Prontiers in Microbiology OPEN ACCESS ctited by: ‘Aina ve Lvs ce Bordon, France Reviewed ty: ut Gone Aor, Intute dl Cas ca a Vi y et iro (GW Sean ‘Carman Bao, rivera vaenoa, Sa “Correspondence: tame Mereno-Garla fined total cor Specialty section: ‘Ts atlas suet 0 Food terot9, a sacton ote ura Frontiers in Mersey ecetved: 10 December 2017 ‘Accepted: 31 ary 2018 Puptsned: 15 Fens 2018 citation Morano-Garla J, arc Maras 7 Mauro JC and Moron (2018) Yeast mation yt for Aéshoie Wine Fermentaions ‘cua Tend and ite Prrspectvs. Font Micro 9:24, ‘a 10.5289/mrics 2078 00241 Yeast Immobilization Systems for Alcoholic Wine Fermentations: Actual Trends and Future Perspectives Jaime Moreno-Garcia"*, Teresa Garcia-Martinez’, Juan C. Mauricio’ and Juan Moreno? Deparment o Meobtogy, Agrtood Canes ot ntatonal Ecalenc COA, Campus ce Faeanaes, Unvesty oF ‘coraana, Cacia, Span “Deparment of Ageutursl Chemin end Saf Secs, Agra Camo terion Escalon ea), Campus ce Rabanat, Unvertyof Corba, Coraba, Span ‘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 methodotogies 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, ike winemaking. The most studied methods for yeast immobilization include the use of natural supports (e.g., fuit pieces), organic supports (e.9,, alginate), inorganic (¢.g., porous ceramics), membrane systems, and mult functional agents. Some advantages of the yeast- immobilization systems include: high cell densities, product yield improvement, lowered risk of microbial contamination, better Control and reproducibility of the processes, as well 2s reuse of the immobilization system for batch fermentations and continuous fermentation technologies. However, these methods have some consequences on the behavior of the yeasts, affecting the final products of the fermentative metabolism. This review compiles current information about cell immobilizer requirements for winemaking purposes, the immobilization methods applied to the production of fermented beverages to date, and yeast physiological consequences of immobilization strategies. Finally, a recent inter-species immobilization methodology has been revised, where yeast cells are attached to the hyphae of a Generally Recognized As Safe fungus and remain adhered following loss of viability of the fungus. The bio-capsules formed with this method open new and promising strategies for alcoholic beverage production (wine and low ethanol content beverages) Keywords: immobilization, win, yeast biocapaules, fermentation, yeast metabolism INTRODUCTION Yeast immobilization offers numerous opportunities for industrial fermentation processes such as beer, cider production, or winemaking. This technology aims to confine intact, active yeast cells to a specific region, thus inereasing the eell density, permitting the enhancement and prolongation of certain metabolites (eg, aromatic) production, allowing better control and stability ofthe yeast strain, providing cell protection against shear forces, and enabling cell recovery/reutilization and Fronts in Mcrtiigy | wines or 1 Fetrury 2078 | Vote 9| re 24Moreno Garcia continuous fermentations, among other advantages (Williams and Munnecke, 1981; Groboillot eta, 1994; Sakura etal, 2000; Kourkoutas et al, 2004b; Baptista et al, 2006; Nedovié et al, 2015), Although this technology reduces process cost and allows customization of wine properties, industrial use of immobilized cells is still limited (Djordjevic et al, 2016; Berbegal et al, 2017). Nedovié 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 up. Moreover, to accomplish crucial factors in winemaking like ‘consumer acceptance, safety issues, and profitability, Kourkoutas et al. (2004b) recommended supports that are abundant in nature, cost-effective, and of food-grade quality for successful industrial application. Nevertheless, questions such as “what particular immobilization system utilize in what wine elaboration process” or “how immobilization affects cell physiology, flavor formation, and wine stability ~ including microbial, chemical, and sensorial” still need to be addressed in order to promote yeast immobilization technologies in wine industrial processes. The overall objective of this review is to compile the most updated information about the requitements of cell immobilizers for winemaking, the immobilization systems applied and proposed to the production of wine (including advantages and drawbacks), and yeast physiological consequences of immobilization strategies. Special attention was placed ‘on interspecies immobilization methodologies, which are considered novel approaches for winemaking and other fields cof applications. This is the case for “yeast biocapsules” which consist of yeast cells attached to the hypha of a dead filamentous fungus cataloged as Generally Recognized As Safe (GRAS), 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, eg, 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 earrier has to comply with certain requirements as follows (Martin and Etievant, 1991): (Big surface, with funetional properties and/or chemical groups favoring cells to adhere. (i) Easy o handle and regenerate. (Gi) High and setained cell viability and operational stability. (iv) Catalytic activity na affected. (¥) Uniform and controllable porosity to allow