REVIEW

published: 15 February 2018

doi: 10.3389/fmicb.2018.00241

Yeast Immobilization Systems for

Alcoholic Wine Fermentations:
Actual Trends and Future
Perspectives
Jaime Moreno-García 1* , Teresa García-Martínez 1 , Juan C. Mauricio 1 and Juan Moreno 2
1
Department of Microbiology, Agrifood Campus of International Excellence (ceiA3), Campus de Rabanales, University of
Cordoba, Cordoba, Spain, 2 Department of Agricultural Chemistry and Soil Science, Agrifood Campus of International
Excellence (ceiA3), Campus de Rabanales, University of Cordoba, Cordoba, Spain

Yeast immobilization is defined as the physical confinement of intact cells to a region

of space with conservation of biological activity. The use of these methodologies for
alcoholic fermentation (AF) offers many advantages over the use of the conventional
free yeast cell method and different immobilization systems have been proposed so
far for different applications, like winemaking. The most studied methods for yeast
immobilization include the use of natural supports (e.g., fruit pieces), organic supports
(e.g., alginate), inorganic (e.g., porous ceramics), membrane systems, and multi-
Edited by:
functional agents. Some advantages of the yeast-immobilization systems include: high
Aline Lonvaud, cell densities, product yield improvement, lowered risk of microbial contamination, better
Université de Bordeaux, France
control and reproducibility of the processes, as well as reuse of the immobilization
Reviewed by:
system for batch fermentations and continuous fermentation technologies. However,
Lucía González-Arenzana,
Instituto de Ciencias de la Vid y del these methods have some consequences on the behavior of the yeasts, affecting the
Vino (ICVV), Spain final products of the fermentative metabolism. This review compiles current information
Carmen Berbegal,
Universitat de València, Spain
about cell immobilizer requirements for winemaking purposes, the immobilization
*Correspondence:
methods applied to the production of fermented beverages to date, and yeast
Jaime Moreno-García physiological consequences of immobilization strategies. Finally, a recent inter-species
[email protected]
immobilization methodology has been revised, where yeast cells are attached to the
Specialty section: hyphae of a Generally Recognized As Safe fungus and remain adhered following loss
This article was submitted to of viability of the fungus. The bio-capsules formed with this method open new and
Food Microbiology,
promising strategies for alcoholic beverage production (wine and low ethanol content
a section of the journal
Frontiers in Microbiology beverages).
Received: 10 December 2017 Keywords: yeast immobilization, wine, yeast biocapsules, fermentation, yeast metabolism
Accepted: 31 January 2018
Published: 15 February 2018
Citation: INTRODUCTION
Moreno-García J, García-Martínez T,
Mauricio JC and Moreno J (2018)
Yeast immobilization offers numerous opportunities for industrial fermentation processes such as
Yeast Immobilization Systems
for Alcoholic Wine Fermentations:
beer, cider production, or winemaking. This technology aims to confine intact, active yeast cells to
Actual Trends and Future a specific region, thus increasing the cell density, permitting the enhancement and prolongation
Perspectives. Front. Microbiol. 9:241. of certain metabolites (e.g., aromatic) production, allowing better control and stability of the yeast
doi: 10.3389/fmicb.2018.00241 strain, providing cell protection against shear forces, and enabling cell recovery/reutilization and

Frontiers in Microbiology | www.frontiersin.org 1 February 2018 | Volume 9 | Article 241

Moreno-García et al. Yeast Immobilization for Winemaking

continuous fermentations, among other advantages (Williams (v) Uniform and controllable porosity to allow free exchange
and Munnecke, 1981; Groboillot et al., 1994; Sakurai et al., 2000; of substrates, products, cofactors, and gases.
Kourkoutas et al., 2004b; Baptista et al., 2006; Nedović et al., (vi) Good mechanical, chemical, thermal, and biological
2015). stability.
Although this technology reduces process cost and allows (vii) Easy, cost-effective, and amenable to scale-up
customization of wine properties, industrial use of immobilized immobilization technique.
cells is still limited (Djordjevic et al., 2016; Berbegal et al., (viii) Not affect product quality.
2017). Nedović et al. (2015) proposed that future investigation
should approach the storage of immobilized cells long-term
and new designs of the processes and bioreactors that are
simple, flexible, and non-expensive and can be readily scaled
IMMOBILIZATION METHODS
up. Moreover, to accomplish crucial factors in winemaking like DEPENDING ON THE YEAST CELL
consumer acceptance, safety issues, and profitability, Kourkoutas LOCALIZATION
et al. (2004b) recommended supports that are abundant in
nature, cost-effective, and of food-grade quality for successful To date, different methods for yeast immobilization have been
industrial application. Nevertheless, questions such as “what developed depending on the mechanism of cell localization
particular immobilization system utilize in what wine elaboration (Figure 1 and Table 1).
process” or “how immobilization affects cell physiology, flavor
formation, and wine stability – including microbial, chemical, Auto-Immobilization
and sensorial” still need to be addressed in order to promote yeast Winemakers benefit from the ability of certain yeasts species
immobilization technologies in wine industrial processes. to auto-immobilize in an innate way. From a biological point
The overall objective of this review is to compile the most of view, immobilization favors yeast cells as it allows cell
updated information about the requirements of cell immobilizers cooperation to fully utilize available resources and maximize
for winemaking, the immobilization systems applied and chances of survival through improved resistance to stress
proposed to the production of wine (including advantages (Honigberg, 2011). Microorganisms, notably Saccharomyces
and drawbacks), and yeast physiological consequences of cerevisiae can perform various multi-cellular manners of
immobilization strategies. Special attention was placed immobilization: adhesion, biofilm formation, filament formation,
on inter-species immobilization methodologies, which are and flocculation. The effect of some of these behaviors on the
considered novel approaches for winemaking and other fields wine quality is widely known to be beneficial and is already
of applications. This is the case for “yeast biocapsules” which applied industrially. This is the case of yeast biofilm formation
consist of yeast cells attached to the hypha of a dead filamentous for biological aging in the elaboration of Sherry wines and
fungus cataloged as Generally Recognized As Safe (GRAS). flocculation for the second fermentation of sparkling wines.
Yeast immobilization in biofilms is formed spontaneously in
the wine-air interface of wines that are stored in barrels during a
CELL IMMOBILIZER REQUIREMENTS
FOR WINEMAKING PURPOSES
Accurate selection of the immobilization method and the carrier
material (with consideration of legality and stability, safety,
operating costs, and product quality) is essential. Among the
production systems that have been the subject of investigations,
some seem to fulfill the above prerequisites and lead to
promotion of aroma formation during the fermentation process
and improvement of the overall sensory characteristics of the
final products, e.g., wine, beer, and cider. However, actions
should be also focused on economical, abundant, non-damaging,
and food-grade immobilization supports, which will ameliorate
quality and provide a singular aroma profile and fine taste to
the final product. In general, for alcoholic beverage production
purposes, the cell carrier has to comply with certain requirements
as follows (Martin and Etievant, 1991):

