Emerging biotechnologies and non-thermal technologies for winemaking in a context of global warming.

In the current situation, wine areas are affected by several problems in a context of global warming: asymmetric maturities, pH increasing, high alcohol degree and flat wines with low freshness and poor aroma profile. The use of emerging biotechnologies allows to control or manage such problems. Emerging non-Saccharomyces as Lachancea thermotolerans are very useful for controlling pH by the formation of stable lactic acid from sugars with a slight concomitant alcohol reduction. Lower pH improves freshness increasing simultaneously microbiological stability. The use of Hanseniaspora spp. (specially H. vineae and H. opuntiae) or Metschnikowia pulcherrima promotes a better aroma complexity and improves wine sensory profile by the expression of a more complex metabolic pattern and the release of extracellular enzymes. Some of them are also compatible or synergic with the acidification by L. thermotolerans, and M. pulcherrima is an interesting biotool for reductive winemaking and bioprotection. The use of bioprotection is a powerful tool in this context, allowing oxidation control by oxygen depletion, the inhibition of some wild microorganisms, improving the implantation of some starters and limiting SO2. This can be complemented with the use of reductive yeast derivatives with high contents of reducing peptides and relevant compounds such as glutathione that also are interesting to reduce SO2. Finally, the use of emerging non-thermal technologies as Ultra High-Pressure Homogenization (UHPH) and Pulsed Light (PL) increases wine stability by microbial control and inactivation of oxidative enzymes, improving the implantation of emerging non-Saccharomyces and lowering SO2 additions. GRAPHICAL ABSTRACT.

Synergetic Effect of Metschnikowia pulcherrima and Lachancea thermotolerans in Acidification and Aroma Compounds in Airén Wines.

On the one hand, the species Lachancea thermotolerans is known for its high genetic diversity, allowing for the existence of strains that produce high concentrations of lactic acid. In contrast, the species Metschnikowia pulcherrima is renowned for its high enzymatic activity capable of producing aromatic esters during fermentation. By enhancing acidity and boosting the concentration of aromatic compounds, both species are currently used to enhance the organoleptic profile of wines. In this regard, ternary fermentations with M. pulcherrima and L. thermotolerans were carried out and the wines produced were further analysed with GC-FID, FTIR, and UV-Vis spectrophotometry. The outcomes showed that the species M. pulcherrima favored an increase in ethyl lactate (between 37 and 41 mg/L) along with an increased concentration of 2-phenylethyl alcohol (between 30 and 35 mg/L), whereas the species L. thermotolerans was able to produce 1 g/L of lactic acid in ternary fermentations. Additionally, pH levels were slightly lower in these fermentations and the color of the white wines produced showed less chemical oxidation as hue values were lower than the control. Finally, the ternary fermentations of L. thermotolerans and M. pulcherrima had higher overall rating in the tasting. In conclusion, ternary fermentations involving these two non-Saccharomyces species are suggested as a substitute for spontaneous fermentations in the production of wines from neutral varieties to express freshness more vividly. This biotechnology may be further favored by the possibility of applying emerging technologies for the removal of microorganisms in grapes and musts.

Emerging Non-Thermal Technologies for the Extraction of Grape Anthocyanins.

Anthocyanins are flavonoid pigments broadly distributed in plants with great potential to be used as food colorants due to their range of colors, innocuous nature, and positive impact on human health. However, these molecules are unstable and affected by pH changes, oxidation and high temperatures, making it very important to extract them using gentle non-thermal technologies. The use of emerging non-thermal techniques such as High Hydrostatic Pressure (HHP), Ultra High Pressure Homogenization (UHPH), Pulsed Electric Fields (PEFs), Ultrasound (US), irradiation, and Pulsed Light (PL) is currently increasing for many applications in food technology. This article reviews their application, features, advantages and drawbacks in the extraction of anthocyanins from grapes. It shows how extraction can be significantly increased with many of these techniques, while decreasing extraction times and maintaining antioxidant capacity.

Non-Saccharomyces as Biotools to Control the Production of Off-Flavors in Wines.

Off-flavors produced by undesirable microbial spoilage are a major concern in wineries, as they affect wine quality. This situation is worse in warm areas affected by global warming because of the resulting higher pHs in wines. Natural biotechnologies can aid in effectively controlling these processes, while reducing the use of chemical preservatives such as SO2. Bioacidification reduces the development of spoilage yeasts and bacteria, but also increases the amount of molecular SO2, which allows for lower total levels. The use of non-Saccharomyces yeasts, such as Lachancea thermotolerans, results in effective acidification through the production of lactic acid from sugars. Furthermore, high lactic acid contents (>4 g/L) inhibit lactic acid bacteria and have some effect on Brettanomyces. Additionally, the use of yeasts with hydroxycinnamate decarboxylase (HCDC) activity can be useful to promote the fermentative formation of stable vinylphenolic pyranoanthocyanins, reducing the amount of ethylphenol precursors. This biotechnology increases the amount of stable pigments and simultaneously prevents the formation of high contents of ethylphenols, even when the wine is contaminated by Brettanomyces.

