Enological Repercussions of Non-Saccharomyces Species 3.0

A special issue of Fermentation (ISSN 2311-5637). This special issue belongs to the section "Fermentation for Food and Beverages".

Deadline for manuscript submissions: closed (30 June 2021) | Viewed by 41299

Special Issue Editor

Special Issue Information

Dear Colleagues,

Since the beginning of this century, non-Saccharomyces yeasts have taken increased relevance in wine processing. Several biotechnological companies now produce non-Saccharomyces yeasts at an industrial level to improve aroma or flavor, stabilize wine, produce biological acidification, or conversely metabolize malic acid. Species like Torulaspora delbrueckii, Metschnikowia pulcherrima, Kloeckera apiculata, Lachancea thermotolerans, Schizosaccharomyces pombe, and several others are common due to the technological applications they have in sensory quality but also in wine ageing and stabilization. Moreover, spoilage non-Saccharomyces yeasts like Brettanomyces bruxellensis, Saccharomycodes ludwigii, and Zygosaccharomyces bailii are becoming important because of the alterations they are able to produce in high-quality wines. New strategies have been developed to control to control these defective yeasts without affecting sensory quality. The knowledge of the physiology, ecology, biochemistry, and metabolomics of these yeasts can help to better use them in controlling traditional problems such as low fermentative power, excessive volatile acidity, low implantation under enological conditions, and sensibility to antimicrobial compounds like sulfites traditionally used in wine processing. The implantation of non-Saccharomyces yeasts can be improved by using emerging non-thermal treatments such as high hydrostatic pressure (HHP), ultra-high-pressure homogenization (UHPH), e-beam irradiation, pulsed electric field (PEF), or pulsed light (PL). Additionally, new applications of non-Saccharomyces yeasts such as their use as nutrients during fermentation, as fast releasers of polysaccharides at the end of fermentation or during ageing on lees, and the application of these species in biocontrol or as bioprotective tools to eliminate or decrease spoilage microorganisms in grapes, open new possibilities in current wine biotechnology. This Special Issue intends to compile current research and revised information on non-Saccharomyces yeasts with enological applications to facilitate the use and the understanding of this biotechnological tool.

The success of the first two Editions can be found at:

https://0-www-mdpi-com.brum.beds.ac.uk/journal/fermentation/special_issues/non-saccharomyces

https://www.mdpi.com/journal/fermentation/special_issues/non_saccharomyces_2

Prof. Dr. Antonio Morata
Guest Editor

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Keywords

  • non-Saccharomyces yeasts
  • wine biotechnology
  • sensory quality
  • color stabilization
  • spoilage yeasts

Published Papers (12 papers)

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Editorial

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3 pages, 203 KiB  
Editorial
Enological Repercussions of Non-Saccharomyces Species 3.0
by Antonio Morata
Fermentation 2022, 8(11), 613; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation8110613 - 08 Nov 2022
Viewed by 971
Abstract
The use of non-Saccharomyces in wine production has increased steadily since the commercial introduction of the first non-Saccharomyces strains produced as dry active yeasts at the beginning of the century by CHR Hansen and Lallemand (2004–2007) [...] Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)

