Carrau, Boido, Ramey AAM 2020

CHAPTER THREE
Yeasts for low input winemaking:

Microbial terroir and flavor
differentiation
Francisco Carraua,∗, Eduardo Boidoa, David Rameyb
a
Área Enologı́a y Biotecnologı́a de Fermentaciones, Departamento de Ciencia y Tecnologı́a de Alimentos,
Facultad de Quı́mica, Universidad de la Republica, Montevideo, Uruguay
b
Ramey Wine Cellars, Healdsburg, CA, United States
∗
Corresponding author: e-mail address: [email protected]
Contents
1. Introduction 90
2. Low input winemaking technology 93
2.1 A quality paradox: The high winemaking technology 94
2.2 Fining agents 95
2.3 Filtration 96
3. Microbial terroir and native yeasts 96
3.1 Biological control of fungi and insects by yeasts in the vineyards 99
3.2 Grape harvest and application of the traditional Saccharomyces yeast at the
winery 101
4. The alternative: Spontaneous fermentation and non-Saccharomyces strains 104
4.1 Friendly yeasts cooperate with the microbial terroir 105
4.2 Nutrient demand and competition in mixed cultures 106
5. Other application of non-Saccharomyces yeasts for low input winemaking 106
5.1 Color stability and synthesis of derived anthocyanin compounds 106
5.2 Increasing or decreasing total acidity by yeasts 107
5.3 Lees contact effects: Fast and slow cell lysis 109
5.4 Yeast strategies for low alcohol formation 110
6. Natural clarification and minimal manipulation before bottling 110
7. Concluding remarks 112
Acknowledgments 114
References 114
Abstract
Vitis vinifera flowers and grape fruits are one of the most interesting ecosystem niches
for native yeasts development. There are more than a 100 yeast species and millions of
strains that participate and contribute to design the microbial terroir. The wine terroir
concept is understood when grape and wine micro-regions were delimited by different
quality characteristics after humans had been growing vines for more than 10,000 years.
Advances in Applied Microbiology, Volume 111 # 2020 Elsevier Inc. 89

ISSN 0065-2164 All rights reserved.
https://doi.org/10.1016/bs.aambs.2020.02.001
90 Francisco Carrau et al.
Environmental conditions, such as climate, soil composition, water management, winds

and air quality, altitude, fauna and flora and microbes, are considered part of the “terroir”
and contribute to a unique wine style. If “low input winemaking” strategies are applied,
the terroir effect will be expected to be more authentic in terms of quality differentia-
tion. Interestingly, the role of the microbial flora associated with vines was very little
study until recently when new genetic technologies for massive species identification
were developed. These biotechnologies allowed following their environmental
changes and their effect in shaping the microbial profiles of different wine regions.
In this chapter we explain the interesting positive effects on flavor diversity and
wine quality obtained by using “friendly” native yeasts that allowed the microbial terroir
flora to participate and contribute during fermentation.
1. Introduction
The concept of low input winemaking was introduced in the 1990s by
a few winemakers with practical experience that discovered that minimal
handling of the grapes and avoiding some oenological practices in the winery
improves wine quality (Ramey, 1995). During the 1970s, there was a great
development and input at the winery of new technologies, stainless steel
tanks, cooling and filtration capacity, grapes pumping and pressing
technology, adjustments of pH by adding tartaric acid, clarification methods,
commercial yeast applications, addition of ammonium salts and other addi-
tives. However, some winemakers started to detect that some of these
winemaking operations were affecting wine composition, color, flavor,
body and palate. The more you manipulate the grape musts and wine,
the more you lose of anthocyanins, flavors and other compounds related
to the structure, such as proteins, mannoproteins, lipids, phenolics and poly-
saccharides. The low input concept comes from the great movement that
started in the 1980s with the reaction to the conventional agriculture that
increased the use of fertilizers and chemicals by three- or four-fold inputs
during the 1960–80 period. Some massive commodities plantations increase
exponentially at that time with many adverse side-effects to the food and
environmental conditions (Schaller, 1993).
Although there is not too much scientific information in the literature
about these topics in relation to grapes and wines, it is clearly known that
in the winemaking environment many oenologists have started to pay more
attention to these topics to obtain increased quality. The quality wine indus-
try is growing exponentially and is considered today to be the biggest brand-
ing category within the food industry (Bellini & Resnick, 2018), with more
Yeasts for low input winemaking 91
than 1 million wine brands on the market. Future winemakers will aim to
differentiate their product and focus on unique regional characteristics (the
“terroir” and “flavor phenotype” concepts). Winemakers and wine scientists
have started to understand that the microbial terroir exists and can shape the
native yeast composition of grapes during harvest time (Gilbert, van der
Lelie, & Zarraonaindia, 2014). The role of native yeasts within the terroir
concept and how they influence the final wines was ignored until recently.
The fact that many native yeasts are not adapted to the grape must or the
winery ecosystem or that they can produce off-flavors has been the topic
of research for many decades. The potential or positive contribution of
non-Saccharomyces yeast was underestimated (Binati et al., 2019; Ciani &
Comitini, 2011; Contreras et al., 2014; Jolly, Varela, & Pretorius, 2014;
Martin, Jose Valera, Medina, Boido, & Carrau, 2018; Mortimer &
Polsinelli, 1999; Perez-Torrado, Barrio, & Querol, 2018) and complete fer-
mentation or avoiding the production of off-flavors was the main objective
for decades. This challenge was clearly improved by the use of commercial
yeast strains of Saccharomyces cerevisiae. There was a conflict in winemakers’
minds between the compromise of standardization or taking the risk
of searching for flavor diversity (Carrau, Gaggero, & Aguilar, 2015).
However, more recently it was discovered that flavor diversity or complex-
ity is not obtained with pure fermentation of very efficient fermentation
strains, where ethanol production is the major capacity of S. cerevisiae.
Many non-Saccharomyces species were identified as good and intense flavor
producers under pure or mixed cultures with other yeasts, compared to the
conventional application of S. cerevisiae. Increasing yeast diversity increases
flavor complexity and might complete grape must fermentation and allow a
winemaker to express the terroir differences that give visibility to their brand
in this huge wine market.
Applications of the same commercial strains in different regions of the
world may be said to result in ordinary products, limiting diversity of the pro-
ducer style of wine. Some winemakers consider this a weakness of today’s
winemaking technology, and they are interested in producing wines of a
unique style, with minimal handling and by avoiding the use of additives.
This winemaking strategy is mainly possible with wines produced from grapes
of optimal concentration of the key compounds related to wine stability and
quality, such as high polyphenol and total extract content. Grape berry com-
position is the primary determinant of must composition prior to fermentation.
Manipulation of grapevines has the potential to influence grape berry
composition and, ultimately, the composition of wine, which, in turn,
can affect its stability against microbial spoilage and allows the use of the
native flora. Many of these technical concepts were called “low input
winemaking” (Ramey, 1995), and, in our opinion, after obtaining high
grape quality, the natural microflora and/or the type of yeast strain inocu-
lated is the second key step in winemaking to increase quality and complex-
ity (Carrau, 2005, 2006). The concept of “terroir” is still very controversial
as was discussed recently by Pretorius (2020). In this review, we will show
the importance of respecting the microbial terroir or native or wild yeasts so
as to differentiate our wines and give them a sense of origin. In our opinion,
human technology should be very precise and knowledgeable to let the
microbial terroir influence your regional end product. It is human technol-
ogy, in certain unique vineyard locations, that developed a regional reputa-
tion based on wine quality.
The native microbial flora as part of the terroir concept was a debated
for many years. Today, it is clearly demonstrated by new generation
sequencing technologies as being one of the key characteristics of a certain
wine region (Bokulich, Thorngate, Richardson, & Mills, 2014; Curtin &
Pretorius, 2014; Gilbert et al., 2014; Liu, Zhang, Chen, & Howell, 2019;
Pretorius, 2020; Wu et al., 2014). The massive use of commercial yeast
of the species Saccharomyces cerevisiae has confused what might be the poten-
tial of leaving native yeasts to do their work in your grapes. Interestingly, it is
known that regional dispersion of native yeast cells by insects such as honey
bees was estimated to be not more than 10 km away from their origin
(Goddard, Anfang, Tang, Gardner, & Jun, 2010). This ensures a restricted
limit for microbiota diversity within a few vineyards of a certain micro-
region. However, birds such as pigeons might fly for about 60 km from their
nests, and migratory birds might disperse up to 300 km, but mainly for resis-
tant strains such as Saccharomyces (Francesca, Canale, Settanni, & Moschetti,
2012). Non-Saccharomyces strains are probably more restricted to local
regional terroirs than Saccharomyces.
In this review, we focus on this specific aspect of the microbial terroir
flora and show, with some examples, how native yeasts might be selected
for the production of highly differentiated unique wines. We will discuss
the use of “friendly” selected yeasts and how they share their fermentation
niche with the natural yeast flora of a certain specific vine site or terroir, all-
owing to obtain increased yeast diversity that will contribute to the flavor
complexity of wine. In contrast, the majority of Saccharomyces strains do
not allow this when used conventionally. These applied microbial concepts
will contribute to help develop new strategies for wine microbiologists and
winemakers to avoid sensory characteristic losses in different wine ecosys-