(i) Big surface, with functional properties and/or chemical

groups favoring cells to adhere.
(ii) Easy to handle and regenerate.
FIGURE 1 | Basic methods of cell immobilization depending on the cell
(iii) High and retained cell viability and operational stability.
localization.
(iv) Catalytic activity not affected.

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 TABLE 1 | Methods of yeast immobilization: brief description, advantages, disadvantages, and examples of applications in winemaking.
Moreno-García et al.

Methods of Brief description Advantages Disadvantages Examples in winemaking (proposed or industrially applied)
immobilization

Auto-immobilization Innate ability of cells to aggregate (i.e., Beneficial effects on wine Sensitive to factors like pH, Biofilms of flor yeasts are traditionally used in biological aging for Sherry
adhesion, biofilm formation, filament quality and industrially used. medium, composition, O2 wine elaboration. Biofilms were also proposed for the reduction of the
formation, and flocculation). content, etc. ethanol content in wines (Moreno et al., 2016). Flocculation is being
used for wine clarification in sparkling wine production.

Frontiers in Microbiology | www.frontiersin.org

Immobilization on a Adsorption of cells to a carrier by cell Cheap carrier materials and Depth and bonding strength of Cellulose covered with Ca-alginate and DEAE-cellulose covered with an
support surface membrane-immobilizer covalent ease of carrying out the the cells are not determined. anion-exchange resin were recommended by Lommi and Advenainen
bonding or by electrostatic forces. process. Potential detachment of yeast (1990) while gluten pellets and delignified cellulosic materials were
cells. recommended for room and low-temperature industrial fermentations
(Bardi and Koutinas, 1994; Bardi et al., 1996a,b, 1997; Mallouchos
et al., 2003). Fruit pieces were investigated for continuous processes
(Kourkoutas et al., 2001, 2002, 2003a,b), and combined
alcoholic-malolactic fermentations (Mallouchos et al., 2002; Genisheva
et al., 2014).
Mechanical Cells are entrapped in microporous or Useful when minimal transfer of Cell loss during mass transfer Glass pellets coated with a membrane of alginates were proposed for
containment behind a ultraporous membrane filters, compounds or cell-free and possible membrane batch and continuous winemaking processes (Ogbonna et al., 1989),
barrier microcapsules or on an interaction products is needed. biofouling. “Millispark” cartridge for sparkling wine production (Ramon-Portugal

3
surface of two immiscible liquids. et al., 2003) and a two-vessel bioreactor system (one operated as a
membrane bioreactor) employed for continuous dry winemaking
(Takaya et al., 2002).
Entrapment in a porous Cells incorporation to rigid networks. Prevention of cell diffusion and High cost, low mechanical, and Ca-alginate gels were promoted for clarification in sparkling winemaking
matrix allowance of transfer of chemical stability. The biomass (Colagrande et al., 1994; Fumi et al., 1988), cell-recycle batch process
substrates and metabolism entrapped in a gel matrix is and optimization of primary must fermentations (Suzzi et al., 1996),
products. critical for usage of increase glycerol of wines (Ciani and Ferraro, 1996; Ferraro et al.,
biotechnological processes 2000), treatment of sluggish and stuck fermentations (Silva et al., 2002),
utilizing viable immobilized removal of ethanol or toxins (Canonico et al., 2016; Farbo et al., 2016),
yeast cells. and simultaneous alcoholic-malolactic wine fermentations (Bleve et al.,
2016).
Natural supports Principle of food-grade purity and used High abundance, low cost, and Degradation process of the Delignified cellulosic material, gluten pellets, grains, and fruit pieces
with slightest or no pre-treatment. food-grade nature. supports not evaluated. were proved effectively for winemaking. Yeast immobilized in a GRAS
Industrial scale-up not fungi (yeast biocapsules) has been tested for white wine, sparkling
described. wine, and natural sweet wine elaboration (Peinado et al., 2004;
Puig-Pujol et al., 2013; García-Martínez et al., 2015). Usage of corn
grains for ambient/low temperature batch fermentations was found
adequate (Kandylis et al., 2012). Delignified cellulosic material was
proven for simultaneously alcoholic-malolactic fermentations (Servetas
et al., 2013).