Research

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15 pages, 1113 KiB  
Article
Use of Lachancea thermotolerans for Biological vs. Chemical Acidification at Pilot-Scale in White Wines from Warm Areas
by Cristian Vaquero, Pedro Miguel Izquierdo-Cañas, Adela Mena-Morales, L. Marchante-Cuevas, José María Heras and Antonio Morata
Fermentation 2021, 7(3), 193; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030193 - 15 Sep 2021
Cited by 16 | Viewed by 2792
Abstract
Climate change is affecting vineyards, resulting in grapes with a low acidity a high pH and sugar at harvest time. The most common procedure so far to improve the acidity and reduce the final pH of wines is to use tartaric acid, but [...] Read more.
Climate change is affecting vineyards, resulting in grapes with a low acidity a high pH and sugar at harvest time. The most common procedure so far to improve the acidity and reduce the final pH of wines is to use tartaric acid, but wine can also be acidified microbiologically using Lachancea thermotolerans yeasts, a natural bio-tool that acidifies gradually during the first stage/days of fermentation. Two strains of L. thermotolerans were compared with one Saccharomyces cerevisiae at a pilot-scale under similar fermentation conditions and in duplicate. A sequential inoculation was performed on the third day for the non-Saccharomyces, producing only about 1 g/L of lactic acid, which was suitable for comparison with the Saccharomyces, to which 1.5 g/L of tartaric acid had been added to lower the final pH. The three fermentations ended with a total acidity without significant differences. A significant and normal feature of the L. thermotolerans yeasts is their higher propane-1,2,3-triol production, which was observed in the Laktia yeast, and the acetic acid was <0.3 g/L. The amount of volatile metabolites was generally higher for non-Saccharomyces and the increase was seen in carbonyl compounds, organic acids, lactones, fumaric compounds, and phenols. Finally, the sensory analysis showed that there were hardly any significant differences, even though the non-Saccharomyces had a higher quantity of volatile metabolites, which could lead to a good acceptance of the product, since biological acidification was used, generating a more natural product. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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12 pages, 1850 KiB  
Article
Wine Aroma Characterization of the Two Main Fermentation Yeast Species of the Apiculate Genus Hanseniaspora
by María José Valera, Valentina Olivera, Eduardo Boido, Eduardo Dellacassa and Francisco Carrau
Fermentation 2021, 7(3), 162; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030162 - 21 Aug 2021
Cited by 16 | Viewed by 2765
Abstract
Hanseniaspora species are the main yeasts isolated from grapes and grape musts. Regarding genetic and phenotypical characterization, especially fermentative behavior, they can be classified in two technological clusters: the fruit group and the fermentation group. Among the species belonging to the last group, [...] Read more.
Hanseniaspora species are the main yeasts isolated from grapes and grape musts. Regarding genetic and phenotypical characterization, especially fermentative behavior, they can be classified in two technological clusters: the fruit group and the fermentation group. Among the species belonging to the last group, Hanseniaspora osmophila and Hanseniaspora vineae have been previously isolated in spontaneous fermentations of grape must. In this work, the oenological aptitudes of the two species of the fermentation group were compared with Saccharomyces cerevisiae and the main species of the fruit group, Hanseniaspora uvarum. Both H. osmophila and H. vineae conferred a positive aroma to final wines and no sensory defects were detected. Wines fermented with H. vineae presented significantly higher concentrations of 2-phenylethyl, tryptophol and tyrosol acetates, acetoin, mevalonolactone, and benzyl alcohol compared to H. osmophila. Sensorial analysis showed increased intensity of fruity and flowery notes in wines vinificated with H. vineae. In an evolutionary context, the detoxification of alcohols through a highly acetylation capacity might explain an adaption to fermentative environments. It was concluded that, although H. vineae show close alcohol fermentation adaptations to H. osmophila, the increased activation of phenylpropanoid metabolic pathway is a particular characteristic of H. vineae within this important apiculate genus. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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20 pages, 1025 KiB  
Article
Selection Process of a Mixed Inoculum of Non-Saccharomyces Yeasts Isolated in the D.O.Ca. Rioja
by Rocío Escribano-Viana, Lucía González-Arenzana, Patrocinio Garijo, Rosa López, Pilar Santamaría and Ana Rosa Gutiérrez
Fermentation 2021, 7(3), 148; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030148 - 10 Aug 2021
Cited by 8 | Viewed by 1889
Abstract
The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use [...] Read more.
The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO2 resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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15 pages, 1522 KiB  
Article
Application of Hanseniaspora vineae Yeast in the Production of Rosé Wines from a Blend of Tempranillo and Albillo Grapes
by Juan Manuel Del Fresno, Iris Loira, Carlos Escott, Francisco Carrau, Carmen González, Rafael Cuerda and Antonio Morata
Fermentation 2021, 7(3), 141; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030141 - 03 Aug 2021
Cited by 9 | Viewed by 2367
Abstract
Hanseniaspora vineae is an apiculate yeast that can be used for the production of interesting commercial wines, due to its contribution of fermentative volatiles. This paper presents a detailed comparative study of the use of H. vineae, compared to pure fermentations of [...] Read more.
