Impact of Saccharomyces cerevisiae metabolites produced during fermentation on bread quality parameters: A review.

Although bread making with the use of Baker's yeast has a long tradition in human history, little attention has been paid to the connection between yeast addition and the final bread quality. Nowadays, bakers mainly use different flour additives such as enzymes (amylases, hemicellulases, and proteases) to change and improve dough properties and/or bread quality. Another strategy is the use of modified industrial Baker's yeast. To date, there is no yeast strain used in the baking industry, which is genetically modified, despite some studies demonstrating that the application of recombinant DNA technology is a possibility for improved strains suitable for baking. However, due to the fact that the majority of consumers in Europe highly reject the use of genetically modified microorganisms in the production of food, other strategies to improve bread quality must be investigated. Such a strategy would be a reconsideration of the selection of yeast strains used for the baking process. Next to the common criteria, the requirement for adequate gas production, more attention should be paid on how yeast impacts flavor, shelf life, color, and the nutritional value of baked products, in a similar way to which yeast strains are selected in the wine and brewing industries.

New rapid PCR protocol based on high-resolution melting analysis to identify Saccharomyces cerevisiae and other species within its genus.

AIMS: Selection projects aiming at the identification of new Saccharomyces strains are always on going as the use of the suitable yeast can strongly improve fermented food production, particularly winemaking. They are mainly targeted on Saccharomyces cerevisiae, but other species in the Saccharomyces genus are of interest. For this reason, more and more efficient molecular techniques for yeast identification able to accelerate yeast selection process are always needed. Among the Saccharomyces genus, four yeasts are widespread in natural environments: S. cerevisiae; S. uvarum; S. kudriavzevii and S. paradoxus. Therefore, among the Saccharomyces species, their discrimination is of great interest. METHODS AND RESULTS: A two-step protocol is proposed. Firstly the Saccharomyces genus identification is achieved by multiplex PCR analysis. Then, the Saccharomyces species is determined by a new method based on high-resolution melting analysis (HRMA). CONCLUSIONS: For HRMA two primer pairs have been proposed. The first was able to achieve the simultaneous identification of the four widespread Saccharomyces species, the second was used for the unambiguous discrimination of S. cerevisiae within its taxonomical genus. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay allowed an easy, rapid and simultaneous discrimination of S. cerevisiae, S. uvarum and S. paradoxus during yeast selection programs.

Exploration of genetic and phenotypic diversity within Saccharomyces uvarum for driving strain improvement in winemaking.

The selection and genetic improvement of wine yeast is an ongoing process, since yeast strains should match new technologies in winemaking to satisfy evolving consumer preferences. A large genetic background is the necessary starting point for any genetic improvement programme. For this reason, we collected and characterized a large number of strains belonging to Saccharomyces uvarum. In particular, 70 strains were isolated from cold-stored must samples: they were identified and compared to S. uvarum strains originating from different collections, regarding fermentation profile, spore viability and stress response. The results demonstrate a large biodiversity among the new isolates, with particular emphasis to fermentation performances, genotypes and high spore viability, making the isolates suitable for further genetic improvement programmes. Furthermore, few of them are competitive with Saccharomyces cerevisiae and per se, suitable for wine fermentation, due to their resistance to stress, short lag phase and fermentation by-products.

Selection of indigenous Saccharomyces cerevisiae strains in Shanshan County (Xinjiang, China) for winemaking and their aroma-producing characteristics.

In order to select potential indigenous Saccharomyces strains, diversity of indigenous Saccharomyces strains in Shanshan County (Xinjiang, China) was preliminarily analyzed. Twenty-one genotypes were found through interdelta fingerprinting analysis. According to this result, representatives of each genotype were chosen to test the enological criteria. After tests of fermentation characteristics and growth ability, eight strains were finally selected as starters to further fermentation of Merlot must for aroma analysis and sensory evaluation at the same testing conditions, with one commercial strain F15 as control. Each strain of Saccharomyces cerevisiae produced individual volatiles in different concentrations and combinations which significantly influenced resulting wine flavour. Except of LFP522, all indigenous isolates produced more concentration of esters than F15. Higher concentrations of linalool, ß-damascenone and citral, associated with S. cerevisiae LFE1809, considerably distinguished this strain from the others. Sensory evaluation present the Merlot wine fermented by LFE1225 isolated from Merlot, had the highest sensory score.