free exchange of substrates, products, cofactors, and gases. Good mechanical, chemical, thermal, and biological stability Easy, cost-effective, and immobilization technique. Not affect product quality. wi) (it amenable to scale-up witty IMMOBILIZATION METHODS DEPENDING ON THE YEAST CELL LOCALIZATION To date, different methods for yeast immobilization have been developed depending on the mechanism of eell localization (Figure 1 and Table 1), Auto-Immobi n ‘Winemakers benefit from the ability of certain yeasts species to auto-immobilize in an innate way. From a biological point cof view, immobilization favors yeast cells as it allows cell cooperation to fully utilize available resources and maximize chances of survival through improved resistance to stress (Honigberg, 2011), Microorganisms, notably Saccharomyces cerevisiae can perform various multicellular manners of immobilization: adhesion, biofilm formation, filament formation, and flocculation, The effect of some of these behaviors on the wine quality is widely known to be beneficial and is already applied industrially. This is the case of yeast biofilm formation for biological aging in the elaboration of Sherry wines and Alocculation 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 Copoper Seose Auto-immobilization Immobilization on a support surface Mechanical containment behind a barrier Entrapment in a porous matrix FIGURE 1 | Base methods of cal lnmabization depending on the cet Fetrury 2078 | Vote 9| re 24‘Yeas immobleaton for Wremakng Moreno Garcia posunsep You dors wasnpy ‘ponsye9 ou syed ns 908 p00, uote 0 04-0 fy josseooc uotepeag FUR sooo, "CUEpUNGE LAH peEN pu Alun pepo} BAO sects ern ‘oro sjoomau pu erences P. sD.9H08 new um vaEngp 0 jo uoWeag “SHOW PBL UONEINDON SQ STON EU RUAN ‘pai ope sarod 09 vsqusw aassod 24-29 0 spureauo ‘aw suexaueulsroogenn ep RUNES pss SEU EURO SSN 199 RALEAPUULILBYK ABE —_-OsnoIodanU pede ae 2) reousuy 960 seo p MADER eel “sssoaed sso sno 440 BuO POUUUGp uA SIe2 aM 2g no Batu p acco aoc sz] ¢ousu- Cub oogre iets Jotfuars6spuoqpueaiog puree werden — jaaqssunoe. vondespy uD uaezou 2p ws “woronaacy pu ui} Zo vensoducs unipau ——posn ENO pu aN wa UOIRLUO} UNO woSaIDE dom soeeja1 ansves SUMUD SHR ROWE — “eTERERBEE OL Spo pANRERUY —_uogERIROMUCIY vonezigounu ‘sobequonpesig sobewerpy wonduoeap ous yospouon upouun u svonordde jo sure pve sebenarpoeD cobeuaNpYUoIEDEEP Joa VORSZIGOUNA HEB IO EON b TEL February 2018 |Youme 9 Arie 265 Fronts in Mcrbicigy | wir Fontes. orMoreno Garcia i & i 2 q | Eas e /Page alg ae E iggeh 2 EGGE iHHE BBEaEE a |88 Bag Hh pone Gah ae zee ua fH pill i ay Bae TABLE 1 | Contres i : 8 Methods ot Immobilization Fronts in Mcrtiigy | wines or Loukas eta, 2000 5 i i ; nero fatnntaton procuctty arc rom, cwarics, porous aes, pohuethane Neterganie mata porcus Inorganic supports ‘Yeas immobleaton for Wremakng process that is known as “biological aging.” This type of biofilm is called “flor” or “velum” ~ formed by special yeast strains known as “flor yeasts” ~ and protects wine from oxidation and 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, acctoin, and 2,3-butanediol and promotes their inclusion as carbohydrates lipids, and proteins into yeast cells via the Krebs Cycle (Martinez et al, 1998; Zara et al., 2010; ‘Moreno and Peinado, 2012; Moreno-Garcia et al, 2013, 2014, 2015a,b, 2017). The resulting wines are characterized by sensorial characteristics known as lor or velum bouquet (Lopez-Alejandre, 2005). Gell flocculation consists of non-sexual aggregation of single-celled organisms in suspension to form a larger unit for 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 several factors like cell wall composition, medium, pH, and dissolved oxygen (Kourkoutas et al, 2004b; Nedovié et al, 2005). It is used in the production of sparkling wines, such as Champagne, performed by the "Champenoise” technique, which consists of a second fermentation in a sealed bottle of a base 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 of removing cell deposit from the bottle, clarifying the wine, and reducing wine losses (a process called dégorgement) (Valles et aly 2008), Simultaneously, yeast immobilization through flocculation reduces the wine production costs as there is less energy expended, thus turning into a greener’ process that could enhance the quality of final products. tisalso used in the brewing industry as packed-bed oF fluidized-bed or even continuous stiered-tank reactors (Kourkoutas et al, 2004) and it affects fermentation productivity and quality, as well as yeast removal and retrieval, Agents or cross-linkers can enhance flocculation of cells that do not spontaneously aggregate. Immobilization on a Support Surface Immobilization on a support surface is defined as the binding of yeast cells to a carvier by covalent bonding between the cell and the support, or by adsorption (ionic bonds o electrostatic forces). Examples of known support surfaces are cellulosic materials 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 Jow cost of used immobilization materials, such as cellulosic and inorganic materials, and the simplicity of achieving the process. However, the depth of the cell biofilm and the bonding strength often vary and are not readily determined. As cells are directly