Hanseniaspora vineae is an apiculate yeast that can be used for the production of interesting commercial wines, due to its contribution of fermentative volatiles. This paper presents a detailed comparative study of the use of H. vineae, compared to pure fermentations of S. cerevisiae in Tempranillo and Albillo rosé wines. Fermentations were carried out in oak barrels and stainless steel barrels. The results indicated that fermentation with H. vineae resulted in wines with residual sugars below 3.4 g/L and similar general characteristics, compared to S. cerevisiae. However, H. vineae wines contain up to 44% more total anthocyanins, resulting in an appreciable improvement in colour. In addition, H. vineae produced up to 65% more 2-phenylethyl acetate in stainless steel barrels and 2.5 times more terpene alcohols in oak barrels. Therefore, the use of H. vineae results in a more attractive colour, as well as fruity and floral organoleptic characteristics of rosé wines. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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15 pages, 37767 KiB  
Article
Temporal Comparison of Microbial Community Structure in an Australian Winery
by Cristian Varela, Kathleen Cuijvers and Anthony Borneman
Fermentation 2021, 7(3), 134; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030134 - 27 Jul 2021
Cited by 2 | Viewed by 4554
Abstract
Most modern fermented foods and beverages are produced in fit-for-purpose facilities which are designed to ensure not only a reliable product, but also one safe for consumption. Despite careful hygiene, microorganisms can colonise these facilities and establish resident populations that can potentially contribute [...] Read more.
Most modern fermented foods and beverages are produced in fit-for-purpose facilities which are designed to ensure not only a reliable product, but also one safe for consumption. Despite careful hygiene, microorganisms can colonise these facilities and establish resident populations that can potentially contribute to the fermentation process. Although some microorganisms may not negatively affect the final product, spoilage microorganisms can be detrimental for quality, generating substantial economic losses. Here, amplicon-based phylotyping was used to map microbial communities within an Australian winery, before, during and after the 2020 vintage. Resident bacterial and yeast populations were shown to change over time, with both relative abundance and location within the winery varying according to sampling date. The bacterial family Micrococcaceae, and the genera Sphingomonas and Brevundimonas were the most abundant bacterial taxonomies, while Naganishia, Pyrenochaeta and Didymella were the most abundant fungal genera. Mapping the spatial distributions of the microbial populations identified the main locations that harboured these resident microorganisms, that include known wine spoilage yeasts and bacteria. Wine spoilage microorganisms, including the genefugura Lactobacillus, Acetobacter, Gluconobacter and Brettanomyces showed very low relative abundance and were found only in a couple of locations within the winery. Microbial populations detected in this facility were also compared to the resident microbiota identified in other fermented food facilities, revealing that microbial population structures may reflect the nature of the product created in each facility. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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16 pages, 2306 KiB  
Article
Growth of Non-Saccharomyces Native Strains under Different Fermentative Stress Conditions
by Margarita García, Julia Crespo, Juan Mariano Cabellos and Teresa Arroyo
Fermentation 2021, 7(3), 124; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030124 - 22 Jul 2021
Cited by 6 | Viewed by 2197
Abstract
The selection of yeast strains adapted to fermentation stresses in their winegrowing area is a key factor to produce quality wines. Twelve non-Saccharomyces native strains from Denomination of Origin (D.O.) “Vinos de Madrid” (Spain), a warm climate winegrowing region, were tested under [...] Read more.
The selection of yeast strains adapted to fermentation stresses in their winegrowing area is a key factor to produce quality wines. Twelve non-Saccharomyces native strains from Denomination of Origin (D.O.) “Vinos de Madrid” (Spain), a warm climate winegrowing region, were tested under osmotic pressure, ethanol, and acidic pH stresses. In addition, mixed combinations between non-Saccharomyces and a native Saccharomyces cerevisiae strain were practised. Phenotypic microarray technology has been employed to study the metabolic output of yeasts under the different stress situations. The yeast strains, Lachancea fermentati, Lachancea thermotolerans, and Schizosaccharomyces pombe showed the best adaptation to three stress conditions examined. The use of mixed cultures improved the tolerance to osmotic pressure by Torulaspora delbrueckii, S. pombe, and Zygosaccharomyces bailii strains and to high ethanol content by Candida stellata, S. pombe, and Z. bailii strains regarding the control. In general, the good adaptation of the native non-Saccharomyces strains to fermentative stress conditions makes them great candidates for wine elaboration in warm climate areas. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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10 pages, 4004 KiB  
Communication
Biological Stoichiometric Analysis during Substrate Utilization and Secondary Metabolite Production by Non-Saccharomyces Yeasts Using Grape Pomace Extract as Fermentation Medium
by Maxwell Mewa-Ngongang, Heinrich W. du Plessis, Seteno K. O. Ntwampe, Enoch A. Akinpelu, Ucrecia F. Hutchinson, Boredi S. Chidi, Vincent I. Okudoh and Neil P. Jolly
Fermentation 2021, 7(2), 89; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7020089 - 02 Jun 2021
Cited by 1 | Viewed by 3826
Abstract
The emerging interest in the search for alternatives to synthetic preservatives has led to various successful research studies exploring the use of yeasts as potential biological control agents and producers of biopreservatives. The findings that yeasts could be used as producers of biopreservatives [...] Read more.