(Continued)

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Yeast Immobilization for Winemaking
Moreno-García et al. Yeast Immobilization for Winemaking

process that is known as “biological aging.” This type of biofilm

from cabernet sauvignon and pinot noir grape varieties (Andrade Neves
wine produced from the tropical fruit cagaita (Oliveira et al., 2011), wine

Gellan particles cross-linked with magnesium acetate were proposed

production (Fumi et al., 1988; Busova et al., 1994; Colagrande et al.,

Mineral kissiris proposed in low temperature winemaking (Bakoyianis

is called “flor” or “velum” – formed by special yeast strains

winemaking at ambient temperatures (Sevda and Rodrigues, 2011),

alcoholic fermentations (Kana et al., 1992; Loukatos et al., 2000).

et al., 1992, 1993), γ-alumina and kissiris for continuous or batch
Examples in winemaking (proposed or industrially applied)

1994). Organic supports have been also successfully applied to:

known as “flor yeasts” – and protects wine from oxidation and

Alginate gels have been commercially applied for sparkling wine

continuous fermentations (Ogbonna et al., 1989), pomegranate

for alcoholic fermentation of grape must (Iurciuc et al., 2016).

influences the sensory properties of Sherry type wines. The yeast
metabolic activity mainly results in a consumption of ethanol
and glycerol – the major carbon sources – and production
of acetaldehyde – the main metabolite liberated into the aged
wine. Additionally, consumption of ethanol raises the contents
of acetic acid, acetoin, and 2,3-butanediol and promotes their
inclusion as carbohydrates, lipids, and proteins into yeast cells
via the Krebs Cycle (Martínez et al., 1998; Zara et al., 2010;
Moreno and Peinado, 2012; Moreno-García et al., 2013, 2014,
2015a,b, 2017). The resulting wines are characterized by sensorial
characteristics known as flor or velum bouquet (López-Alejandre,
2005).
Cell flocculation consists of non-sexual aggregation of
et al., 2014).

single-celled organisms in suspension to form a larger unit

or aggregates of many cells known as flocs (Jin and Speers,
1998). The large size of the flocs makes their potential use
in reactors feasible. It is considered the simplest and cheapest
immobilization technique although it is easily influenced by
metabolism and viability. High
High costs, low mechanical,

several factors like cell wall composition, medium, pH, and

concentrations of mineral

dissolved oxygen (Kourkoutas et al., 2004b; Nedović et al.,

Strong changes in cell
and chemical stability.

2005). It is used in the production of sparkling wines, such as

Disadvantages

Champagne, performed by the “Champenoise” technique, which

consists of a second fermentation in a sealed bottle of a base
residues.

wine previously obtained by fermentation of a grape must. In

the last phase of this course, the bottles are turned down and
yeast cells deposit on the neck of the bottle. Here, the utilization
of flocculent yeast cells is important as it eases the process
conditions and form spherical

of removing cell deposit from the bottle, clarifying the wine,

contamination and inhibitory
beads that protects against

productivity and efficiency.

Usually abundant and can
substances while favoring

and reducing wine losses (a process called dégorgement) (Valles

enhancing stability, flavor
substrate utilization and

productivity and aroma.

Ability to gel under mild

et al., 2008). Simultaneously, yeast immobilization through

improve fermentation

flocculation reduces the wine production costs as there is less

energy expended, thus turning into a ‘greener’ process that could
Advantages

enhance the quality of final products. It is also used in the brewing

industry as packed-bed or fluidized-bed or even continuous
stirred-tank reactors (Kourkoutas et al., 2004b) and it affects
fermentation productivity and quality, as well as yeast removal
extracted from natural sources by more

and retrieval. Agents or cross-linkers can enhance flocculation of

ceramics, porous glass, polyurethane
complex processes (e.g., polymeric

cells that do not spontaneously aggregate.

Synthetically made (e.g., plastic) or

Not organic materials like porous

hydrogels) regardless of their

Immobilization on a Support Surface

Immobilization on a support surface is defined as the binding of
yeast cells to a carrier by covalent bonding between the cell and
Brief description

food-grade purity.

the support, or by adsorption (ionic bonds or electrostatic forces).

Examples of known support surfaces are cellulosic materials
foam, etc.

like diethylaminoethyl-cellulose (DEAE-cellulose), delignified

sawdust, sawdust, and wood; or inorganic materials like
hydromica, montmorillonite, palygorskite, porous glass, and
porous porcelain. This method has been widely applied due to
TABLE 1 | Continued

low cost of used immobilization materials, such as cellulosic and

Inorganic supports

inorganic materials, and the simplicity of achieving the process.

Organic supports
immobilization

However, the depth of the cell biofilm and the bonding strength
Methods of

often vary and are not readily determined. As cells are directly
exposed to the solution, detachment and relocation are possible
while yeast growth.

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Moreno-García et al. Yeast Immobilization for Winemaking