exposed to the solution, detachment and relocation are possible while yeast growth. Fetrury 2078 | Vote 9| re 24Moreno Garcia Among the cellulosic material, fruit pieces, delignified cellulosic materials (DCMs), and gluten pellets (GPs) have been applied in winemaking. Fruit pieces ease the immobilization methods required. Apple and quince constitute abundant and low price supports of food-grade purity of immobilization that were found suitable for continuous processes and lead to production of improved sensory traits (Kourkoutas etal, 2001, 2002, 2003a,b). Further, grape skins were used to immobilize S. cerevisiae because of easy application, increased productivity, and positive influence on wine aroma compared to free cells (Mallouchos et al, 2002). This support was established by these authors as suitable for winemaking and proposed for future investigation to their utilization in combined alcoholic {fermentation (AF) and malolactc fermentations (MLFs). On the other hand, DCM and GP were considered effective in carrying ‘out fermentations at both room and low-temperature as well as increasing rates and improving organoleptic quality compared to free cells (Bardi and Koutinas, 1994; Bardi et al., 1996a,5, 1997; Mallouchos et al., 2003). DCM and GP were proposed to use at industrial levels because they are inexpensive and abundant supports of food-grade purity that are easy to produce industrially. In comparison with other natural supports, they lower fermentation rates, present a longer operational stability, are suitable for low-temperature winemaking, and also accepted by consumers. Yeast cells immobilized with DCM and GP were found to fit commercialization objectives through freeze-drying techniques as the freeze-dried immobilized yeasts produced wines of similar quality to those made by fresh immobilized yeast cells and of enhanced properties in comparison with free cells (conomopoulou et al., 2000, 2002, 2003; Bekatorou et al., 2001). ‘This last feature makes DCM and GP attractive for industrial use Inorganic support surfaces (eg. palygorskite, hydromica, and, porous porcelain) were shown to have mainly few advantages in winemaking (Hamdy et al, 1990; Colagrande et al, 1994). Researchers recommended the utilization of cellulose and DEAE-cellulose (as main carrier) covered with Ca-alginate and an anion-exchange resin, respectively, as immobilization supports for winemaking; the first for continuous winemaking purposes (Lommi and Advenainen, 1990). Increases of calcium fon contents and off-flavors due to the use of alginate or DEAE-cellulose, respectively, must be considered in winemaking processes Mechanical Containment behind a Barrier ‘The most common are the microporous or ultraporous ‘membrane filters and the microcapsules. They are utilized when the minimal transfer of compounds or cel-free products is necessary (Park and Chang, 2000). This cell immobilization type can be attained by three methods: (i) by utilization of microporous membrane filters, (ii) by entrapment of ells in a microcapsule, or (iii) by cell immobifization on to an interaction surface of two immiscible liquids. It has been used in winemaking, and however, its use is limited because of Joss during mass transfer (Lebeat et al, 1998) and potential membrane biofouling caused by cell growth (Gryta, 2002), “Milispark” cartridge developed by Millipore is an example ‘Yeas immobleaton for Wremakng utilized for secondary fermentation in a bottle of sparkling ‘winemaking (Ramon-Portugal et al., 2003). S. cerevisiae and Schizosaccharomyces pombe were co-immobilized on glass pellets coated with a membrane of alginates to further use them for batch and continuous winemaking processes (Ogbonna et al, 1989), Wines with similar features to those produced with free cells were obtained, Takaya et al. (2002) reported that 1 system consisting of two-vessel bioreactor (one operating as a continuous stirred tank reactor and the other one as the membrane bioreactor), where cells were entrapped by a cross-flow type microfilter, was suitable for continuous dey ‘winemaking and had 28-fold higher production than a batch system, Moreover, microfiltration and ultrafiltration membranes as well as silicon, ceramic, and other membranes have been employed. Entrapment in a Porous Matrix Entrapment in a porous matrix is attained when cells are incorporated in a rigid network which prevents them from diffusing into the neighboring medium while still admitting ‘mass transfer of substrates and metabolism products, They are divided in two methods: (i cells infiltrate into the porous matrix until their motility is interfered by other cells and (i) the porous material is assembled in situ into a culture of cells, Some examples are polysaccharide gels like alginates, K-carrageenan, agat, chitosan, and polygalacturonic acid or other polymeric ‘matrixes like gelatine, collagen, and polyvinyl alcohol (Norton and D'Amore, 1994; Park and Chang, 2000). One ofthe problems ‘of this technique is cell release when located on the outer surface cof the matrix. To bypass this possiblity, double layer beads have been used (Tanaka et al, 1989; Taillandier et al, 1994; Ramon. Portugal et al, 2003). In general, the use of polysaccharide hydrogels and alginates is not a suitable industrial