tems or terroir regions and the risk of using uncontrolled native yeast at
the winery level.
2. Low input winemaking technology

From a practical point of view, there are many examples that can show
the importance of minimal handling of the grapes and musts before and after
vinification. Many physical and chemical phenomena affect wine quality at
the pre-fermentation steps, and this together with the microbial interactions
during the complete process will affect the sensorial characteristics of the end
product. We have demonstrated, with two model strains of Saccharomyces, an
interesting example of how di-ammonium phosphate (DAP) additions in
the 1970s were increased to solve the high nitrogen demand characteristics
of a few commercial strains, such as Montrachet 522 (Carrau, 2005, 2006).
The question is why we have some selected Saccharomyces strains that, even
with yeast assimilable nitrogen levels below 100 mgN/L, produce a pleasant
flavor phenotype (Carrau et al., 2008). Interestingly, within Saccharomyces
strains, we have shown that the commercial strain M522 increases ester pro-
ductions by DAP addition and that a native strain KU1 decreases the pro-
duction above 125–180 mgN/L. The same must will result in different
wines depending on the yeast strain utilized. Low input winemaking strat-
egies must look for preventing methods such as starters that do not demand
nutrient additions. Such would be the use of a strain selected for low YAN
processes, like strain KU1 of our model, which is adapted to the natural
environment of low nitrogen grape musts. There are many examples of
aroma compounds that are decreased when yeast assimilable nitrogen is high
or DAP is added to a grape must. We have demonstrated this for many
compounds produced by Saccharomyces, such as higher alcohols, some esters,
lactones, sulfur compounds, benzenoids, sesquiterpenes and other phen-
ylpropanoids (Carrau, Boido, & Dellacassa, 2017; Martin et al., 2016)
(Fig. 1). More recently, it was also shown a decrease for some important sul-
fur derived compounds, such as the varietal thiols precursors, which is typical
in Sauvignon blanc (Alegre, Cullere, Ferreira, & Hernández-Orte, 2017).
However, some inconsistent results about the advantage of working with
low YAN levels and avoiding DAP addition are found in the literature.
This is due to there being much experimental data that does not consider
the nitrogen availability level or the competitive, mixed, natural flora that
appear in real winemaking grape musts when commercial dry yeasts are
Fig. 1 The main flavor compounds synthesis is affected negatively by DAP additions.
The opposite situation only occurred for some high-nitrogen demand Saccharomyces
strains that were adapted to higher YAN levels, probably before they were commercially
available (Carrau et al., 2008). The figure shows that above 180 mgN/L of YAN some wine
S. cerevisiae, considered low nitrogen demand strains, reduce flavor synthesis, probably
favoring the primary ethanol fermentation (Carrau et al., 2017).
applied (Carrau et al., 2017). We have designed a fermentation synthetic

grape medium for selecting native yeast strains and for a rapid flavor screen-
ing that has 100 mgN/L of YAN (Carrau et al., 2015) as well as a simple
sensory analysis.
2.1 A quality paradox: The high winemaking technology

Sometimes, accelerated processes for finishing a wine also results in quality
compound losses. The low input winemaking strategy should be to select a
patient way for minimal manipulation of the wine to prepare its final bot-
tling. Today, it should be easier for the winemaker than for our ancestors to
understand and improve the magnificent tools we have available. Cooling
and heating systems, better barrels, tanks and new inert materials for matur-
ing wines, inert gas alternatives for manipulating finished wines, new
methods for easy measures at the lab, turbidity, infrared methods, color,
microbial stability, improved bottling facilities, etc.
For example, color and limpidity are appreciated by consumers and con-
tribute to the purchasing decision. The natural clarification process is slow,
and it was possible to improve the process by adding different agents to
obtain better limpidity in less time. For this reason, in high volume produc-
tions, the use of processes such as clarification and filtration is necessary prior
to bottling to ensure wine clarity in the bottle (Dumitriu, Lopez de Lerma,
Cotea, & Peinado, 2018; McRae et al., 2017). These processes affect wine
flavor and mouthfeel characteristics. Macromolecules in wines, mainly in