Among the cellulosic material, fruit pieces, delignified utilized for secondary fermentation in a bottle of sparkling
cellulosic materials (DCMs), and gluten pellets (GPs) have been winemaking (Ramon-Portugal et al., 2003). S. cerevisiae and
applied in winemaking. Fruit pieces ease the immobilization Schizosaccharomyces pombe were co-immobilized on glass pellets
methods required. Apple and quince constitute abundant and coated with a membrane of alginates to further use them for
low price supports of food-grade purity of immobilization batch and continuous winemaking processes (Ogbonna et al.,
that were found suitable for continuous processes and lead to 1989). Wines with similar features to those produced with
production of improved sensory traits (Kourkoutas et al., 2001, free cells were obtained. Takaya et al. (2002) reported that
2002, 2003a,b). Further, grape skins were used to immobilize a system consisting of two-vessel bioreactor (one operating
S. cerevisiae because of easy application, increased productivity, as a continuous stirred tank reactor and the other one as
and positive influence on wine aroma compared to free cells the membrane bioreactor), where cells were entrapped by a
(Mallouchos et al., 2002). This support was established by cross-flow type microfilter, was suitable for continuous dry
these authors as suitable for winemaking and proposed for winemaking and had 28-fold higher production than a batch
future investigation to their utilization in combined alcoholic system. Moreover, microfiltration and ultrafiltration membranes
fermentation (AF) and malolactic fermentations (MLFs). On the as well as silicon, ceramic, and other membranes have been
other hand, DCM and GP were considered effective in carrying employed.
out fermentations at both room- and low-temperature as well as
increasing rates and improving organoleptic quality compared Entrapment in a Porous Matrix
to free cells (Bardi and Koutinas, 1994; Bardi et al., 1996a,b, Entrapment in a porous matrix is attained when cells are
1997; Mallouchos et al., 2003). DCM and GP were proposed incorporated in a rigid network which prevents them from
to use at industrial levels because they are inexpensive and diffusing into the neighboring medium while still admitting
abundant supports of food-grade purity that are easy to produce mass transfer of substrates and metabolism products. They are
industrially. In comparison with other natural supports, they divided in two methods: (i) cells infiltrate into the porous matrix
lower fermentation rates, present a longer operational stability, until their motility is interfered by other cells and (ii) the
are suitable for low-temperature winemaking, and also accepted porous material is assembled in situ into a culture of cells. Some
by consumers. Yeast cells immobilized with DCM and GP were examples are polysaccharide gels like alginates, k-carrageenan,
found to fit commercialization objectives through freeze-drying agar, chitosan, and polygalacturonic acid or other polymeric
techniques as the freeze-dried immobilized yeasts produced matrixes like gelatine, collagen, and polyvinyl alcohol (Norton
wines of similar quality to those made by fresh immobilized yeast and D’Amore, 1994; Park and Chang, 2000). One of the problems
cells and of enhanced properties in comparison with free cells of this technique is cell release when located on the outer surface
(Iconomopoulou et al., 2000, 2002, 2003; Bekatorou et al., 2001). of the matrix. To bypass this possibility, double layer beads have
This last feature makes DCM and GP attractive for industrial use. been used (Tanaka et al., 1989; Taillandier et al., 1994; Ramon-
Inorganic support surfaces (e.g., palygorskite, hydromica, and Portugal et al., 2003). In general, the use of polysaccharide
porous porcelain) were shown to have mainly few advantages hydrogels and alginates is not a suitable industrial choice for
in winemaking (Hamdy et al., 1990; Colagrande et al., 1994). several reasons: (i) high prices, (ii) low mechanical and chemical
Researchers recommended the utilization of cellulose and stability that causes cell and residues release in wine, and (iii)
DEAE-cellulose (as main carrier) covered with Ca-alginate biomass entrapped in a gel matrix that is critical for utilization
and an anion-exchange resin, respectively, as immobilization of biotechnological processes using viable immobilized cells
supports for winemaking; the first for continuous winemaking (Kourkoutas et al., 2004b).
purposes (Lommi and Advenainen, 1990). Increases of calcium Salts like Na-, Ca-, or Ba-alginate have been extensively used
ion contents and off-flavors due to the use of alginate or for cell immobilization, and among them, Ca-alginate gels are
DEAE-cellulose, respectively, must be considered in winemaking the most advisable for AF (Colagrande et al., 1994). Notably,
processes. Ciani and Ferraro (1996) proposed a system entrapping Candida
stellata in Ca-alginate gels as an attractive system to increase
Mechanical Containment behind a glycerol content in wine. These authors reported a 30-fold
Barrier improvement in fermentation rate (g of CO2 /day) in comparison
The most common are the microporous or ultraporous with free cells and twofold production of ethanol and a reduction
membrane filters and the microcapsules. They are utilized when in acetaldehyde and acetoin production. Moreover, Ferraro et al.
the minimal transfer of compounds or cell-free products is (2000) attempted to scale up the immobilization systems to pilot
necessary (Park and Chang, 2000). This cell immobilization and industrial scales. They revealed an interesting flavor profile
type can be attained by three methods: (i) by utilization of of wines produced when co-inoculated with S. cerevisiae, and
microporous membrane filters, (ii) by entrapment of cells however, the wild wine microbiota was not completely repressed.
in a microcapsule, or (iii) by cell immobilization on to an Ca-alginate beads have also been recommended to entrap highly
interaction surface of two immiscible liquids. It has been used flocculent S. cerevisiae strains to perform cell-recycle batch
in winemaking, and however, its use is limited because of process and optimize primary must fermentations (Suzzi et al.,
loss during mass transfer (Lebeau et al., 1998) and potential 1996). Another application of Ca-alginate cell entrapment is
membrane biofouling caused by cell growth (Gryta, 2002). the secondary fermentation in sparkling winemaking for easy
“Millispark” cartridge developed by Millipore is an example clarification and removal of cells. S. cerevisiae strains are being