Improvement of Must Fermentation from Late Harvest cv. Tempranillo Grapes Treated with Pulsed Light.

Pulsed light irradiation is a nonthermal technology currently used for the elimination of pathogens from a diverse range of food products. In the last two decades, the results obtained using PL at laboratory scale are encouraging wine experts to use it in the winemaking industry. PL can reduce native yeast counts significantly, which facilitates the use of starter cultures, reducing SO2 requirements at the same time. In this experimental set up, Tempranillo grapes were subjected to pulsed light treatment, and the fermentative performance of non-Saccharomyces yeasts belonging to the species Schizosaccharomyces pombe, Lachancea thermotolerans, Torulaspora delbrueckii, Metschnikowia pulcherrima and Hanseniaspora vineae was monitored in sequential fermentations against spontaneous fermentation and pure culture fermentation with the species Saccharomyces cerevisiae. The experimental analyses comprised the determination of anthocyanin (High performance liquid chromatography with photodiode array detector-HPLC-DAD), polyphenol index and colour (Ultraviolet-visible spectroscopy-UV-Vis spectrophotometer), fermentation-derived volatiles (Gas chromatography with flame ionization detector-GC-FID), oenological parameters (Fourier transform Infrared spectroscopy-FT-IR) and structural damage of the skin (atomic force microscopy-AFM). The results showed a decrease of 1.2 log CFU/mL yeast counts after pulsed light treatment and more rapid and controlled fermentation kinetics in musts from treated grapes than in untreated samples. The fermentations done with treated grapes allowed starter cultures to better implant in the must, although a larger anthocyanin loss (up to 93%) and an increase in hue values (1 unit) towards more yellow hues were observed for treated grapes. The development of biomass was larger in musts from treated grapes. The profile of volatile compounds and oenological parameters reveals that fermentations carried out with untreated grapes are prone to deviations from native microbiota (e.g., production of lactic acid). Finally, no severe damage on the skin was observed with the AFM on treated grapes.

Pulsed Electric Fields to Improve the Use of Non-Saccharomyces Starters in Red Wines.

New nonthermal technologies, including pulsed electric fields (PEF), open a new way to generate more natural foods while respecting their organoleptic qualities. PEF can reduce wild yeasts to improve the implantation of other yeasts and generate more desired metabolites. Two PEF treatments were applied; one with an intensity of 5 kV/cm was applied continuously to the must for further colour extraction, and a second treatment only to the must (without skins) after a 24-hour maceration of 17.5 kV/cm intensity, reducing its wild yeast load by up to 2 log CFU/mL, thus comparing the implantation and fermentation of inoculated non-Saccharomyces yeasts. In general, those treated with PEF preserved more total esters and formed more anthocyanins, including vitisin A, due to better implantation of the inoculated yeasts. It should be noted that the yeast Lachancea thermotolerans that had received PEF treatment produced four-fold more lactic acid (3.62 ± 0.84 g/L) than the control of the same yeast, and Hanseniaspora vineae with PEF produced almost three-fold more 2-phenylethyl acetate than the rest. On the other hand, 3-ethoxy-1-propanol was not observed at the end of the fermentation with a Torulaspora delbrueckii (Td) control but in the Td PEF, it was observed (3.17 ± 0.58 mg/L).

Biocompatibility in Ternary Fermentations With Lachancea thermotolerans, Other Non-Saccharomyces and Saccharomyces cerevisiae to Control pH and Improve the Sensory Profile of Wines From Warm Areas.