red wines, contribute to the mouthfeel, texture and color of the wine.
These macromolecules include polyphenols, particularly procyanidins from
grape skins and seeds (Boido et al., 2011), which contribute to color stability
by formation of pigment polymers with anthocyanin (Boido, Alcalde-Eon,
Carrau, Dellacassa, & Rivas-Gonzalo, 2006) and astringency via interaction
with oral proteins (Payne, Bowyer, Herderich, & Bastian, 2009). Another
important group of macromolecules is polysaccharides, originating from
yeast fermentation (e.g., mannoproteins) and extracted from grapes (e.g.,
arabinogalactan proteins and rhamnogalacturonans) (Bindon, Varela,
Kennedy, Holt, & Herderich, 2013; Riou, Vernhet, Doco, & Moutounet,
2002). On the other hand, procyanidins and polysaccharides can interact
by hydrogen bonding to form colloids that contribute to wine texture
(Riou et al., 2002). In our experience, a natural clarification process of a
new red wine in an oak barrel (300 L) is obtained in about 1 week and in
a stainless steel tank (5000 L), in about 40 days, both at 14 °C and measured
with a turbidimeter to reach under 5 NTU (nephelometric turbidity units).
There is a big timing difference, but both processes were without any addi-
tives and resulted in very good quality styles, short barrel aging and an
unoaked red wine. It is known by many winemakers that had contributed
consistently in the last 30 years to improve phenol maturity of red grapes,
viticulture management has resulted in increased tannins maturity and sen-
sory softness of wines. Barrel aging might be shortened or avoided to show
less oaky wines and increased flavors from the grape variety and a clean
terroir expression will be expected. This example is interesting for under-
standing that barrel aging and flavor effect after this process is not a terroir
expression. If a Californian and a Uruguayan winemaker use the same sup-
plier of barrels, they will be less differentiated if they overaged a certain wine.
It makes sense to use barrels for a shorter time of aging and obtain a natural
clarification to avoid deep filtration processes before bottling.
2.2 Fining agents

The most popular fining agent is bentonite, which consists of montmoril-
lonite clay that swells in water and produces a sheet like structure on which
cation exchange, hydrogen bonding and adsorption can occur (Marchal &
Jeandet, 2009). Although it is mainly use for removing unstable proteins
during or after fermentation, a decrease in the color of fining wines with
bentonite was reported (Stankovic, Jovic, Zivkovic, & Pavlovic, 2012)
and might affect the flavor of white wines. Many other fining agents were
developed in the last two decades, just to give other examples, in the case of
protein based fining agents such as white eggs or vegetal proteins, the astrin-
gency and bitterness of wine can decline by interaction with procyanidin
(Oberholster, Carstens, & Du Torr, 2013). In addition, residues of some
clarifiers such as cow’s milk casein have been detected mainly in white wines
and may cause allergic reactions in sensitive consumers (Zelenakova,
Fikselova, Ziarovska, Kolesarova, & Jurcaga, 2019). Low input winemaking
demands that these operations with additives be well understood before
applying and that they might be curative and not preventive for quality.
2.3 Filtration
Another well-known manipulation in the wine industry is the cleaning
operation through filtration systems. The colloids formed by hydrogen
bonding between procyanidins and polysaccharides might be removed by
filtration processes, causing negative sensory impacts (El Rayess et al.,
2011, 2012).
The type of membrane used in the filtration affects polyphenols reten-
tion. The most classic nylon membranes used in the wine industry contain
multiple amide bounds that can bind anthocyanins or other polar molecules
by hydrogen bonding (Bowyer, Edwards, & Eyre, 2013). The nylon mem-
brane was replaced in many applications by the polyethersulfone (PES)
membrane, with minimal sites for hydrogen bonding and minor loss of
anthocyanins.
Recent studies show that filtration over a plate filter with cellulose sheets
causes a significant reduction of mannoproteins, homogalocturonans and
polysaccharides rich in arabinose and galactose in red wines (Martı́nez-
Lapuente, Guadalupe, & Ayestarán, 2017). In this work it was shown that
cross-flow microfiltration produces large losses of polyphenols and polysac-
charides but also increases the ratio arabinose over galactose polysaccharides
affecting the sensory characteristics of the wines (Martı́nez-Lapuente
et al., 2017).
3. Microbial terroir and native yeasts

We have discussed the concepts of terroir and low input winemaking
strategies. Now, we will focus on native yeasts and how they can interact to
improve quality with particular characteristics in a certain region. Today, it is
of general knowledge that winemaking starts in the vineyard. This process
could be improved with the selection of the right grape varieties and a vine-
yard management focused on fruit quality. This management and the soil
and site characteristics are going to influence the grape microflora that would
enter the winery (Tempère et al., 2018). As the winemaking process is not
under sterile conditions, the winemaker must understand the great biodiver-
sity that appears in the grape juice and, on the other hand, learn how to use
commercial yeasts or conduct a spontaneous fermentation to reach quality
flavors in the final wine.
Fermented foods were originally developed by our ancient predeces-
sors as a biological way to preserve different fresh agricultural products,
such as fruit juices, milk or meat. The challenge in those times was to
extend the conservation of these foods for a few months to take advantage
of them after the harvest time without adding mineral products such as salt
or vinegar. In addition, centuries of masters in fermentation were needed
to understand how to manage what some chemists in the 19th century
called “enzyme activities,” which decrease pH or generate alcohol as
inhibitors of microbial contamination for more safe foods. Interestingly,
the discovery of the true fermentation was demonstrated by the practical
masters of fermentation in the beer or wine industry at the end of the 18th
century and not in the academy (Anderson, 1989). Now the role of yeasts
in the fermentation of sugars into alcohol and carbon dioxide has been
known for more than two centuries. However, well over a half of the
19th century elapsed before Pasteur established the role of yeast strain in
the production of different wines and beers in 1866. There have been great
advances in selection of strains of Saccharomyces cerevisiae—the widely used
yeast for beer, bread, cider and wine production. Yeast biology had
advanced due to the knowledge developed with Saccharomyces cerevisiae,
and there are still very limited studies on other yeasts. The good news is
that there is a wild and enormous stock of genetic and phenomic reserve
within non-Saccharomyces strains. In relation to the food industry, this fact
will increase available yeast diversity for obtaining flavor and sensory com-
plexity for applied fermentation processes (Carrau et al., 2015; Cordente,
Curtin, Varela, & Pretorius, 2012). On the other hand, from the yeast
technology point of view, the progress in molecular characterization
was fundamental for strain identification and differentiation within
Saccharomyces and will be fundamental for non-Saccharomyces strain discrim-
ination and follow complex populations during non-sterile fermentations
such as wine or cider. These methods, together with the massive sequenc-
ing for defining the microbiome and their correlations with the
Fig. 2 Native yeast selections by flavor traits is a key strategy to ensure quality initial
fermentation, avoiding the risk of free spontaneous processes. Here we showed natural
tools used to understand and improve selected strains and their interactions with the
microbial terroir. Wine microbiologists now have many biotechnology alternatives to
genome modification approaches. Friendly yeast starters are explained as strains that
are not so aggressive with the natural flora, as happens with Saccharomyces cerevisiae
strains. These starters should allow other strains and yeast species to contribute to the
final flavor profile. They work in a consortia style of vinification, and it is expected that
this process with increase yeast diversity and increase flavor complexity, microbial
stability and wine differentiation in the market. Low input winemaking strategies are
favored by increased yeast diversity.
environment or microbial interactions in the fruit or during vinification,

will allow us to understand complex metabolic processes, as will the
mixed cultures and consortia yeast fermentation. In Fig. 2, a breeding strat-
egy for yeast selections is shown, where non-genomic modified organisms
are applied and the natural diversity of millions of yeast strains can be
explored to obtain superior starters for winemaking. Obviously many
molecular and metabolic knowledge needs to be developed to understand
the microbial interactions within a consortia system, as it is the wine
fermentation.
3.1 Biological control of fungi and insects by yeasts in the