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Moreno-García et al. Yeast Immobilization for Winemaking

immobilized for this purpose and commercially applied in for the utilization of alginate gels for the second fermentation
winemaking processes (Fumi et al., 1988; Colagrande et al., 1994). in order to improve the technology of sparkling wine and have
S. cerevisiae encapsulated in Ca-alginate were also utilized with been commercially applied (Busova et al., 1994; Colagrande et al.,
success for the treatment of sluggish and stuck fermentations and 1994; Fumi et al., 1988). Immobilization of yeasts in organic
revealed better results than the traditional free cells method – supports has also been successfully applied to the following:
the system attained a decrease rate of 2.8 g/L × day of reducing mead production (Pereira et al., 2014), pomegranate winemaking
sugar with a viable cell concentration of 5 × 106 /mL and no (Sevda and Rodrigues, 2011), wine made from the tropical fruit
increase in off-flavor content or volatile acidity (Silva et al., 2002). cagaita (Oliveira et al., 2011), wine from Cabernet Sauvignon or
According to the winemakers, one of the major drawbacks of Pinot noir grape varieties (Andrade Neves et al., 2014), green
calcium salt-based systems is the high Ca2+ content provoked by beer production (Wang et al., 1989), stout beer production
the low solubilization of calcium tartrate in the bottled wine. (Almonacid et al., 2012), lager-beer (Naydenova et al., 2013), and
cider (Nedovic et al., 2000).

IMMOBILIZATION METHODS Inorganic Supports

DEPENDING ON THE CHEMICAL Several inorganic materials such as porous ceramics, porous glass,
COMPOSITION OF THE CARRIER polyurethane foam, etc., have been introduced as yeast cell carrier
materials for many fermentation processes: beer production
The materials used as immobilization supports (carriers), can (Virkajärvi and Pohjala, 2000; Virkajärvi et al., 2002; Kourkoutas
be divided, based on their origin, into natural materials and et al., 2004b) and wines (Ogbonna et al., 1989; Bakoyianis et al.,
artificially treated materials; according to Kourkoutas et al. (2010) 1992, 1993; Kana et al., 1992; Loukatos et al., 2000; Bonin and
and Nedović et al. (2010), they can be categorized as shown in Skwira, 2008). However, even though they are usually abundant
Table 1. and can improve fermentation productivity and aroma, they
can experience strong changes in metabolism and viability as
Natural Supports the cells used in artificial immobilization methods are not in
Carrier materials are mainly of food-grade purity and are their natural form. Also, they are usually considered undesirable
used with minimal to no pre-treatment; like brewer’s spent for winemaking due to high concentrations of mineral residues
grains, DCM, GP, pieces of fruit, sawdust, wood, etc. Their found in the product. Nonetheless, their use in immobilization
abundance, low cost and food-grade composition have made systems can be regarded as promising for use in bioethanol or
them an interesting way to enhance the aroma character of many distillates production.
products, e.g., wine, beer. The utilization of natural supports
such as DCM, GP, grains, and fruit pieces, for immobilization, Other Materials
was proved effective for winemaking as previously mentioned Other methods of immobilization such as membrane systems,
(see the section “Immobilization on a Support Surface”). Natural entrapment by various types of interaction (i.e., Van der
materials with certain food-grade meet the prerequisites for the Waals’ forces, ionic bonds, hydrogen bridges) and multi-
selection of the carrier and result in promoting aroma formation functional agents – several functionalities integrated into a single
and advancement of the sensory features of the final fermented miniaturized device (i.e., glutaraldehyde-based system) – are
product. S. cerevisiae cells immobilized in corn grains were scarcely treated. As earlier cited, Takaya et al. (2002) revealed
considered a good candidate system because it was efficient for a membrane-based bioreactor as a good candidate system for
fermentations at ambient and low temperatures during repeated continuous dry wine production. Ligno-cellulosic materials from
batch fermentations of grape must (Kandylis et al., 2012). agricultural wastes can be valuable substrates for immobilization,
after removal of the lignin fraction from the cellulose matrix by an
Organic Supports alkaline treatment in view to create tubular cellulose–based (TC)
Organic supports are artificially made (e.g., plastic) or obtained nanoestructures.
from natural sources by more complicated techniques (e.g.,
polymeric hydrogels) regardless of their food-grade composition.
Natural or synthetic polymers have been widely researched most YEAST PHYSIOLOGICAL
probably due to their gel-forming ability under gentle conditions CONSEQUENCES OF IMMOBILIZATION
and the capacity to form spherical beads that protect yeast cells STRATEGIES
against contamination and inhibitory substances while favoring
substrate utilization and improving stability, flavor production, Cell growth, physiology, and metabolic alterations may be
and efficiency (Nedović et al., 2010). Most used are those induced by immobilization although they are hard to predict
comprised of alginates, cellulose, carrageenan, agar, pectic acid, (Melzoch et al., 1994; Norton and D’Amore, 1994; Walsh and
and chitosan. Ca-alginate gels among them are more convenient Malone, 1995; Djordjevic et al., 2016).
for AF (Colagrande et al., 1994), and however, the use of alginates Assays comparing immobilized and free cells have revealed
and polysaccharide hydrogels generally did not offer a favorable effects on increase in stored polysaccharides, altered growth
industrial alternative as previously explained (see the section rates, lower yield of fermentation by-products, activation of
“Entrapment in a Porous Matrix”). Most attempts were made yeast energetic metabolism, increased substrate uptake and