choice for several reasons: (i) high prices, (i) low mechanical and chemical stability that causes cell and residues release in wine, and (ii) biomass entrapped in a gel matrix that is critical for utilization ‘of biotechnological processes using viable immobilized cells (Kourkoutas et al, 20040). Salts like Na-, Ca-, or Ba-alginate have been extensively used. {or cell immobilization, and among them, Ca-alginate gels are the most advisable for AF (Colagrande et al, 1994). Notably, Ciani and Ferraro (1996) proposed a system entrapping Candida stellata in Ca-alginate gels as an attractive system to increase lycerol content in wine. These authors reported a 30-fold ‘improvement in fermentation rate (g of COp/day) in comparison with free cells and twofold production of ethanol and a reduction in acetaldehyde and acetoin production. Moreover, Ferraro etal (2000) attempted to scale up the immobilization systems to pilot and industrial scales. They revealed an interesting flavor profile ‘of wines produced when co-inoculated with S. cerevisiae, and however, the wild wine microbiota was not completely repressed. Cavalginate beads have also been recommended to entrap highly Aocculent S. cerevisiae strains to perform cell-recycle batch process and optimize primary must fermentations (Suzzi et al, 1996). Another application of Ca-alginate cell entrapment is the secondary fermentation in sparkling winemaking for easy clarification and removal of cells, , cerevisiae strains are being Fetrury 2078 | Vote 9| re 24Moreno Garcia immobilized for this purpose and commercially applied in winemaking processes (Fumi etal, 1988; Colagrande etal, 1994), S. cerevisiae encapsulated in Ca-alginate were also utilized with ‘success for the treatment of sluggish and stuck fermentations and revealed better results than the traditional free cells method — the system attained a decrease rate of 2.8 g/L x day of reducing sugar with a viable cell concentration of 5 x 10%/mlL and no {nerease in off-avor content ot volatile acidity (Silva etal, 2002) According to the winemakers, one of the major drawbacks of calcium salt-based systems is the high Ca content provoked by the low solubilization of calcium tartrate in the bottled wine IMMOBILIZATION METHODS DEPENDING ON THE CHEMICAL COMPOSITION OF THE CARRIER ‘The materials used as immobilization supports (carriers), can be divided, based on their origin, into natural materials and artificially treated materials; according to Kourkoutas etal (2010) and Nedovié et al. (2010), they can be categorized as shown in ‘Table 1 Natural Supports Carrier materials are mainly of food-grade purity and are used with minimal to no pre-treatment like brewer spent grains, DCM, GR, pivces of fruit, sawdust, wood, ete. Their abundance, low cost and food-grade composition have made them an interesting way to enhance the aroma character of many products, eg, wine, beer. The utilization of natural supports Such as DCM, GP, gras, and fruit pieces, for immobilization, ‘was proved effective for winemaking as previously mentioned {see the section “Immobilization on a Support Surface"). Natural materials with certain food-grade meet the prerequisites forthe selection ofthe cartier and result in promoting aroma formation and advancement of the sensory features ofthe final fermented product. S. cerevisiae cells immobilized in corm grains were considered a good candidate system because it was ecient for fermentations at ambient and low temperatures during repeated batch fermentations of grape must (Kandylis tal, 2012) Organic Supports Organic supports are atlicially made (eg, plastic) or obtained from natural sources by more complicated techniques (© polymeric hydrogels) regardless oftheir food-grade composition Natural or synthetic polymers have been widely researched most probably due to their gl-forming ability under gentle conditions fnd the capacity to form spherical beads that protect yeast cells agxinst contamination and inhibitory substances while fvoring substrate utilization and improving stability, favor production. and effcioney (Nedovie et al, 2010), Most wsed are those comprised of alginates, xulose, carrageenan, agar, pectic acid, Sand chitosan. Cavalginate gels among them are ore convenient for AB (Colagrande etl, 1994), and however the use of alginate, and polysaccharide hydrogels generally di not offer a favorable industrial alternative as previously explained (see the section “Entrapment in a Porous Matrix"), Most stempts were made ‘Yeas immobleaton for Wremakng for the utilization of alginate gels for the second fermentation in order to improve the technology of sparkling wine and have been commercially applied (Busova etal, 1994 Colagrande etal, 1994; Fumi et al., 1988). Immobilization of yeasts in organic supports has also been successfully applied to the following: ‘mead production (Pereira etal, 2014), pomegranate winemaking (Sevda and Rodrigues, 2011), wine made from the tropical fruit cagaita (Oliveira et al, 2011), wine from Cabernet Sauvignon or Pinot noir grape varieties (Andrade Neves et al, 2014), green beer production (Wang et al, 1989), stout beer production (Almonacid et al, 2012) lager-beer (Naydenova etal, 2013),and cider (Nedovic eta, 2000), Inorganic Supports Several inorganic materials