vineyards
Within the low input winemaking strategy, the increase of beneficial flora in
the vineyard should be taken into account as a key way to decrease fungal
diseases. It is well known that wine grape cuticles are a primary source of
non-Saccharomyces yeasts that could interact with other microorganisms like
filamentous fungi and bacteria, determining a complex microbial ecosystem
(Barata, Malfeito-Ferreira, & Loureiro, 2012; Fleet, 2003) (see Fig. 3). Some
of the yeast species associated with grapes have been shown to have a broad
spectrum of antagonistic activity against fungal pathogens (McLaughlin,
Wilson, Droby, Ben, & Chalutz, 1992; Rabosto et al., 2006; Spadaro &
Droby, 2016; Suzzi, Romano, Ponti, & Montuschi, 1995).
Native yeasts associated with the grape berry
pruine pedicel
skin brush
Epiphytes Endophytes
Hanseniaspora/ Kloeckera Rhodotorula Cryptococcus
Metschnikowia Aureobasidium Debaryomyces
Issatchenkia Hortaea peripheral
Rhodosporidium
Pichia beams
Yarrowia
Saccharomyces
central
beams
seeds
pulp
Fig. 3 The grape berry structure is shown in this scheme along with the main yeast gen-
era identified as epiphytes and endophytes that were found in the Vitis vinifera Tannat
using microbial and transcriptomic methods (Godoy et al., 2018). Interestingly, endo-
phytes were mainly from the basidiomycetes yeast group. Only 5–10% of this flora
was found to be antagonistic against pathogenic fungi, and a similar percentage was
proved to contribute with superior flavors to the quality of wine. A low input
winemaking strategy would be to increase this beneficial flora in the vineyard by selec-
tion and application in the vines.
The strategy of increasing the natural antagonistic population of yeasts

present in vineyards to prevent fungal diseases is shown as a “low-input
viticulture” practice, decreasing the demand of chemical treatments. On
the other hand, some of these yeasts strains would contribute to wine sen-
sory complexity after harvest in a winery. Several grape varieties and soils
of different Uruguayan vineyards were investigated for the presence of
microorganisms that are antagonistic to the phytopathogenic fungus
B. cinerea “in vitro” (Rabosto et al., 2006). Among 223 isolates of yeasts
and bacteria, eight non-Saccharomyces yeast strains and four bacteria showed
more than 50% effectiveness in vitro against Botrytis cinerea compared with
the growth of the control. A species of Bacillus subtilis and an isolate of the
yeast Hanseniaspora uvarum showed high, in vitro, antagonistic capability
against the pathogen, and both organisms were very effective (100% and
90%, respectively) in controlling rot development on grape clusters.
These two strains were selected for further studies using laboratory and
vineyard applications. As this microbial flora will enter at harvest time with
the grapes, flavor selection by sensory analysis of the antagonistic strains
was applied. After this sensory screening test, strains of Metschnikowia
fructicola; M. pulcherrima and H. vineae were selected that might contribute
to wine quality at the beginning of fermentation in the winery. The role of
endophytes in Vitis plants is still not known, due to very limited studies
(Godoy, Martin, Da Silva, Medina, & Carrau, 2018); however, 100%
of antagonistic efficiency was detected in strains of the endophyte
Aureobasidium. Further research of when they are integrated into the grape
berry will be necessary to understand when they can be applied during in
the growth season. To resume, as this antagonistic yeast flora is very scarce
in the natural composition of the microbiota, the strategy of selection and
enrichment of it by spraying should be considered a benefit to the mature
period of the grapes.
Some bacteria and viruses are well known as insect antagonistic, such as
Bacillus thuringiensis (Melo, Soccol, & Soccol, 2016). However, there are
some yeasts that can control insect populations (Stefanini, 2018).
Usually yeasts are dispersed by insects, and this give an advantage to the
native flora survival or to yeast strains that might increase hybridization
as was proved for native Saccharomyces cerevisiae (Stefanini et al., 2012).
However, although studies are very limited, some yeasts might affect insect
nutrition or are used to attract them into flavor trap systems to decrease the
natural population.
3.2 Grape harvest and application of the traditional

Saccharomyces yeast at the winery
Before application of a commercial yeast, winemakers should control the
process of harvest, as the microbial composition of the grape must will be
affected by the method of harvest. Hand-picked or mechanical harvest, size
of grape recipients, temperature and timing of transport to the winery, vari-
ety and ripeness, sulfite addition, etc., should be considered. Healthy grapes
with a well applied harvest process will naturally contain about 1–5 104
cells per mL in the final grape must, where 99.9% are usually non-
Saccharomyces species. In the cellar, many details of the process will also influ-
ence the natural flora, hygiene, and a rapid cooling system immediately at
the crushing facility and before pressing are key factors to prevent aeration
and the growth of undesired native yeasts that might consume some nutri-
ents such as vitamins and amino acids. After many years of yeast selection of
native flora during harvest time, strains that produce quality flavor (or are not
producers of off-flavors) are very scarce (Carrau et al., 2015). According to
our data of native yeast selection, we found about 5–8% of strains that pro-
duce superior flavors in Uruguayan wine regions (Medina, Martin, Boido, &
Carrau, 2019) compared to Saccharomyces reference strains. A number of
researchers and winemakers have found that spontaneous fermentations
are associated with greater wine body, unusual or odd aromas and flavors,
creamy texture and greater complexity (Carrau, 2006; Fleet, 2003;
Medina et al., 2013; Ramey, 1995; Varela et al., 2009). However, the
alternative of spontaneous fermentation processes might be risky for a wine-
maker, as he will not have enough time to make a precise microbial control
during vintage time (Boulton, Singleton, & Bisson, 1996). Many years ago it
was the “pied de cuve” or the “mother yeast” that was the alternative for
conducting a partial spontaneous vinification at the beginning of the harvest
(Peynaud, 2006; Ribereau-Gayon, Peynaud, & Lafourcade, 1951). This
might still be an interesting alternative. Starting with about 100 or 200 k
of grapes of a known vineyard with perfect sanity, the grapes were crushed
with small addition of sulfites (about 30 mg/L to avoid some bacteria), and
their fermentation was started in a small batch. The objective at that time was
to ensure a complete fermentation and the absence of off-flavors such as the
hydrogen sulfide gas. Naturally, with this process, native Saccharomyces strains
were selected as they are resistant to sulfites and reductive conditions. After a
couple of days, if the flavors were good and pleasant, the winemaker
increased the batch and prepared the first inocula for the start of the new
vintage. Usually by this method, they select Saccharomyces strains after the
pre-fermentation process. After a second round of yeast enrichment in
the following tank, yeast diversity also starts to decrease. Although through
this mechanism we start to work with native Saccharomyces flora that are dif-
ferent from the commercial strains, final yeast diversity was low due to their
dominance against non-Saccharomyces native flora. An advantage of these
native Saccharomyces yeasts is that they are better adapted to industrial con-
ditions, such as low assimilable nitrogen, than commercial strains, producing
pleasant aromas without di-ammonia phosphate DAP additions. However,
after a few generations, you will be working with one or two strains in the
system. It is well known that Saccharomyces strains are very dominant and
exclude from the fermentation medium all non-Saccharomyces and the major-
ity of Saccharomyces strains.
The first commercially available strains started to appear in the 1960s, and
today, there are many alternatives, although some strains might be the same
with different producer brands. It was shown that vineyards close to a winery
could be affected by the commercial S. cerevisiae strains used in the winery,
and it was demonstrated that after only 3 years of using a commercial
Saccharomyces strain in a winery, the strain will start to appear in the vineyards
around the winery, interacting with the native flora (Valero, Schuller,
Cambon, Casal, & Dequin, 2005). The commercial strains added in a winery
could be affecting the unique wine styles of a determined region. It is clearly
known that where a strain of Saccharomyces is inoculated in a certain levels of
population as suggested for commercial strains, the fermentation will be
carry on with a single yeast fermentation. In a few hours, the native flora
present in the grapes will be excluded from the medium as we demonstrated
with a Chardonnay barrel-fermented compared to spontaneously fermented
(Medina et al., 2013) (Fig. 4). This behavior is what we would like to use to
define a non-friendly strain; a natural adaptation in the Saccharomyces genus is
to compete efficiently with other yeasts in the fermentation winery niche,
which is very rich in sugars (there are very few species defined as Crabtree-
positive yeasts). In the environmental winery ecosystem, the grape must is
where Saccharomyces is the best-adapted to, high osmotic pressure by sugars,
low pH at 3.0–3.5, presence of sulfites and anaerobic conditions. As it was
demonstrated in a few hours, these species will build a more aggressive envi-
ronment to exclude other microbial competitors, such as by increasing tem-
perature, producing CO2 and increasing reduction and production of
ethanol as a microbial antiseptic compound (Goddard & Greig, 2015).
day 3 day 10
day 3 day 10
100% 100%
Scnative 4 Non -
Saccharomyces
80% 80%
Scnative 3
60%
60%
Scnative 1 Commercial
40%
40%
Commercial AIG 804 20%
20%
0%
0%
Commercial Commercial
Spontaneous Spontaneous
day 3 day 10
day 3 day 10
Lall/laffort non-Sacch
Lall/laffort
100% 100%
Scnative 3
Scnative 4
80% 80%
Scnative 2 Scnative 3
60% 60%
Scnative 2
Scnative 1 40%
40% Scnative 1
20% H.vineae 20% H.vineae
0% 0% Commercial AIG 804