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Moreno-García et al. Yeast Immobilization for Winemaking

product yield, higher intracellular pH, increased resistance for preference of the produced wines, where scores were higher
against toxic and inhibitory compounds, and increased invertase when using immobilized cells compared to free cells (Tsakiris
activity (Norton and D’Amore, 1994). Immobilization of yeast et al., 2004a,b). These authors also found that temperature is an
to various solid surfaces affects intrinsic cell growth rate, important factor for wines elaborated with immobilized yeasts
which either increased (Bandyopadhyay and Ghose, 1982) or at lower temperatures, which were preferred by the consumers.
decreased (Doran and Bailey, 1986). The pH in immobilized Another aim of yeast immobilization in winemaking is the
S. cerevisiae cells in alginate beads is lower than in free yeasts, removal of the off-flavor aroma compounds, as well as the de-
6.8 and 6.9, respectively, which was attributed to increased acidification of wines to enhance the organoleptic features of the
permeability of the cell membrane for protons, leading to a final product (Genisheva et al., 2012, 2013, 2014; Vilela et al.,
higher ATP utilization and activating glycolysis and glucose 2013). Nedović et al. (2015) suggested more attention to be
uptake (Galazzo and Bailey, 1990). This results in an increased placed on evaluation of sensory quality of wine produced by
enzyme activity and thus more substrate channeled to biomass free and immobilized cells with a trained panel or consumers
and ethanol production. Norton and D’Amore (1994) reported along with instrumental analyses in order to assess the quality of
an enhanced ethanol resistance, a partial removal of substrate final products and further support the development of acceptable
inhibition by cell immobilization, and higher tolerance to toxic products before being marketed.
compounds. These authors suggested that an increased ethanol
tolerance might be due to a modification in concentration of
membrane fatty acid because of oxygen diffusion limitations or NEW TRENDS OF YEAST
simply due to cell encapsulation by a protective layer of the IMMOBILIZATION IN WINEMAKING
immobilization material. On the other hand, the tolerance to
toxic compounds can be indirectly related to osmotic stress that During the last few years, novel yeast cell immobilization systems
leads to intracellular production of compounds like polyols that have been designed to adapt to winemaking purposes. New
regulate pressure, which also leads to diminished water activity materials such as spherical gellan particles cross-linked with
and consequently increased tolerance to toxic chemicals (Norton magnesium acetate were found suitable for AF of grape must
and D’Amore, 1994). Finally, Lodato et al. (1999) showed higher (Iurciuc et al., 2016). In this way, Kumar et al. (2016) studied the
thermal stability in immobilized yeasts. use of nano-/micro-porous of cellulosic materials (TC) produced
In immobilized cell fermentations, increased ester and by delignification of mango (Mangifera indica L.), sal (Shorea
decreased fusel alcohols formation and the ratio of esters to robusta G.) sawdust and rice husk (Oryza sativa L.) in various
alcohols have the highest influence on beverage technology (Bardi food bioprocesses. They conclude that the porous structure of
and Koutinas, 1994; Mallouchos et al., 2003). Some trials have TC renders it suitable for use as carrier for yeast immobilization
been attempted to model the accumulation of dominant yeast in AF and also as filter material in microorganism removal
metabolites produced by free and immobilized cells (Vassilev processes. The TCs used as S. cerevisiae cells immobilization
et al., 2013). Nagarajana et al. (2014) observed a permanent carriers for AF of grape must and glucose media at 15◦ C
pattern of gene expression different from starving planktonic provide satisfying fermentation rates, high ethanol content and
cells: highly expressing genes in cell wall reassembling and productivity, and volatile by-products production. In addition,
stress tolerance, glycolysis, but decreasing transcription of genes advanced applications in winemaking and co-immobilization of
that regulate the cell cycle and in the tricarboxylic acid cycle. different organisms in different carriers, same carrier, or among
Consequently, changes in concentrations of metabolites are each other (exploiting adherence properties of organisms) were
observed when using entrapped or adsorbed yeast cells. Special recently described.
attention has to be given to compounds such as alcohols (ethanol, Canonico et al. (2016) co-immobilized non-Saccharomyces
higher alcohols), carbonyl compounds (acetaldehyde, vicinal yeasts in Ca-alginate to perform sequential fermentations
diketones), esters (acetate esters, medium-chain fatty acid esters), coupled with a final inoculation of free S. cerevisiae cells to
organic acids (medium-chain fatty acids), and sulfur compounds reduce ethanol content in wine. The yeasts immobilized were
(hydrogen sulfide, sulfur dioxide, dimethyl sulfide) as they are Crabtree negative (sugar consumption by respiration and low
those that most affect flavor during fermentation (Dufour et al., ethanol yield) and naturally present on grapes and winemaking
2003; Nedović et al., 2015). In fermented beverages, the greatest equipment. The strategy resulted in high reaction rates, avoidance
aroma impact is due to increased esters, decrease of fusel alcohol of contamination where the sugar content was reduced to a 50%
concentrations, and ratio of esters/alcohols from fermenting in 3 days and the ethanol up to 1.6% v/v, and in less prolonged
yeast metabolism (Bardi and Koutinas, 1994; Mallouchos et al., time than in non-immobilized formats. An enhancement of
2003; Nedović et al., 2015). In white wine production, it was the analytical profile of wine was observed for most of the
detected a significant difference in sensory properties among free yeasts immobilized. Although this produced promising results,
and immobilized cells (Mallios et al., 2004; Tsakiris et al., 2004a,b; Canonico et al. (2016) recommended further research because
Genisheva et al., 2012). Kourkoutas et al. (2004b) noted a stronger the system submitted uses high inoculation levels and expensive
flavor and aroma in semi-sweet wines when immobilizing yeasts. immobilization procedures, which increases the costs of the
Kourkoutas et al. (2004a), Tsakiris et al. (2004a,b), and Gonzalez- fermentation process. Then, Moreno et al. (2016) proposed
Pombo et al. (2014) did not report an important influence on the to use the S. cerevisiae ability to auto-immobilize in biofilms
pleasantness of wine. A slight difference was revealed in the scores (i.e., flor velum) to further consume ethanol from a red wine.