such as porous ceramics, porous glass, polyurethane foam, ete, have been introduced as yeast cell carier Iaterials for many fermentation processes: beer production (Wirkajirvi and Pohjala, 2000; Vickjiriet al, 2002; Kourkoutas et al, 20045) and wines (Ogbonna eal, 1989: Bakoyianis et al, 1992, 1993; Kana etal, 1992 Loukatos etal, 2000; Bonin and Skwira, 2008), However even though they are usually abundant and can improve fermentation prodvctvity and arom, they an experience strong changes in metabolism and viability a the cells used in articial immobilization methods are notin ‘heir natural form. Also, they are usually considered undesirable for winemaking due to high concentrations of mineral residues found in the product. Nonetheles, thir use in immobilization systems can be regarded as promising for use in bioethanol or distilates production, Other Materials Other methods of immobilization such as membrane systems, entrapment by various types of interaction (ie, Van der Waals) forces, ionic bonds, hydrogen bridges) and mult Jianetional agents ~ several functionalities integrated into a single ‘miniaturized device (ie,, glutaraldehyde-based system) ~ are scarcely treated. As earlier cited, Takaya et al. (2002) revealed 1a membrane-based bioreactor as a good candidate system for continuous dry wine production, Ligno-cellulosic materials from agricultural wastes can be valuable substrates for immobilization, alter removal ofthe lignin fraction from the cellulose matrix by an alkaline treatment in view to create tubular cellulose-based (TC) nnanoestructures. YEAST PHYSIOLOGICAL CONSEQUENCES OF IMMOBILIZATION STRATEGIES Call growth, physiology, and metabolic alterations may be induced by immobilization although they are hard to predict (Melzoch et al, 1994 Norton and D'Amore, 1994; Walsh and Malone, 1995; Djordjevic etal, 2016). Assays comparing immobilized and free cells have revealed elfects on increase in stored polysaccharides, altered growth rates, lower yield of fermentation by-products, activation of yeast energetic metabolism, increased substrate uptake and Fetrury 2078 | Vote 9| re 24Moreno Garcia product yield, higher intracellular pH, increased resistance against toxic and inhibitory compounds, and increased invertase activity (Norton and D’Amore, 1994), Immobilization of yeast to various solid surfaces affects intrinsic cell growth rate, Which either increased (Bandyopadhyay and Ghose, 1982) or decreased (Doran and Bailey, 1986). The pH in immobilized S. cerevisiae cells in alginate beads is lower than in free yeasts, 68 and 6.9, respectively, which was attributed to. increased permeability of the cell membrane for protons, leading to a higher ATP utilization and activating glycolysis and glucose uptake (Galazzo and Bailey, 1990). This results in an increased ‘enzyme activity and thus more substrate channeled to biomass and ethanol production. Norton and D’Amore (1994) reported aan enhanced ethanol resistance, a partial removal of substrate inhibition by cell immobilization, and higher tolerance to toxic ‘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 simply due to cell encapsulation by a protective layer of the immobilization material. On the other hand, the tolerance to toxic compounds can be indirectly related to osmotic stress that leads to intracellular production of compounds like polyols that regulate pressure, which also leads to diminished water activity and consequently increased tolerance to toxic chemicals (Norton and D'Amore, 1994) Finally, Lodato etal. (1999) showed higher ‘thermal stability in immobilized yeasts, In immobilized cell fermentations, increased ester and decreased fusel alcohols formation and the ratio of esters to alcohols have the highest influence on beverage technology (Bardi and Koutinas, 1994; Mallouchos et a, 2003). Some trials have been attempted to model the accumulation of dominant yeast metabolites produced by free and immobilized cells (Vassiley et aly, 2013). Nagarajana et al. (2014) observed a permanent pattern of gene expression different from starving planktonic cells: highly expressing genes in cell wall reassembling and stress tolerance, glycolysis, but decreasing transcription of genes that regulate the cell cycle and in the tricarboxylie acid eyele Consequently, changes in concentrations of metabolites are observed when using entrapped or adsorbed yeast cells. Special attention has to be given to compounds such as alcohols (ethanol, higher alcohols), carbonyl compounds (acetaldehyde, vicinal dliketones), esters (acetate esters, medium-chain fatty acid esters), ‘organic acids (medium-chain fatty acids), and sulfur compounds (hydrogen sulfide, sulfur dioxide, dimethyl sulfide) as they are those that most affect flavor during fermentation (Dufour et al, 2003; Nedovié et al, 2015). In fermented beverages, the greatest aroma impact is due to increased esters, decrease of fusel alcohol concentrations, and ratio of esters/alcohols ftom fermenting yeast metabolism (Bardi and Koutinas, 1994; Mallouchos et al, 2003; Nedovié et al, 2015). In white wine production, it was slotected a significant difference in sensory properties among free and immobilized cells (Mallios etal, 2004; Tsakiris etal, 20043,05 Genisheva etal, 2012). Kourkoutas etal. (200ab) noted