Commercial
AIG 804 H.vineae H.vineae
H.vineae + Com H.vineae + Com
Fig. 4 This figure shows the concept of “friendly starters”, where the comparison of four different procedures of inoculation in Chardonnay
barrel-fermented trials at winery level can be seen. Spontaneous, commercial, a sequential inoculation with H. vineae + Commercial and a
single inoculation with H. vineae strain T205F. An increase in yeast diversity of the processes was obtained with this last treatment, as
H. vineae let the natural or winery flora participate during fermentation. Genetic identification data obtained and adapted from Medina, K.,
Boido, E., Fariña, L., Gioia, O., Gomez, M.E., Barquet, M. et al., 2013. Increased flavour diversity of Chardonnay wines by spontaneous fermentation
and co-fermentation with Hanseniaspora vineae. Food Chemistry 141. 2513–21, doi:10.1016/j.foodchem.2013.04.056.
4. The alternative: Spontaneous fermentation and

non-Saccharomyces strains
These arguments lead scientists and winemakers to search for non-
Saccharomyces strains as a way to increase diversity and flavor complexity
of regional wines and differentiate from the massive market. Mixed cultures
was the main strategy and was utilized by adding a Saccharomyces strain after a
non-Saccharomyces was inoculated (sequential inoculation) to finish fermen-
tation when alcohol is above approximately 10% volume. It has been
demonstrated that the initial effect of diverse non-Saccharomyces strains
was significantly and positively influencing the final wine flavor. A few
publications have provided support to prove these results at laboratory scale
with some yeast genera, such as Hanseniaspora, Pichia, Torulaspora and
Metschnikowia (Anfang, Brajkovich, & Goddard, 2009; Carrau, 2003;
Ciani, Beco, & Comitini, 2006; Ciani & Comitini, 2011; Egli, Edinger,
Mitrakul, & Henick-Kling, 1998; Fleet, 2003; Herraiz, Reglero, Herraiz,
Martin-Alvarez, & Cabezudo, 1990; Jolly, Augustyn, & Pretorius, 2003;
Perez et al., 2011; Taillandier, Lai, Julien-Ortiz, & Brandam, 2014;
Seguinot et al., 2020), and under real winemaking conditions (Lleixà
et al., 2016; Medina et al., 2013).
Initial growth of non-Saccharomyces yeasts in the fermentation had an
inhibitory effect on the subsequent growth of S. cerevisiae. A slower fermen-
tation profile was noted in the majority of the reported mixed culture pro-
cesses. Although this might be interesting for quality and low energy
demand for fermentation temperature control, this inhibition could be
implicated in sluggish or stuck fermentations. Such problems can be circum-
vented by adding of some key vitamins or yeast cell extracts when
Saccharomyces is inoculated or starts to grow naturally in the middle of the
fermentation and nutrients are consumed by the initial flora (Gobert,
Tourdot-Marechal, Sparrow, Morge, & Alexandre, 2019; Martin et al.,
2018; Medina, Boido, Dellacassa, & Carrau, 2012). In our experience, a
limited bound or total SO2 in juice of about 30 mg/L exerts an inhibitory
effect on bacteria, functioning as a biostat (not biocide), to keep malolactic
bacteria in check. If the SO2 is not there, the bacteria start the malolactic fer-
mentation, scavenging nutrients from the yeast, which then slow down and
the consequent formation of acetic acid or stuck fermentation might occur.
There is considerable controversy concerning the effect of leaving spon-
taneous growth of native yeasts on the organoleptic quality of wines, due to
the risk of production of large amounts of ethyl acetate, acetic acid or hydro-
gen sulfide. According to Romano, Fiore, Paraggio, Caruso, and Capece
(2003), the synthesis of secondary products is an individual and reproducible
strain characteristic. In our experience using sensory analysis, we screened
native yeasts isolated from healthy Tannat grapes, and results showed that
about 10% of strains were producers of pleasant or good sensory character-
istics. This fact explained why spontaneous fermentation is not a popular
alternative for winemakers today. Within the limited selected strains by this
method, we discovered many strains of Hanseniaspora vineae, Metschnikowia
pulcherrima and M. fructicola that produced superior flavors. Intense fruity
aromas that contributed to increasing the varietal character of this variety
were obtained in a sequential mixed culture with Saccharomyces (Medina,
Boido, Dellacassa, & Carrau, 2005). Wines obtained at the industrial level
were fruitier, with higher color and similar volatile acidity and alcohol
content compared to the pure Saccharomyces vinification (Medina, Boido,
Dellacassa, & Carrau, 2018). Similar results were obtained with barrel-
fermented Chardonnay fermentations (Medina et al., 2013). During our
experiments with spontaneous fermentations, we noted that some inocula-
tions with non-Saccharomyces strains started to slowly ferment and behaved
similar to the spontaneous treatments but always with pleasant aromas.
Molecular identification methods confirmed that even these treatments have
more diversity of yeast strains in the process than the spontaneous process
(Martin et al., 2018; Medina et al., 2013) (Fig. 4). We started to think about
a friendly way to share the fermentation medium if our starters allowed other
yeast species to grow and contribute with diverse metabolites compared to a
single strain fermentation.
4.1 Friendly yeasts cooperate with the microbial terroir