Developing a novel selection method for alcoholic fermentation starters by exploring wine yeast microbiota from Greece.

The selection of native yeast for alcoholic fermentation in wine focuses on ensuring the success of the process and promoting the quality of the final product. The purpose of this study was firstly to create a large collection of new yeast isolates and categorize them based on their oenological potential. Additionally, the geographical distribution of the most dominant species, Saccharomyces cerevisiae, was further explored. Towards this direction, fourteen spontaneously fermented wines from different regions of Greece were collected for yeast typing. The yeast isolates were subjected in molecular analyses and identification at species level. RAPD (Random Amplified Polymorphic DNA) genomic fingerprinting with the oligo-nucleotide primer M13 was used, combined with Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. All yeast isolates were scrutinized for their sensitivity to killer toxin, production of non-desirable metabolites such as acetic acid and H2S, ß-glucosidase production and resistance to the antimicrobial agent; SO2. In parallel, S. cerevisiae isolates were typed at strain level by interdelta - PCR genomic fingerprinting. S. cerevisiae strains were examined for their fermentative capacity in laboratory scale fermentation on pasteurized grape must. Glucose and fructose consumption was monitored daily and at the final point a free sorting task was conducted to categorize the samples according to their organoleptic profile. According to our results, among the 190 isolates, S. cerevisiae was the most dominant species while some less common non-Saccharomyces species such as Trigonopsis californica, Priceomyces carsonii, Zygosaccharomyces bailii, Brettanomyces bruxellensis and Pichia manshurica were identified in minor abundancies. According to phenotypic typing, most isolates were neutral to killer toxin test and exhibited low acetic acid production. Hierarchical Cluster Analysis revealed the presence of four yeast groups based on phenotypic fingerprinting. Strain level typing reported 20 different S. cerevisiae strains from which 65% indicated fermentative capacity and led to dry wines. Sensory evaluation results clearly discriminated the produced wines and consequently, the proposed yeast categorization was confirmed. A novel approach that employs biostatistical tools for a rapid screening and classification of indigenous wine yeasts with oenological potential, allowing a more efficient preliminary selection or rejection of isolates is proposed.

Enological Suitability of Indigenous Yeast Strains for 'Verdejo' Wine Production.

The use of indigenous yeasts for the production of wines is a tool to defend the typicity of a particular region. The selection of appropriate indigenous yeasts ensures the maintenance of oenological characteristics by simulating spontaneous alcoholic fermentation (AF) while avoiding the risks of stuck or sluggish fermentations. In this study, autochthonous yeasts from Verdejo grape juice (Appellation of Origin Rueda) were selected, identified, and characterized to exploit the characteristics of the 'terroir'. The fermentation capacity of seven strains was studied individually at the laboratory scale. The most suitable strains (Saccharomyces cerevisiae: Sacch 1, Sacch 2, Sacch 4, and Sacch 6) and Sacch 6 co-inoculated with Metschnikowia pulcherrima were characterized at the pilot scale. The fermentation kinetics, bioproduct release, volatile composition, and sensory profile of the wines were evaluated. Significant differences were found, especially in the aroma profile. In particular, Sacch 6 and Sacch 6 co-inoculated with M. pulcherrima produced higher amounts of ethyl esters and acetates and lower amounts of higher alcohols than the spontaneous AF. Wines inoculated with indigenous yeasts had higher sensory scores for fruit aromas and overall rating. The selection of indigenous yeasts improved the aroma of Verdejo wines and could contribute to determining the wine typicity of the wine region.

Fermentation conditions for yeast selection and effect of yeast-bacterial interaction in developing a starter culture for tequila fermentation.