a stronger flavor and aroma in semi-sweet wines when immobilizing yeasts. Kourkoutas etal. (2004a), Tsakiris etal. (2004a,b), and Gonzalez. Pombo etal. (2014) did not report an important influence on the pleasantness of wine, A slight difference was revealed in the scores ‘Yeas immobleaton for Wremakng for preference of the produced wines, where scores were higher ‘when using immobilized cells compared to free cells (Tsakiris et al, 2004a,b). ‘These authors also found that temperature is an important factor for wines elaborated with immobilized yeasts at lower temperatures, which were preferred by the consumers Another aim of yeast immobilization in winemaking is the removal of the off-flavor aroma compounds, as well as the de- acidification of wines to enhance the organoleptic features of the final product (Genisheva et al., 2012, 2013, 2014; Villa et al, 2013). Nediovie et al. (2015) suggested more attention to be placed on evaluation of sensory quality of wine produced by free and immobilized cells with a trained panel or consumers along with instrumental analyses in order to assess the quality of final products and further support the development of acceptable products before being marketed. NEW TRENDS OF YEAST IMMOBILIZATION IN WINEMAKING During the last few years, novel yeast cll immobilization systems hhave been designed to adapt to winemaking purposes. New ‘materials such as spherical gellan particles cross-linked with ‘magnesium acetate were found suitable for AF of grape must (lurciuc etal, 2016). In this way, Kumar etal. (2016) studied the use of nano-/micro:porous of cellulosic materials (TC) produced by delignification of mango (Mangifera indica L.), sal (Shorea robusta G.) savedust and rice husk (Oryza sativa L.) in various food bioprocesses. They conclude that the porous structure of ‘TC renders it suitable for use as carrier for yeast immobilization in AF and also. as filler material in microorganism removal processes, The TCs used as S, cerevisiae cells immobilization carriers for AF of grape must and glucose media at 15°C provide satisiying fermentation rates, high ethanol content and productivity, and volatile by-products production. In addition, advanced applications in winemaking and co-immobilization of different organisms in different carriers, same carrer, or among each other (exploiting adherence properties of organisms) were recently described, Canonico et al. (2016) co-immobilized non-Saccharomyces yeasts in Ca-alginate to perform soquential_ fermentations coupled with a final inoculation of free S. cerevisiae cells to reduce ethanol content in wine. The yeasts immobilized were Crabtree negative (sugar consumption by respiration and low ethanol yield) and naturally present on grapes and winemaking ‘equipment. The strategy resulted in high reaction rates, avoidance ‘of contamination where the sugar content was reduced to a 50% in 3 days and the ethanol up to 1.6% v/v, and in less prolonged time than in non-immobilized formats. An enhancement of the analytical profile of wine was observed for most of the yeasts immobilized. Although this produced promising results, Canonico et al. (2016) recommended further research because the system submitted uses high inoculation levels and expensive immobilization procedures, which increases the costs of the fermentation process. Then, Moreno et al. (2016) proposed to use the S. cerevisiae ability to auto-immobilize in biofilms (ie, flor velum) to further consume ethanol from a red wine. Fetrury 2078 | Vote 9| re 24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 L-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 claboration of new wine types in an inexpensive manner. Another application of yeast immobilization (Candida intermedia yeast cells encapsulated in Ca-alginate and magnetic Co-alginate ‘beads) was discovered by Farbo etal. (2016) objectfied to remove the mycotoxin ochratoxin A from rotten grape juice. Although they obtained significant reductions of over 80%, these authors “observed a slow release ofthe mycotoxin by the yeast carriers. By using the same cartier, acid bacteria Oenococcus een were confined to operate simultancously AF and. MLF with the purpose of enhancing safety and quakty of wine (Gervetas et al, 2013; Bleve et al, 2016). In this way, Servetas et al. (2013) co-immobilized S. cerevisiae and O. oeni in DCM carriers covered with starch gel composite and observed a high elfcieney at low temperature fermentations (10°C) obtaining wine characterized by an increased ester formation and lower higher alcohols. Bleve et al. (2016) co-immobilized the same microorganisms in Ca-alginate beads revealing an efficient performance of Negroamaro must fermentation with a decrease Of the time needed to complete AF and MLF, low production of volatile acidity and similar organoleptic traits of the wine obtained than with those using sequential AF-MLF in free cell formats. The yeast and bacteria cells immobilized were reused up to three times with no activity loss. Also, & cerevisiae and O, en entrapped cells into grape stems/skins were used in sequential AF and MLF obtaining wines with sweet and fruity flavors (Genisheva et al, 2014). Nevertheless it must be considered that simultaneous AF and MLF could also have severe