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Moreno-García et al. Yeast Immobilization for Winemaking

In this work, the authors observed a decrease in the ethanol

content (also 1.6% v/v) and volatile acidity, favorable effect on
the color and astringency, and differences in the content of
1-propanol, isobutanol, acetaldehyde, 1,1-diethoxiethane, and
ethyl lactate after a short time (40 days) under velum aging
conditions. From a sensory analysis, wines were well accepted
by the younger consumers in a panel thus, concluding that
flor yeasts auto-immobilization in form of biofilms can be
used as fining agents supporting new perspectives for the
elaboration of new wine types in an inexpensive manner. Another
FIGURE 2 | Yeast biocapsules (3–5 mm in diameter) formed with
application of yeast immobilization (Candida intermedia yeast
Saccharomyces cerevisiae and Penicillium chrysogenum (Left) and section of
cells encapsulated in Ca-alginate and magnetic Ca-alginate the inner wall of a fresh biocapsule exhibiting intact hyphae (filamentous form)
beads) was discovered by Farbo et al. (2016) objectified to remove with yeast cells (oval shape) photographed in a scanning electron microscope
the mycotoxin ochratoxin A from rotten grape juice. Although SEM (Right).
they obtained significant reductions of over 80%, these authors
observed a slow release of the mycotoxin by the yeast carriers.
By using the same carrier, S. cerevisiae and lactic acid bacteria so they attach to each other thus eliminating the need of
Oenococcus oeni were confined to operate simultaneously AF and external supports and decreasing the final price of the process.
MLF with the purpose of enhancing safety and quality of wine Future research on the function of the FLO11 gene as well as
(Servetas et al., 2013; Bleve et al., 2016). In this way, Servetas other genes involved in biofilm formation, in flor yeast will
et al. (2013) co-immobilized S. cerevisiae and O. oeni in DCM help boost cell-immobilization methodologies by decreasing the
carriers covered with starch gel composite and observed a high release of yeast cells to the external medium (Nedović et al.,
efficiency at low temperature fermentations (10◦ C) obtaining 2015).
a wine characterized by an increased ester formation and García-Martínez et al. (2011) demonstrated the death of
lower higher alcohols. Bleve et al. (2016) co-immobilized the the fungus when the yeast biocapsules were incubated in
same microorganisms in Ca-alginate beads revealing an efficient media supporting yeast fermentation by effect of direct contact
performance of Negroamaro must fermentation with a decrease between its hyphae and yeast cells, and to endure as a mere,
of the time needed to complete AF and MLF, low production but highly inert and stable support for yeast cells, which
of volatile acidity and similar organoleptic traits of the wine can ease their reuse. Because of these characteristics, yeast
obtained than with those using sequential AF-MLF in free cell biocapsules have already been utilized in production of white
formats. The yeast and bacteria cells immobilized were reused up wine, sparkling wine, and natural sweet wine as well as for
to three times with no activity loss. Also, S. cerevisiae and O. oeni bioethanol from starch and molasses (Peinado et al., 2005, 2006;
entrapped cells into grape stems/skins were used in sequential García-Martínez et al., 2012, 2013, 2015; Puig-Pujol et al., 2013)
AF and MLF obtaining wines with sweet and fruity flavors in a lab-scale and have been considered a promising technique
(Genisheva et al., 2014). Nevertheless, it must be considered that for industrial-scale fermentation purposes. Comparison of white
simultaneous AF and MLF could also have severe drawbacks grapes juice fermentations conducted by yeast biocapsules vs.
sometimes leading to spoilage wines. free yeasts showed higher amounts of acetaldehyde produced
Novel concepts of organism co-immobilization without the by the biocapsules (84 vs. 63 mg/L, respectively), isobutanol
need of an external support have arose. This kind of methodology (217 vs. 194 mg/l), L-proline (7.7 vs. 6.5 mM), and aspartic
exploits the ability of the organisms used to adhere to external acid (0.42 vs. 0 mM) in final wine. All of these analyzed
bodies. This is the case of the co-immobilization of yeasts compounds ranged between the limits of concentration values
and filamentous fungus categorized as GRAS. It consists of described in the literature and no existence of off-flavors were
the attachment of yeast cells to the mycelium of filamentous reported (Peinado et al., 2005). López de Lerma et al. (2012)
fungus (e.g., Rhizopus sp., Aspergillus niger, and Penicillium sp.) and García-Martínez et al. (2013) used osmotolerant S. cerevisiae
(Peinado et al., 2004, 2005, 2006; Nyman et al., 2013) that can strains to form biocapsules to elaborate sweet wine from raisin
be regarded as a natural immobilization system matrix and must to overcome the lag phase of yeasts under osmotic stress.
complies with several required features for the promotion of Fermentations resulted in high concentrations of compounds
industrial application: abundant, cheap, storable for long-terms, related to osmoregulation like glycerol, acetaldehyde, acetoin
non-destructive, food-grade, etc. Co-immobilizing Penicillium among others, leading to an increased complexity of wine
chrysogenum and yeast cells results in the formation of spherical aroma (García-Martínez et al., 2013). Biocapsule immobilization
bodies that are hollow, known as “yeast biocapsules” (Figure 2). was also compared to Ca-alginate beads for sparkling wine
The system minimizes changes to the yeast metabolism and/or elaboration, producing the first wine with lower calcium ion
yeast viability and enables diffusion of nutrients/products to content and improved enological characteristics (Puig-Pujol
and from the biocapsules due to the porous structure of the et al., 2013).
hypha framework (García-Martínez et al., 2011). The yeast In wine sensory quality analyses, results vary from different
biocapsule methodology exploits the natural adhesion properties studies (Mallios et al., 2004; Tsakiris et al., 2004a,b; Genisheva
of yeast (i.e., biofilm formation) and filamentous fungus cells et al., 2012; Gonzalez-Pombo et al., 2014). Tsakiris et al. (2004a,b)