Similar to other beverages, tequila fermentation can be improved using selected native strains. During fermentation, yeast and bacteria frequently act together and can improve product quality. However, their influence during tequila fermentation is not fully understood. Three Saccharomyces cerevisiae strains isolated in a previous study were used to determine their fermentation parameters. Fermentation of agave juice by a selected yeast strain in the presence of seven native bacterial species (individually and in consortium) was also evaluated. Optimal temperature, initial fructose, and pH were determined using a rotatory central composite design, and concomitant fermentative parameters were used to select the best tequila yeast strain. The yeast strain Teq-199 presented the best fructose consumption (91.0%), ethanol production (33.5 g/L), and yield (72%), as well as produced low concentrations of acetic acid (0.2 g/L). The optimal fermentation temperature was 32°C, similar to that used at distilleries. During fermentation of agave juice by strain Teq-199 in the presence of individual native bacteria, the yeast dominated fermentation, while bacterial species impaired its quality due to lactic acid production (>3.3 g/L). In the presence of a bacterial consortium, strain Teq-199 also dominated fermentation; however, a high concentration of acetic acid (6.5 g/L) was generated, reducing the quality of the fermentation parameters. A comparison with a commercial wine strain K1-V1116, revealed that the strain Teq-199 exhibited the best fermentation parameters, indicating its potential use as an inoculant in industrial processing. Since bacteria were detrimental, distilleries should exert efforts to reduce their impact. PRACTICAL APPLICATION: The optimal fermentation conditions for pre-selected yeast strains isolated during tequila production were determined, which led to the selection of the best-adapted yeast strain to fulfill the actual requirements and conditions in the tequila industry. This study allows the selection of yeast strains that could be used by local producers. In addition, this yeast was able to overcome a well-adapted bacterial consortium, showing dominance throughout the fermentation process. This study is unique since it explored bacterial interactions during fermentation process and proposes a method to assess the effect of these concomitant microorganisms to properly select and design a starter culture.

Screening of Saccharomyces and Non-Saccharomyces Wine Yeasts for Their Decarboxylase Activity of Amino Acids.

The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids-such as arginine, proline, serine, and tyrosine-that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health.

Volatile Compounds in Monovarietal Wines of Two Amarone Della Valpolicella Terroirs: Chemical and Sensory Impact of Grape Variety and Origin, Yeast Strain and Spontaneous Fermentation.

Aroma profiles of withered Corvina and Corvinone wines from two different Valpolicella terroirs were investigated in relationship to yeast strain and use of spontaneous fermentation. The results indicated that volatile chemical differences between wines were mainly driven by grape origin, which was associated with distinctive compositional profiles. Wine content in terpenes, norisoprenoids, benzenoids and C6 alcohols, as well as some fermentative esters, were indeed significantly affected by grape origin. Conversely, yeast strain influence was mainly associated with fermentation-derived esters. Sensory analysis, besides confirming the major role of grape origin as driver of wine differentiation, indicated that spontaneous fermentations reduced the sensory differences associated with grape origin and variety, mainly due to high content of acetic acid and ethyl acetate.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Selection of Saccharomyces cerevisiae isolates for ethanol production in the presence of inhibitors.

Eight yeast isolates identified as Saccharomyces cerevisiae were recovered from molasses-using Cuban distilleries and discriminated by nucleotide sequence analysis of ITS locus. The isolates L/25-7-81 and L/25-7-86 showed the highest ethanol yield from sugarcane juice, while L/25-7-12 and L/25-7-79 showed high ethanol yield from sugarcane molasses. The isolate L/25-7-86 also displayed high fermentation capacity when molasses was diluted with vinasse. In addition, stress tolerance was evaluated on the basis of growth in the presence of inhibitors (acetic acid, lactic acid, 5-hydroxymethylfurfural and sulfuric acid) and the results indicated that L/25-7-77 and L/25-7-79 congregated the highest score for cross-tolerance and fermentation capacity. Hence, these isolates, especially L/25-7-77, could serve as potential biological platform for the arduous task of fermenting complex substrates that contain inhibitors. The use of these yeasts was discussed in the context of second-generation ethanol and the environmental and economic implications of the use of vinasse, saving the use of water for substrate dilution.

The Different Physical and Chemical Composition of Grape Juice and Marc Influence Saccharomyces cerevisiae Strains Distribution During Fermentation.