A simil grape must medium chemically defined with low nitrogen was
developed to make possible the selection of new non-Saccharomyces and
Saccharomyces strains of low nitrogen demand. Furthermore, this medium
was shown to be suitable for comparing the abilities of commercial strains
to administrate nitrogen from a winemaking point of view (Carrau,
2003). This method contributes to the selection of a new generation of
native yeast strains adapted for low input winemaking strategies, where
the addition of DAP can be avoided and the natural amino acid profile of
a certain variety can be respected (Carrau et al., 2015). During winemaking
trials with Chardonnay barrel-fermented with Hanseniaspora vineae strains
(Medina et al., 2013), we noted that conventional inoculated barrels with

S. cerevisiae ALG804 were immediately dominated by this strain after 3 days
and totally at day 10. As is shown in Fig. 4, in spontaneous trials four different
strains were found sharing the process at day 10, in H. vineae mixed culture
with S. cerevisiae six different strains were identified at day 10, and in
H. vineae single culture more than eight different strains were identified at
day 10 (see Fig. 4).
4.2 Nutrient demand and competition in mixed cultures

One of the main problems in the wine industry is the formation of H2S and
other sulfur compounds during winemaking (Bisson, 1991; Henschke &
Jiranek, 1993; Lambrechts & Pretorius, 2000; Rapp & Versini, 1991).
The majority of the commercial yeast strains available usually need an
addition of ammonium phosphate because they are not well adapted to
the natural conditions of many grape musts of high quality vineyards that
usually vary between 100 and 250 mgN/L of assimilable nitrogen
(Carrau, 2003). Today winemakers are trying to decrease the use of nitrogen
additions, measuring the nitrogen content of grape musts in the winery with
improved and simple methods (Gump, Zoecklein, Fugelsang, & Whiton,
2002; Zoecklein, Fugelsang, & Gump, 2010). However, the availability
of strains of low nitrogen demand is still missing to improve this process.
Some of the reasons why an excess of nitrogen should be avoided are
understood now: (1) formation of health risk compounds like the ethyl car-
bamate, (2) increase of residual amino acids like histidine precursor hista-
mine (Medina et al., 2019), (3) an excess of residual nitrogen facilitating
the contamination of wines during aging with yeast strains such as
Brettanomyces, (4) an excess of nitrogen making fermentations more rapid
and demanding more energy for cooling as well as losing more aromas dur-
ing the process, and (5) increased addition of ammonium, changing the typ-
ical amino acid profiles of some varieties like Sauvignon blanc and many
reds, which changes the varietal typicity (Carrau, 2003). See review of these
topics in Bell and Henshcke (2005).
5. Other application of non-Saccharomyces yeasts for

low input winemaking
5.1 Color stability and synthesis of derived anthocyanin
compounds
Selection of strains with a major capacity for synthesis of anthocyanin deriv-
atives is something that was not well understood until recently. Anthocyanin
composition is an important quality parameter for red grapes because of the

significance of these compounds in determining color and aging potential of
many red wines. Because of the importance of color in red wines and the
amount of pigment removed by yeast, we have been studying the effect
of different Saccharomyces cerevisiae strains on anthocyanins extracted from
Tannat wines in Uruguay. A model red grape juice (RGJ medium) was
developed using an anthocyanin extract from grape skins of Vitis vinifera
cv. Tannat (Medina et al., 2005). This cultivar, widely grown in
Uruguay, is one of the richest varieties in polyphenolic compounds
(Boido et al., 2011; Carrau et al., 2011; Da Silva et al., 2013). The RGJ
medium allowed us to standardize fermentation conditions and achieve
reproducible yeast-anthocyanin interactions during vinification. This strat-
egy also reduced anthocyanin losses due to other factors not-yeast related,
such as grape skin adsorption. This method represents an improved approach
to specifically study the effect of yeast-anthocyanin interactions. The capac-
ity of yeast to modify anthocyanin concentration during red wine fermen-
tation showed no correlation between color intensity and total anthocyanin
concentration after fermentation. However, HPLC-MS analysis allowed
identification of anthocyanin derivatives, and the sum of these compounds
showed a direct correlation with the color intensity obtained with each
strain, thus explaining the color variability observed (Medina et al.,
2005). More recently, this method was applied to the screening of non-
Saccharomyces anthocyanin derived compound synthesis, where an increased
intra- and inter-specific diversity was detected (Medina et al., 2018).
Selecting the best strains in terms of color stability and those with low sulfites
input processes it is possible to optimize red wine vinification. Anthocyanin
derived compounds were proved to be related to secondary metabolic prod-
ucts of yeast, such as acetaldehyde and pyruvic acid. Both compounds are
usually blocked with sulfite addition, not allowing reaction with anthocya-
nins to produce derivatives such as the vitisins A and B and ethyl linked
anthocyanin flavanol pigments (Asenstorfer, Markides, Iland, & Jones,
2003; Eglinton et al., 2004; Lee, Swinny, & Jones, 2004; Morata,
Calderón, González, Gómez-Cordoves, & Suárez, 2007; Morata,
Gómez-Cordoves, Colomo, & Suárez, 2003).
5.2 Increasing or decreasing total acidity by yeasts

Is it important for the wine industry to modulate acidity without the addition
of chemical products, and introduce the use of microorganisms to increase
wine acidity (biological acidification) or decrease it (biological deacidification).
The traditional method for biological deacidification of wine is the use of