The emerging interest in the search for alternatives to synthetic preservatives has led to various successful research studies exploring the use of yeasts as potential biological control agents and producers of biopreservatives. The findings that yeasts could be used as producers of biopreservatives lacked some engineering considerations regarding cost-effective process design for scale-up, although partial process optimization using renewable agro-waste has been achieved. This study investigated the biological stoichiometry and bioenergetic parameters during yeast growth and secondary metabolites production i.e., biopreservatives from non-Saccharomyces yeasts using grape pomace extract (GPE), a type of agro-waste, as a fermentation medium. This was achieved by reconfirming the optimum production conditions previously found for Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164 in GPE broth as a fermentation medium, conditions under which a high amount of yeast cells were obtained. High-density cell cultures were produced, from which the yeast cell pellets were harvested, dried, and combusted for the determination of elemental analysis, heat of combustion, biological stoichiometry, and bioenergetic parameters. This work generated biological stoichiometric models and bioenergetics information that could assist in the design of yeast biochemical conversion system when GPE is used as fermentation medium, thereby, addressing the biochemical engineering aspects that were lacking in a previous biopreservative production study using Candida pyralidae Y1117, Pichia kluyveri Y1125, and Pichia kluyveri Y1164. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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15 pages, 973 KiB  
Article
Effect of the Addition of Non-Saccharomyces at First Alcoholic Fermentation on the Enological Characteristics of Cava Wines
by Ana María Mislata, Miquel Puxeu, Immaculada Andorrà, Noelia Espligares, Sergi de Lamo, Montserrat Mestres and Raúl Ferrer-Gallego
Fermentation 2021, 7(2), 64; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7020064 - 27 Apr 2021
Cited by 3 | Viewed by 2390
Abstract
Background: Cava is considered to be a high-quality wine internationally. Hence, it has undergone consistent improvement and/or the preservation of its aromatic qualities, bouquet, color, and foamability, throughout its elaboration and aging. Methods: This study investigates the use of different Saccharomyces and non- [...] Read more.
Background: Cava is considered to be a high-quality wine internationally. Hence, it has undergone consistent improvement and/or the preservation of its aromatic qualities, bouquet, color, and foamability, throughout its elaboration and aging. Methods: This study investigates the use of different Saccharomyces and non-Saccharomyces yeasts strains (Torulaspora delbrueckii and Metschnikowia pulcherrima) in Chardonnay and Xarel.lo cava wines. The usual enological parameters, the volatile composition, the protein contents, and foamability were determined, and sensory analyses were also performed for all of the vinifications (both before tirage and after 18 months of aging on the lees). Results: the protein and foamability results show that there is a direct relationship between both parameters, with better foam persistence achieved in some non-Saccharomyces fermentation. M. pulcherrima base wines showed a high protein content, improving foamability and foaming persistence. In addition, the results of the aromatic composition and the sensory analysis showed that the use of T. delbrueckii at first fermentation produced interesting cavas from an aromatic perspective. These cavas showed the highest values of ethyl isovalerate (120–126 µg/L), providing aromatic fruity notes, especially fresh green apple. Conclusions: the use of non-Saccharomyces yeasts in the base wine fermentation can be an alternative to produce cavas with differentiated aromatic characteristics and interesting foaming ability. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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23 pages, 9829 KiB  
Article
Microbial and Chemical Analysis of Non-Saccharomyces Yeasts from Chambourcin Hybrid Grapes for Potential Use in Winemaking
by Chun Tang Feng, Xue Du and Josephine Wee
Fermentation 2021, 7(1), 15; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7010015 - 21 Jan 2021
Cited by 11 | Viewed by 5284
Abstract
Native microorganisms present on grapes can influence final wine quality. Chambourcin is the most abundant hybrid grape grown in Pennsylvania and is more resistant to cold temperatures and fungal diseases compared to Vitis vinifera. Here, non-Saccharomyces yeasts were isolated from spontaneously [...] Read more.
Native microorganisms present on grapes can influence final wine quality. Chambourcin is the most abundant hybrid grape grown in Pennsylvania and is more resistant to cold temperatures and fungal diseases compared to Vitis vinifera. Here, non-Saccharomyces yeasts were isolated from spontaneously fermenting Chambourcin must from three regional vineyards. Using cultured-based methods and ITS sequencing, Hanseniaspora and Pichia spp. were the most dominant genus out of 29 fungal species identified. Five strains of Hanseniaspora uvarum, H. opuntiae, Pichia kluyveri, P. kudriavzevii, and Aureobasidium pullulans were characterized for the ability to tolerate sulfite and ethanol. Hanseniaspora opuntiae PSWCC64 and P. kudriavzevii PSWCC102 can tolerate 8–10% ethanol and were able to utilize 60–80% sugars during fermentation. Laboratory scale fermentations of candidate strain into sterile Chambourcin juice allowed for analyzing compounds associated with wine flavor. Nine nonvolatile compounds were conserved in inoculated fermentations. In contrast, Hanseniaspora strains PSWCC64 and PSWCC70 were positively correlated with 2-heptanol and ionone associated to fruity and floral odor and P. kudriazevii PSWCC102 was positively correlated with a group of esters and acetals associated to fruity and herbaceous aroma. Microbial and chemical characterization of non-Saccharomyces yeasts presents an exciting approach to enhance flavor complexity and regionality of hybrid wines. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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Review