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Moreno-García et al. Yeast Immobilization for Winemaking

asked consumers to calculate the pleasantness of red wine of yeast cell adherence to the carriers of current immobilization
samples elaborated with yeast cells immobilized and they technologies, (iii) high investments (economic and time) to
noted scores slightly higher for immobilization although integrate these technologies into traditional practices without
not statistically different. Knowledge about the aroma and a secure outcome, (iv) lack of advertisement on immobilized
flavor profile provided by wine yeasts combined with the yeasts, and (v) limited knowledge in winemakers about the yeast
utilization of mixed non-Saccharomyces/Saccharomyces starters immobilization techniques and their benefits.
in immobilized formats for sequential inoculations allow This review shows how studies concerning immobilization
winemakers to use them in a scientifically controlled way to craft supports and matrix properties, such as their solubility, chemical
wine types that match consumer preferences in a diversified range and mechanical stability, their degradation in different culture
of market sectors. The utilization of non-Saccharomyces yeast broths and physico-chemical conditions during their use in a
combined to S. cerevisiae (to avoid stuck fermentations) has been bioreactor, should be addressed more in depth. In this respect,
recommended to improve the quality and complexity of wine the spontaneous and inter-species biological co-immobilization
(Jolly et al., 2014; Capozzi et al., 2015). Hence, the utilization system between a GRAS fungus and an industrial yeast strain
of controlled multi-starter fermentation using previously selected open new perspectives as a new carrier for improvement of yeast
cultures of non-Saccharomyces and S. cerevisiae yeast strains has immobilization systems. Furthermore, for the implementation
been encouraged (Ciani and Comitini, 2011; Comitini et al., to an industrial scale, a higher scientific knowledge is necessary
2011; Domizio et al., 2011; Magyar and Tóth, 2011; Di Maio regarding the influence of immobilization on the physiology
et al., 2012; Morata et al., 2012; Jolly et al., 2014). Yeast cell of industrial yeast strains and about the metabolites excreted,
immobilization will ease sequential inoculations of these yeasts especially those directly related with the sensorial attributes of the
where the selected non-Saccharomyces and Saccharomyces yeasts obtained beverages.
are in high concentrations and active; and ferment for a given
amount of time one after the other until S. cerevisiae is added to
conclude the fermentation (Canonico et al., 2016). This practice CONCLUSION
will allow the non-Saccharomyces yeast longer time to express
We think that immobilization systems used for yeast cells provide
their particular metabolic footprint that would otherwise be
a revolutionary perspective for the next future in production
inhibited by the stress of Saccharomyces competition.
of wine and other beverages. Their use to carry out addressed
In the last few years, Kandylis et al. (2010), Kourkoutas
and controlled fermentation processes can contribute to innovate
et al. (2010), and Tsaousi et al. (2010, 2011) have proven
the production technology and the design and making of new
the potential of elongated periods of storage for thermally
and differentiated supreme quality products for consumers. The
dried immobilized yeast cells in different carriers (delignified
scarceness of industrial applications that exist in the present
brewer’s spent grains and DCM, GP, and freeze dried wheat)
does not mean that research on yeast immobilization techniques
with neither loss of viability nor fermentation activity and
should be abandoned. Aversely, investigation should be boosted
making wines with similar organoleptic characteristics to those of
in order to find the right immobilization technique for the right
fresh inocula, thereby accentuating the commercial potential for
application in winemaking in order to exploit all the potential of
industrial usage. For these reasons, immobilization of microbial
these promising techniques.
cells can improve cell metabolism even under stress conditions
(e.g., high sugar content, low and high temperatures) and
can be used for biological removal of detrimental compounds AUTHOR CONTRIBUTIONS
(i.e., de-acidification) or controlled liberation of flavor-active
compounds, all of which improve the ability of the overall process JM-G performed the necessary literature searches, data
and the quality of the end products. Indeed, the long-term compilation, and wrote the manuscript. TG-M, JCM, and
storage of immobilized cells and their utilization at higher scales JM coordinated the work and critically revised the manuscript
will boost the industrialization of immobilized technology in before submission.
winemaking.
FUNDING
FINAL CONSIDERATIONS This work was supported by the “XXII Programa Propio de
Fomento de la Investigación 2017” (MOD.4.1 P.P.2016 J.J.
Studies evidence the advantages of immobilized yeast cells
MORENO V.) from the University of Cordoba (Spain); funding
in comparison with free yeast cells. Cell immobilization has
from the Spain’s Ministry of Education, Culture and Sport, Grants
been proven to be an interesting strategy to overcome some
for training university teachers (FPU) (JM-G).
important inconveniences in fermented alcoholic beverages
production. However, though many benefits are described and
new technologies are still arising, there are not many applications ACKNOWLEDGMENTS
for winemaking at an industrial level. Potential reasons could be
as follows: (i) lack of feasibility at the cellar scale – some of the The authors thank Minami Ogawa for her assistance with English
methods may be difficult to up-grade, (ii) insufficient effectivity language editing.

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Moreno-García et al. Yeast Immobilization for Winemaking

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of fluidized bed bioreactor containing immobilized yeast cells and the diacetyl Copyright © 2018 Moreno-García, García-Martínez, Mauricio and Moreno. This
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immobilized yeast cells. Biotechnol. Bioeng. 23, 1813–1825. doi: 10.1002/bit. is permitted, provided the original author(s) and the copyright owner are credited
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independent biofilm formation by a flor wine yeast strain of Saccharomyces comply with these terms.

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