lactic acid bacteria (for malolactic fermentation) to consume malic acid
and produce the soft lactic acid (Kunkee, 1968; Vilela, 2019). Recently, a
new winemaking method was proposed combining the use of two non-
conventional yeasts as an alternative to the traditional malolactic fermenta-
tion (Benito, Calderón, Palomero, & Benito, 2015). These authors use
Schizosaccharomyces pombe to consume malic acid and produce alcohol
(malo-alcoholic fermentation. See Amerine & Kunkee, 1968). The decrease
in acidity is very important compared to the bacterial malolactic fermenta-
tion, as this yeast does not produce lactic acid. The combined use S. pombe
with Lachancea thermotolerans that produces lactic acid from sugars was con-
sidered in order to modulate the acidity. However, even when similar levels
of acidification are produced compared to the bacterial malolactic fermen-
tation, further experiments are needed to improve the sensory profile and
reach more round complexity (Lerm, Engelbrecht, & du Toit, 2010).
However, in regions where global climate change had increased warm
conditions, high alcohol content and reduction of total acidity of wines
is affecting wine palate balance. The application of L. thermotolerans might
be an interesting way to correct these characteristics through the production
of lactic acid and glycerol, decreasing alcohol yield (Balikci, Tanguler,
Jolly, & Erten, 2016; Gobbi et al., 2013; Kapsopoulou, Kapaklis, &
Spyropoulos, 2005). L. thermotolerans has a moderate fermentative power
and ethanol tolerance of 5–9% v/v (Morata et al., 2018), and some isolates
resist 10–20 mg/L of free SO2 (Aponte & Blaiotta, 2016). Its use in
winemaking produces wines with greater “spicy” and acidic notes
(Balikci et al., 2016; Garcı́a, Esteve-Zarzoso, Crespo, Cabellos, &
Arroyo, 2017), improving the overall quality and fruitiness, probably
because it produces high levels of ethyl isobutyrate (strawberry nuances).
This yeast has been described as a producer of β-glucosidase (Rosi,
Vinella, & Domizio, 1994) and cysteine lyase (Zott et al., 2011)—important
enzymes involved in the release of the aroma compound from varietal
precursors. In fermentation with this yeast, significant amounts of
4-mercapto-4-methylpentan-2-one were released (Zott et al., 2011), which
increased the contents of nerol and terpinen-4-ol (Rosi et al., 1994).
Interestingly, L. thermotolerans is also capable of developing a biological
deacidification of wines with high volatile acidity, as it was reported that it
might consume 94.6% of the initial acetic acid under aerobic conditions
and in a high glucose medium (Vilela-Moura, Schuller, Mendes-Faia, &
C^ orte-Real, 2008).
In cooler regions when it is necessary to carry out a deacidification before

vinification, Schizosaccharomyces pombe has characteristics that make it widely
used for this purpose. This yeast has high ethanol tolerance (around 10–15% v/v)
(Loira, Morata, Palomero, González, & Suárez-Lepe, 2018; Suárez-Lepe,
Palomero, Benito, Calderón, & Morata, 2012) but has limitations for use in
winemaking because it shows a slow growth rate, excessive hydrogen sulfide
and volatile acidity production (Callejo, González, & Morata, 2017;
Rankine, 1968). However, some strains of S. pombe have recently been isolated
with good properties for use in winemaking, producing wines with low acetic
acid and ethyl carbamate and an appropriate volatile aroma profile (Benito et al.,
2016). Biological deacidification of wine by this yeast is related to its ability to
transform malic acid into ethanol and carbon dioxide (Volschenk, van
Vuuren, & Viljoen-Bloom, 2003). Furthermore, recently it was shown that
S. pombe improves color stability of red wine due to its high capacity of formation
of vitisin A and anthocyanin-vinyl phenol derivatives. This might be explained
by the high production of pyruvic acid during fermentation and a strong hydro-
xycinnamate decarboxylase activity that increase the vinylphenol compound
(Morata et al., 2012).
5.3 Lees contact effects: Fast and slow cell lysis

The contribution of cell lysis to palate and body of the wines is well known.
In white or sparkling wines, it improves body and bubbles persistence. Some
white varieties that are to light in body are improved with a lees contact pro-
cess, where yeast cells are broken and integrated with the wine, increasing
protein and polysaccharide content. Cell mannoproteins and other cell com-
ponents, such as glutathione in white wines (Dubourdieu & Lavigne-
Cruege, 2004), that improve palate might also increase oxide-reduction
and protein stability of the final wine, avoiding the use of stabilizers or fur-
ther treatments. Usually this process with a conventional wine yeast of
Saccharomyces cerevisiae takes about 6–9 months in barrel/batonnage or even
more in bottle/sparkling processes (Richard Goeffroy, personal communi-
cation). However, yeast species might vary in their lysis rate process, and this
is an interesting trait for shortening the wine aging process. A rapid cell lysis
process will reduce barrel aging time in white and red wines and might
decrease the financial costs of bottle or barrel aging for the winery.
Furthermore, a short barrel aging process will decrease the overly oaky sen-
sory characteristics of many white wines of certain varieties. Recently, we
discovered that some strains of Hanseniaspora vineae resulted in a fivefold
increase of cell lysis and a fast loss of membrane integrity compared to

Hanseniaspora uvarum and Saccharomyces cerevisiae (Carrau et al., 2019). It was
noted that the lysis process was exponentially accelerated when sugars were
exhausted from the fermentation medium immediately after the end of fer-
mentation. Further studies about this topic are carried on at winery level.
5.4 Yeast strategies for low alcohol formation

In the past, one of the main parameters of quality in wine was its alcoholic
degree; therefore, when selecting a yeast, the main characteristic to be taken
into account was the efficiency that it presents in the production of ethanol
with respect to the amount of sugar consumed. However, today the viticul-
ture aims for the production of quality wines to improve other important
aspects related to flavor. Vineyard productions with lower yields obtain high
quality grapes since the fruits are concentrated with the secondary metabolites
(mainly polyphenols and volatile compounds), improving organoleptic qual-
ity of the resulting wine. On the other hand, phenolic maturity is one of the
fundamental parameters for harvest time determination, which, in many cases,
delays the grape harvest date, resulting in an increased sugar concentration. All
these factors result in wines with too high alcohol potential (15–16% v/v).
Wines with a high ethanol content cause a series of inconveniences both
to the producer and the consumer. High content of ethanol in a wine
increases the sensation of heat in the mouth and has a negative impact on
the perception of volatile components (Contreras et al., 2014; Mira de
Orduña, 2010). The current trend in the world is to consume beverages with
low alcohol content and increased antioxidants concentration (Da Silva
et al., 2013); (OMS, 2014; Quiroz, Rojas, Gonzalez, & Morales, 2014).
Furthermore, in many countries, higher taxes are associated with alcohol
percentage, increasing significantly above 14% (Contreras et al., 2014).
It was shown that non-Saccharomyces yeasts are less efficient in the pro-
duction of ethanol, either in pure or mixed fermentations with
Saccharomyces cerevisiae producing other secondary metabolites such as glyc-
erol (Gonzalez, Quirós, & Morales, 2013). The use of mixed fermentations
is a trend that is used, at present, for these purposes of increasing a secondary
metabolites alternative to ethanol.
6. Natural clarification and minimal manipulation