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23 pages, 956 KiB  
Review
Commercially Available Non-Saccharomyces Yeasts for Winemaking: Current Market, Advantages over Saccharomyces, Biocompatibility, and Safety
by Ricardo Vejarano and Angie Gil-Calderón
Fermentation 2021, 7(3), 171; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7030171 - 28 Aug 2021
Cited by 43 | Viewed by 6517
Abstract
About 42 commercial products based on non-Saccharomyces yeasts are estimated as available on the market, being mostly pure cultures (79%), with a predominance of Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima. The others are multi-starter consortia that include non-Saccharomyces [...] Read more.
About 42 commercial products based on non-Saccharomyces yeasts are estimated as available on the market, being mostly pure cultures (79%), with a predominance of Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima. The others are multi-starter consortia that include non-Saccharomyces/Saccharomyces mixtures or only non-Saccharomyces species. Several commercial yeasts have shown adequate biocompatibility with S. cerevisiae in mixed fermentations, allowing an increased contribution of metabolites of oenological interest, such as glycerol, esters, higher alcohols, acids, thiols, and terpenes, among others, in addition to a lower production of acetic acid, volatile phenols, biogenic amines, or urea. Multi-starter inoculations are also reviewed here, which show adequate biocompatibility and synergy between species. In certain cases, the aromatic profile of wines based on grape varieties considered neutral is improved. In addition, several yeasts show the capacity as biocontrollers against contaminating microorganisms. The studies conducted to date demonstrate the potential of these yeasts to improve the properties of wine as an alternative and complement to the traditional S. cerevisiae. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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15 pages, 1564 KiB  
Review
Genetic, Physiological, and Industrial Aspects of the Fructophilic Non-Saccharomyces Yeast Species, Starmerella bacillaris
by María Laura Raymond Eder and Alberto Luis Rosa
Fermentation 2021, 7(2), 87; https://0-doi-org.brum.beds.ac.uk/10.3390/fermentation7020087 - 01 Jun 2021
Cited by 12 | Viewed by 3810
Abstract
Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast species, frequently found in enological ecosystems. Peculiar aspects of the genetics and metabolism of this yeast species, as well as potential industrial applications of isolated indigenous S. bacillaris strains worldwide, have recently [...] Read more.
Starmerella bacillaris (synonym Candida zemplinina) is a non-Saccharomyces yeast species, frequently found in enological ecosystems. Peculiar aspects of the genetics and metabolism of this yeast species, as well as potential industrial applications of isolated indigenous S. bacillaris strains worldwide, have recently been explored. In this review, we summarize relevant observations from studies conducted on standard laboratory and indigenous isolated S. bacillaris strains. Full article
(This article belongs to the Special Issue Enological Repercussions of Non-Saccharomyces Species 3.0)
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