drawbacks sometimes leading to spoilage wines. Novel concepts of organism co-immobilzation without the need of an external support have arose. This kind of methodology exploits the ability of the organisms used to adhere to external bodies. This is the case of the co-immobilization of yeasts and filamentous fungus categorized as GRAS. It consists of the attachment of yeast cells to the mycelium of filamentous fungus (eg, Rhizopus sp. Aspergillus niger, and Penicillium sp.) (Peinado et al, 2004, 2005, 2006; Nyman et al., 2013) that can be regarded as a natural immobilization system matrix and complies with several required features for the promotion of industrial application: abundant, cheap, storable for long-terms, non-destructive, food-grade, etc. Co-immobilizing Penicillium chrysogenum and yeast cells results in the formation of spherical bodies that are hollow, known as “yeast biocapsuiles” (Figure 2) ‘The system minimizes changes to the yeast metabolism and/or yeast viability and enables diffusion of nutrients/products to and from the biocapsules due to the porous structure of the hypha framework (Garcia-Martinez et al, 2011). The yeast Diocapsule methodology exploits the natural adhesion properties of yeast (i.e, biofilm formation) and filamentous fungus cells cerevisiae and lat FIGURE 2] Yost iocapsues (2-5 renin dams fone wi Saccharomyces cose ae Poncitim crysogerur (Left ane secon of the rner wal ean bocapaue existing ict ryphas(eamanicus fer) wah yeast cal (ova shape) photoraphedin a scartng eaten mixoscope SEN (igh so they attach to each other thus eliminating the need of external supports and decreasing the final price of the process. Puture research on the function of the FLOI1 gene as well as other genes involved in biofilm formation, in flor yeast will help boost cel-immobilization methodologies by decreasing the release of yeast cells to the external medium (Nedovie et al, 2015). Garcia-Martinez et al, (2011) demonstrated the death of the fungus when the yeast biocapsules were incubated in ‘media supporting yeast fermentation by effect of direct contact between its hyphae and yeast cells, and to endure as a mere, bout highly inert and stable support for yeast cells, which can ease their reuse, Because of these characteristics, yeast biocapsules have already been utilized in production of white wine, sparkling wine, and natural sweet wine as well as for bioethanol from starch and molasses (Peinado et a, 2005, 20065 Gatcia-Martinez et al, 2012, 2013, 2013; Puig-Pujol et al, 2013) in a lab-scale and have been considered a promising technique {or industrial-scae fermentation purposes. Comparison of white grapes juice fermentations conducted by yeast biocapsules vs. fee yeasts showed higher amounts of acetaldehyde produced by the biocapsules (84 ys. 63 mg/l, respectively), isobutanol 217 ys. 194 mg/D, L-proline (7.7 vs. 6.5 mM), and aspartic acid (0.42 vs. 0 mM) in final wine. All of these analyzed compounds ranged between the limits of concentration values dlescribed in the literature and no existence of off-lavors were reported (Peinado et al, 2005). Lopez de Lerma et al. (2012) and Garcia-Martines etal (2013) used osmotolerant S. cerevisiae strains to form biocapsules to elaborate sweet wine from raisin ust to overcome the lag phase of yeasts under osmotic stress. Fermentations resulted in high concentrations of compounds rolated to osmoregulation like glycerol, acetaldehyde, acetoin among others, leading to an increased complexity of wine aroma (Garcia Martinez et al, 2013). Biocapsule immobilization ‘was also compared to Ca-alginate beads for sparkling wine claboration, producing the first wine with lower calcium ion content and improved enological characteristics (Puig-Pujol etal, 2013), In wine sensory quality analyses, results vary from different studies (Mallios et al. 2004; Tsakiris et al, 2004a,b; Genisheva etal, 2012; Gonzalez-Pombo et al, 2014). Tsakiris et al. (2004a,b) Fetrury 2078 | Vote 9| re 24Moreno Garcia asked consumers to calculate the pleasantness of red wine samples elaborated with yeast cells immobilized and they noted scores slightly higher for immobilization although not statistically different. Knowledge about the aroma and flavor profile provided by wine yeasts combined with the utilization of mixed non-Saecharomyees/Saccharomyces starters in immobilized formats for sequential inoculations allow winemakers to use them in a scientifically controlled way to cra ‘wine types that match consumer preferences in a diversified range ‘of market sectors. The utilization of non-Saccharomyces yeast combined to S. cerevisiae (to avoid stuck fermentations) has been recommended to improve the quality and complexity of wine olly et al, 20145 Capozzi et al, 2015). Henee, the utilization of controlled multi-starter fermentation using previously selected cultures of non-Saccharomyces and S. cerevisiae yeast strains has been encouraged (Ciani and Comitini, 2011; Comitini et al, 2011; Domizio et al, 2011; Magyar and Téth, 2011; Di Maio et al, 2012; Morata et al., 2012; Jolly et al, 2014). Yeast cell immobilization will ease sequential inoculations of these yeasts where the selected non Saccharomyces and Saccharomyces yeasts 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 etal, 