before bottling
We have heard very frequently about the advantages of unfiltered
wines, and this is another important topic to discuss in this chapter, as we
have experienced many low quality standards of wines with minimal filtra-
tion or that are unfiltered. We believe it was a fashion in the late 1990s and
2000s for many wines defined as organic to be bottled with a “romantic”
idea but poor microbial technology. This was also very frequently the case
with our ancestors, where in some years, the vintage was excellent and in
some years, it was not. The bottling process for optimal quality is a key
moment for the winemaker. He might have done excellent work during
vineyard management, harvest and fermentation and aging, but if any prob-
lem appears at bottling that was not previewed, the image of the product will
be poor and all the previous work underestimated. These points are affirma-
tions to make stand out the importance of this topic. Here some ideas are
given on how to obtain microbial stability for many years in a bottle. If
we are not patient and willing to bottle a young wine after vinification,
in our opinion, a good sterile filtration and the protein stability tests, mainly
for white and rose wines, should be the way to ensure flavor quality and tur-
bidity stability. In Fig. 5, we show an example of a red wine that was barrel-
aged for a short time to finish malolactic fermentation and two key analytical
Fig. 5 Searching for microbial and wine stability before bottling. When a wine is ready
for bottling, you can avoid filtration by some simple analysis of the blend in a tank with
temperature control (10–15 °C). This is a 50 hL tank example of a red wine over 40 days.
Free SO2 variations are an indirect indicator of microbial stability and an easy way to
follow this process at a winery. A turbidimeter also indicates the natural clarification
at the time to define the optimal time for bottling without filtration; data are indicated
in NTU.
parameters that can be followed to ensure quality before bottling with min-
imal handling, free SO2 stability and turbidity. Stability of new blends or
wines that were racked from barrels always need about 40–80 days of har-
monization in a temperature control tank before bottling to preserve flavors
(Ramey & Ough, 1980). After unfiltered bottling, we followed microbial
stability sampling bottles every 15 days. After 90 days of laying bottles for
aging in a cellar, microbial growth was not found by plating tests. The
exception that many winemakers have found is the potential appearance
of Brettanomyces yeast cells if the wine has been in contact with them during
the winemaking process (see recent review Cibrario et al., 2019). This
should be tested with some commercial kits for Brett detection before bot-
tling without filtration.
Alternative low input winemaking strategies are being studied to
improve or avoid the use of additives. For example, the selection of many
new strains with protease activity for fermenting white wines will avoid or
decrease the bentonite doses for reaching protein stability. Although the
crystals from the tartaric acid stability are easier to understand for the con-
sumer, some strains that produce increase levels of mannoproteins will
improve the prevention of crystal formation in the bottle of a young wine.
The other excellent tool we have today for bottling and to decrease or avoid
the use of SO2 is the availability of inert gasses of very good quality such as
argon, N2 and CO2. Manipulation of the wines after vinification and during
bottling under these gasses will improve all the steps of preventing flavor oxi-
dation and microbial stability.
7. Concluding remarks
In this chapter, we try to attract attention to the important concept of
low input technologies to protect flavor of the main fermented beverage,
wine. Many of the concepts discussed here are well applied to other fer-
mented beverages such as cider, beer, sake or yogurt. Flavors are very light
molecules, and it is easy to understand that the less we manipulate the pro-
cesses during fermentation and the finished end product, the less flavors we
will lose. Fermented foods are growing all around the planet, as they are
proved to be healthier than many non-fermented products. This is not only
due to the increase of probiotic flora that contribute to human and animal
microbiota but also because of the fact that microbes are growing in a raw
material and this ensures the absence of potential risky agrochemicals.
Furthermore and not less important, the sensory characteristics of the raw
Fig. 6 Understanding the terroir and low input winemaking technologies will allow us
to show and search for the true particular characteristics of our regional wines. As we
discussed, although we can be close to other vineyards, our natural resources and sit-
uations are different, as we explained in this figure with some examples of climate, air,
soil, etc., and showing that the natural microflora might be affected by commercial yeast
additions, as one of the effects of human technology inputs. In this chapter, we have
presented some ideas for working with friendly native selected yeasts that will lead
a particular microbiota to grow and influence the sensory profile of a wine. As it was
recently discussed by Pretorius (2020), we can classify yeast into five distinct groups:
native (naturally occurring in your vines), settlers (colonizing your winery), nomads
(commercial starters), mannequins (lab yeasts models) and avatars (semisynthetic or
genetically modified).
harvest food are enriched with many secondary compounds, mainly flavors
produced by the microbial flora that participate in the fermentation process.
So, there are healthy and sensory explanations—both the most important
traits and the buying forces that, today, are considered by consumers to enjoy
food. In Fig. 6, we show a general scheme of the concept of terroir and low
input winemaking. We have shown many examples of how the principles of
low input winemaking can be understood and how a winemaker can
improve his strategy for competing in this massive wine market—by leaving
the natural terroir to express itself in wines though its flavor phenotype.
Integration of the knowledge developed by many interdisciplinary innova-

tion processes, such as agricultural biodynamics, organic viticulture, sustain-
able management of the vines and wines, microbial selection, etc., will result
in an intensive sensorial differentiation of what it can be defined as
“terroir wines.”
Acknowledgments
This work was funded by the Comisión Sectorial de Investigación Cientı́fica (CSIC-UdelaR)
Group Project no. 656 and the CSIC Productive Sector, Project no. 602 of UdelaR,
Uruguay, the Agencia Nacional de Investigación e Innovación (ANII) Hanseniaspora
vineae FMV 6956 project and the Alianza Project ANII with Lage y Cia Uruguay/
Oenobrands France, for the application of new non-Saccharomyces yeasts. We would like
to thank our family wineries that made it possible to use their facilities for many years of
experimentation and practical winemaking and to dedicate part of our time and
knowledge to the academic wine community (F.C. Bodega Cerro Chapeu-Castel Pujol;
E.B. Bodega Bouza Boutique; Ramey Wine Cellars).
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CHAPTER THREE

Yeasts for low input winemaking:

Advances in Applied Microbiology, Volume 111 # 2020 Elsevier Inc. 89

Environmental conditions, such as climate, soil composition, water management, winds

winemakers to avoid sensory characteristic losses in different wine ecosys-

2. Low input winemaking technology

applied (Carrau et al., 2017). We have designed a fermentation synthetic

2.1 A quality paradox: The high winemaking technology

flavor and mouthfeel characteristics. Macromolecules in wines, mainly in

2.2 Fining agents

3. Microbial terroir and native yeasts

environment or microbial interactions in the fruit or during vinification,

3.1 Biological control of fungi and insects by yeasts in the

Native yeasts associated with the grape berry

The strategy of increasing the natural antagonistic population of yeasts

3.2 Grape harvest and application of the traditional

20% H.vineae 20% H.vineae

0% 0% Commercial AIG 804

4. The alternative: Spontaneous fermentation and

4.1 Friendly yeasts cooperate with the microbial terroir

(Medina et al., 2013), we noted that conventional inoculated barrels with

4.2 Nutrient demand and competition in mixed cultures

5. Other application of non-Saccharomyces yeasts for

composition is an important quality parameter for red grapes because of the

5.2 Increasing or decreasing total acidity by yeasts

The traditional method for biological deacidification of wine is the use of

In cooler regions when it is necessary to carry out a deacidification before

5.3 Lees contact effects: Fast and slow cell lysis

increase of cell lysis and a fast loss of membrane integrity compared to

5.4 Yeast strategies for low alcohol formation

6. Natural clarification and minimal manipulation

Integration of the knowledge developed by many interdisciplinary innova-

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