During white-grape winemaking, grape marc is separated from juice immediately after crushing. Both mark and juice are obtained from the same grapes, but they differ strongly for their physical and chemical properties. Marc is mainly composed of solid residues. Its pH is usually higher than that of the juice and Saccharomyces cerevisiae strains are largely present. Therefore, it can be considered as a potential alternative environment for the selection of industrial yeasts. In order to evaluate the effect of different pH and physical state of the two matrices on grapes yeast population composition, the isolation of S. cerevisiae, from both grape juice and marc during simultaneous fermentations, was performed. After yeast identification and genotyping, strains present at high frequencies were tested in fermentation at different pH values. Biofilm production was also tested to evaluate strain ability to develop on a solid matrix. Genotype analysis showed that high-frequency strains were always more abundant in one of the two environments, suggesting the existence of a selective effect. Generally, fermentations at different pH revealed that the best fermentation performance of each strain, in terms of CO2 production, was in the pH range of its original environment. Only one strain, mostly present in grape marc, produced a high biofilm level. Therefore, biofilm production does not seem to favor strain adaptation to grape marc condition. PRACTICAL APPLICATION: These results demonstrate that grape juice and marc represent two different environments able to influence yeast strains distribution. The pH level can be included among the selection factors acting on yeast strains distribution. Grape marc can be considered a yeasts reservoir and its fermentation can be used for the development and isolation of new strains, genetically and physiologically different from those present in the grape juice.

Selection of Yeast Strains for Tequila Fermentation Based on Growth Dynamics in Combined Fructose and Ethanol Media.

The high concentration of fructose in agave juice has been associated with reduced ethanol tolerance of commercial yeasts used for tequila production and low fermentation yields. The selection of autochthonous strains, which are better adapted to agave juice, could improve the process. In this study, a 2-step selection process of yeasts isolated from spontaneous fermentations for tequila production was carried out based on analysis of the growth dynamics in combined conditions of high fructose and ethanol. First, yeast isolates (605) were screened to identify strains tolerant to high fructose (20%) and to ethanol (10%), yielding 89 isolates able to grow in both conditions. From the 89 isolates, the growth curves under 8 treatments of combined fructose (from 20% to 5%) and ethanol (from 0% to 10%) were obtained, and the kinetic parameters were analyzed with principal component analysis and k-means clustering. The resulting yeast strain groups corresponded to the fast, medium and slow growers. A second clustering of only the fast growers led to the selection of 3 Saccharomyces strains (199, 230, 231) that were able to grow rapidly in 4 out of the 8 conditions evaluated. This methodology differentiated strains phenotypically and could be further used for strain selection in other processes. PRACTICAL APPLICATION: A method to select yeast strains for fermentation taking into account the natural differences of yeast isolates. This methodology is based on the cell exposition to combinations of sugar and ethanol, which are the most important stress factors in fermentation. This strategy will help to identify the most tolerant strain that could improve ethanol yield and reduce fermentation time.

Unraveling the Enzymatic Basis of Wine "Flavorome": A Phylo-Functional Study of Wine Related Yeast Species.

Non-Saccharomyces yeasts are a heterogeneous microbial group involved in the early stages of wine fermentation. The high enzymatic potential of these yeasts makes them a useful tool for increasing the final organoleptic characteristics of wines in spite of their low fermentative power. Their physiology and contribution to wine quality are still poorly understood, with most current knowledge being acquired empirically and in most cases based in single species and strains. This work analyzed the metabolic potential of 770 yeast isolates from different enological origins and representing 15 different species, by studying their production of enzymes of enological interest and linking phylogenetic and enzymatic data. The isolates were screened for glycosidase enzymes related to terpene aroma release, the ß-lyase activity responsible for the release of volatile thiols, and sulfite reductase. Apart from these aroma-related activities, protease, polygalacturonase and cellulase activities were also studied in the entire yeast collection, being related to the improvement of different technological and sensorial features of wines. In this context, and in terms of abundance, two different groups were established, with α-L-arabinofuranosidase, polygalacturonase and cellulase being the less abundant activities. By contrast, ß-glucosidase and protease activities were widespread in the yeast collection studied. A classical phylogenetic study involving the partial sequencing of 26S rDNA was conducted in conjunction with the enzymatic profiles of the 770 yeast isolates for further typing, complementing the phylogenetic relationships established by using 26S rDNA. This has rendered it possible to foresee the contribution different yeast species make to wine quality and their potential applicability as pure inocula, establishing species-specific behavior. These consistent results allowed us to design future targeted studies on the impact different non-Saccharomyces yeast species have on wine quality, understanding intra and interspecific enzymatic odds and, therefore, aiming to predict the most suitable application for the current non-Saccharomyces strains, as well as the potential future applications of new strains. This work therefore contributes to a better understanding of the concept of wine microbiome and its potential consequences for wine quality, as well as to the knowledge of non-Saccharomyces yeasts for their use in the wine industry.