher alcohols: positive or negative effect on wine aroma
  • involves degradation of amino acids
Carbonyl Compounds
  • acetaldehyde: 10-75 mg/L produced (bruised apple; oxidation)
  • diacetyl: small amount (0.2-0.3 mg/L) produced by yeast (butter )
Volatile Phenols
  • off-odours: medicinal, barnyard
  • vinyl-phenols: stabilise colour in red wine
  • Brettanomyces: ethyl-phenol (negative sensory impact)
Esters
  • influence fruity and floral aromas
  • dependant on: yeast strain, fermentation temp., precursors
  • acetate esters: ethyl acetate (fruity); iso-amyl acetate (banana, pear); 2-phenylethyl acetate (honey, rose, flower)
  • ethyl esters: ethyl hexanoate and ethyl octanoate (apple
Volatile Sulphur Compounds
  • low sensory threshold (generally negative to wine quality)
  • positive: thiols (grape-derived compounds modified by yeast)
  • guava, passion fruit, grapefruit, gooseberry (Sauvignon blanc)
  • release and modification is yeast strain dependant
Monoterpenes
  • grape-derived: aromatic (free) and non-aromatic glucose-bound
  • free form: fruity and floral
  • yeast release bound form via β-glucosidase activity; add to aroma

 

It has also been shown that chemical changes that occur as a result of ageing, either bottle or wood, may also alter the wine composition and quality (1). During the ageing period, compounds are extracted from wood (oak lactones) and these add to aroma complexity. Certain compounds are also transformed and/or liberated from bound forms, which mean they can then play a role in the aroma perception of the wine.

Due to the fierce competition in the wine industry, wine producers are being forced to investigate and understand consumer preferences and expectations and produce wine accordingly. This has become a market-driven industry whereby winemakers are challenged with responding to consumer sentiments and preferences (3). One of the tools in a winemaker’s arsenal that is available to address this challenge is the selection of the microbial populations that will be responsible for fermentation. Therefore the yeast and bacteria strain(s) can be seen as a flavour-impact tool to produce a certain style of wine. This will only be possible with an understanding of the impact aroma compounds and the role the selection of the correct yeast and bacteria can play in the production and or modification of these compounds. This is the reason for the extensive and careful research that goes into the development of all Anchor yeast and bacteria cultures. This way we ensure not only optimal fermentation, but also optimal contributions to the final aroma profile.

So take a big whiff…

References:
1. Wine aroma-important aspect of wine quality. www.newworldwinemaker.com
2. Sensory perception. www.newworldwinemaker.com
3. Swiegers J.H., Bartowsky E.J., Henschke P.A. & Pretorius I.S., 2005. Yeast and bacterial modulation of wine aroma and flavour. The Australian Journal of Grape and Wine Research, 11, 139-173.
4. The complete A-G understanding to waking up your wine. www.newworldwinemaker.com
5. The impact of yeast on the sensory quality of wine. www.newworldwinemaker.com
6: Cordente A.G., Curtin C.D., Varela C. & Pretorius I.S., 2012. Flavour-active wine yeasts. Applied Microbiology and Biotechnology. DOI 10.1007/s00253-012-4370-z

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