Global warming is causing serious problems, especially, in warm regions, where musts with excess sugars and high pH produce wines with decreased freshness and unstable evolution. This study aimed to determine biocompatibility between yeast species, the capacity for microbiological acidification, and the aromatic profile produced in ternary fermentations in which Lachancea thermotolerans has been co-inoculated with Hanseniaspora vineae, Torulaspora delbrueckii, or Metschnikowia pulcherrima, and the fermentation process is subsequently completed with sequential inoculation of Saccharomyces cerevisiae. For this purpose, different cell culture media and instruments were used such as infrared spectroscopy, enzymatic autoanalyzer, chromatograph coupled with a flame ionization detector, spectrophotometric analysis, among others. The behavior of these yeasts was evaluated alone and in co-inoculation, always finishing the fermentation with sequential inoculation of S. cerevisiae, at a stable temperature of 16°C and with a low level of sulfites (25 mg/L) in white must. Significant results were obtained in terms of biocompatibility using population counts (CFU/ml) in differential plating media that permitted monitoring. Quantification of the five species was studied. Concerning acidification by L. thermotolerans in co-inoculations, we showed some metabolic interactions, such as the inhibition of acidification when H. vineae/L. thermotolerans were used, generating just over 0.13 g/L of lactic acid and, conversely, a synergistic effect when M. pulcherrima/L. thermotolerans were used, achieving 3.2 g/L of lactic acid and a reduction in pH of up to 0.33. A diminution in alcohol content higher than 0.6% v/v was observed in co-inoculation with the L. thermotolerans/M. pulcherrima yeasts, with total sugar consumption and very slow completion of fermentation in the inoculations with H. vineae and T. delbrueckii. The aromatic composition of the wines obtained was analyzed and a sensory evaluation conducted, and it was found that both L. thermotolerans and co-inoculations retained more aromatic esters over time and had a lower evolution toward the yellow tones typical of oxidation and that the best sensory evaluation was that of the Lt + Mp co-inoculation. Lachancea thermotolerans and co-inoculations produced wines with low levels of volatile acidity (<0.4 g/L). This work shows that good consortia strategies with binary and ternary fermentations of yeast strains can be a powerful bio-tool for producing more complex wines.

The Impact of Hanseniaspora vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), ß-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

White wine processing by UHPH without SO2. Elimination of microbial populations and effect in oxidative enzymes, colloidal stability and sensory quality.

The use of UHPH sterilization in the absence of SO2 has been used to eliminate wild microorganisms and inactivate oxidative enzymes. A white must of the Muscat of Alexandria grape variety was continuously processed by UHPH at 300 MPa (inlet temperature: 23-25 °C). The initial microbial load of the settled must was 4-log CFU/mL for both yeast and moulds, and slightly lower for bacteria. After UHPH processing, no microorganisms were detected in 1 mL. UHPH musts remain without fermentative activity for more than 60 days. Concentrations of the thermal markers indicated the absence of thermal damage in the UHPH-treated musts, since 5-hydroxymethylfurfural was not detected. In addition, the must treated by UHPH keeps terpene concentrations similar to those of the untreated controls. A strong inactivation of the oxidative enzymes was observed, with no browning at room temperature for more than 3 days. The antioxidant value of the UHPH-treated must was 156% higher than the control.

Industrial Performance of Several Lachancea thermotolerans Strains for pH Control in White Wines from Warm Areas.

In the current scenario of climatic warming, the over-ripening of grapes increases the sugar content, producing flat and alcoholic wines with low acidity, high pH and low freshness. Additionally, a high pH makes wines more chemically and microbiologically unstable, requiring a higher sulphite content for preservation. Some strains of Lachancea thermotolerans can naturally lower the pH of wine by producing lactic acid from sugars; this pH reduction can reach 0.5 units. The industrial performance of four selected strains has been compared with that of two commercial strains and with that of Saccharomyces cerevisiae. The yeasts were assessed under variable oenological conditions, measuring lactic acid production and fermentative performance at two fermentation temperatures (17 and 27 °C), and in the presence or absence of sulphites (25 and 75 mg/L). Lactic acid production depends on yeast populations, with higher concentrations being reached when the microbial population is close to or above 7-log CFU/mL. A temperature effect on acidification can also be observed, being more intense at higher fermentation temperatures for most strains. Ethanol yield ranged from 7-11% vol., depending on the fermentation conditions (temperature and SO2) at day 12 of fermentation, compared with 12% for the S. cerevisiae control in micro-fermentations. The production of fermentative esters was higher at 27 °C compared with 17 °C, which favoured the production of higher alcohols. Volatile acidity was moderate under all fermentation conditions with values below 0.4 g/L.

Evolution of the Phenolic Fraction and Aromatic Profile of Red Wines Aged in Oak Barrels.

Various changes occur in red wine during aging in oak barrels. Among these, the variation of the polyphenolic fraction and the transfer of aromatic compounds to the wine from oak wood are of great importance. The aim of the present work is to compare the chemical composition of wines aged in different new oak barrels with similar commercial denominations. During 8 months, the total polyphenol index (TPI), color parameters, anthocyanins and pyranoanthocyanins, and wood aromatic compounds were periodically evaluated. The measurement of the TPI and color parameters was similar in all wines, but significant differences were found in total anthocyanin and vitisin content and in certain aromatic compounds belonging to volatile phenols, furanic compounds, and phenolic aldehydes. The results obtained indicate the need for the winemaker to carry out preliminary tests in order to be able to choose the wood that best suits the sensorial profile of the wine.