2016). This practice will allow the non-Saccharomyces yeast longer time to express their particular metabolic footprint that would otherwise be inhibited by the stress of Saccharomyces compe In the last few years, Kandylis et al. (2010), Kourkoutas et al. (2010), and Tsaousi et al. (2010, 2011) have proven the potential of elongated periods of storage for thermally dried immobilized yeast cells in different carriers (delignified brewer's spent grains and DCM, GP, and freeze dried wheat) with neither loss of viability nor fermentation activity and _making wines with similar organoleptic characteristics to those of Jresh inocula, thereby accentuating the commercial potential for industrial usage. For these reasons, immobilization of microbial cells can improve cell metabolism even under stress conditions (eg, high suger content, low and high temperatures) and can be used for biological removal of detrimental compounds (ie. de-acidification) oF controlled liberation of flavor-active compounds, all of which improve the ability of the overall process and the quality of the end products, Indeed, the long-term storage of immobilized cells and their utilization at higher scales will boost the industsalization of immobilized technology in winemaking FINAL CONSIDERATIONS. Studies evidence the advantages of immobilized yeast cells in comparison with free yeast cells. Cell immobilization has ‘been proven to be an interesting strategy to overcome some important inconveniences in fermented alcoholic beverages production. However, though many benelits are described and new technologies are still arising, there are not many applications for winemaking at an industrial level. Potential reasons could be as follows: (i) lack of feasibility atthe cellar scale ~ some of the methods may be dificult to up-grade, (i) insufficient effectivity ‘Yeas immobleaton for Wremakng of yeast cell adherence to the carriers of current immobilization technologies, (ii) high investments (economic and time) to integrate these technologies into traditional practices without a secure outcome, (iv) lack of advertisement on immobilized yeasts, and (v) limited knowledge in winemakers about the yeast immobilization techniques and their benefits This review shows how studies concerning immobilization supports and mateix properties, such as their solubility, chemical and mechanical stability, their degradation in different culture broths and physico-chemical conditions during their use in a bioreactor, should be addressed more in depth. In this respect, the spontaneous and inter-species biological co-immobilization system between a GRAS fungus and an industrial yeast strain ‘open new perspectives asa new carrier for improvement of yeast immobilization systems. Furthermore, for the implementation to an industrial scale, a higher scientific knowledge is necessary regarding the influence of immobilization on the physiology of industrial yeast strains and about the metabolites excreted, especially those directly related with the sensorial attributes of the obtained beverages. CONCLUSION ‘We think that immobilization systems used for yeast cells provide a revolutionary perspective for the next future in production ‘of wine and other beverages. Their use to carry out addressed and controlled fermentation processes can contribute to innovate the production technology and the design and making of new and differentiated supreme quality products for consumers. The scarceness of industrial applications that exist in the present does not mean that research on yeast immobilization techniques should be abandoned. Aversely, investigation should be boosted in order to find the right immobilization technique for the right application in winemaking in order to exploit all the potential of these promising techniques. AUTHOR CONTRIBUTIONS IM-G performed the necessary literature searches, data compilation, and wrote the manuscript. TG-M, JCM, and JM coordinated the work and critically revised the manuscript before submission, FUNDING ‘This work was supported by the “XXII Programa Propio de Fomento de la Investigaciin 2017" (MOD.4.1 P.P.2016 J.J. MORENO V,) ftom the University of Cordoba (Spain) funding from the Spain's Ministry of Education, Culture and Sport, Grants for training university teachers (FPU) (JM-G). 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MK, Zta,G., Budron M, and Bakalinky, A. 2010)-Ethanl Independent biol formation by a Hor wine yeast sta of Sacharompecs Fronts in Mcrbilgy | wor tenors or ‘Yeas immobleaton for Wremakng cerevisiae, Appl Environ Micobiok 76, 4089-4091, doi: 101128/AEM. onto ‘Confit of Ioterest Statement: The authors dedae tht the earch was ‘conducted in the absence of any commercial or fancial elaonshps that could be contrac av potential conic of interet Copyright © 2018 Moreno Garcia, Garci- Martine, Mauricio and Moreno. This fen open-access ate dstibted under the terms of the Cri Commons Attbution License (CC BY). The ws disribuon or reproduction in other forums 'spermitod, provided the original authors) and the eapright owner are eee ‘and thatthe original plction in thisjoural ited, macordance with aceped ‘cade practic. No ws, dsrbuton or epraduction is permite which docs not campy wi these ters. 0 February 2018 | You 9| Arie 24