Use of fumaric acid to control pH and inhibit malolactic fermentation in wines.

Fumaric acid is an additive allowed by the Codex Alimentarius and under evaluation by the International Organisation of Vine and Wine (OIV) that can be used for wine acidification but also to inhibit malolactic fermentation (MLF). The use of 300-900 mg/L of fumaric acid can inhibit MLF in red wines decreasing pH by 0.2 units or more depending on the buffer capacity. When MLF was running with populations of either 7 or 8 log CFU/mL strain alpha (Oenococcus oeni) the application of 600 mg/L of fumaric acid stopped the process for more than 50 days and cells were undetected in specific media. In triangular tastings, fumaric acid was not detected at 300-600 mg/L (p < .05). In subsequent preference tests, some tasters perceived more acidity and body. Fumaric acid is a useful technological additive to improve wine microbiological stability and freshness, also allowing reduction of SO2 levels.

Contribution of Non-Saccharomyces Yeasts to Wine Freshness. A Review.

Freshness, although it is a concept difficult to define in wines, can be understood as a combination of different circumstances. Organolepticwise, bluish red, floral and fruity, more acidic and full-bodied wines, are perceived as younger and fresher by consumers. In traditional winemaking processes, these attributes are hard to boost if no other technology or biotechnology is involved. In this regard, the right selection of yeast strains plays an important role in meeting these parameters and obtaining wines with fresher profiles. Another approach in getting fresh wines is through the use of novel non-thermal technologies during winemaking. Herein, the contributions of non-Saccharomyces yeasts and emerging technologies to these parameters are reviewed and discussed.

Influence of Saccharomyces and non-Saccharomyces Yeasts in the Formation of Pyranoanthocyanins and Polymeric Pigments during Red Wine Making.

Yeast are able to modulate many sensory parameters of wines during red must fermentation. The effect on color and on the formation of derived pigments during fermentation has been studied thoroughly since the 90s. Yeast can increase grape anthocyanin's color by acidification by hyperchromic effect (increase of flavylium molecules). Recent studies with non-Saccharomyces species, as Lachancea thermotolerans, described the intense effect of some strains on anthocyanin's color, and subsequent, stability, by strongly reducing wine's pH during fermentation. Moreover, selected yeast strains of Saccharomyces have been shown to release metabolites such as pyruvic acid or acetaldehyde that promote the formation of vitisin A and B pyranoanthocyanins during must fermentation. Schizosaccharomyces pombe, because of its specific metabolism, can produce higher concentrations of pyruvate, which enhances the formation of vitisin A-type derivatives. The hydroxycinnamate decarboxylase activity that some Saccharomyces strains express during fermentation also promotes the formation of vinylphenolic derivatives. Some non-Saccharomyces species, such as S. pombe or P. guilliermondii can also improve the production of these derivatives compared to selected strains of Saccharomyces cerevisiae. Lastly, some yeasts are also able to modulate the formations of polymeric pigments between grape anthocyanins and flavonoids, such as catechins and procyanidins.

Sonication of Yeast Biomasses to Improve the Ageing on Lees Technique in Red Wines.

Ageing on Lees (AOL) is a technique to improve the aromatic and gustatory complexity of wine, mainly by improving its body and reducing its astringency. However, the autolytic process is slow, resulting in high production costs. This work evaluated the effect of adding sonicated lees and combining it with oak chips, as a new technique to accelerate the AOL process and improve the aromatic quality of aged red wine. Cell disruption due to sonication was verified by optical microscopy. Volatile acidity, total polyphenol index, color intensity, tonality, dissolved oxygen, anthocyanins, and fermentative volatiles were monitored throughout the ageing of the wines. Sensory analysis was performed at the end of the ageing process. Polysaccharides released from the cell walls and the oxygen consumption, was quantified using a hydroalcoholic solution. The results indicated a 20% increase of the polysaccharide content and suggested an increase in the antioxidant capacity of the lees. No significant changes were observed in the fermentative volatile compounds and the total polyphenol index (TPI), except for those wines in contact with wood. The sonication of lees had some protective effect on the total anthocyanins content, however, color intensity was significantly lower in the sonicated treatments. The sonication of the lees did not cause any defect at the sensory level. Therefore, sonication could allow a reduction in the SO2 addition to wine, as well as a shortening of the ageing times.

Application of ultrasound to improve lees ageing processes in red wines.

Ageing on lees (AOL) is a technique that increases volatile compounds, promotes colour stability, improves mouthfeel and reduces astringency in red wines. The main drawback is that it is a slow process. Several months are necessary to obtain perceptible effects in wines. Different authors have studied the application of new techniques to accelerate the AOL process. Ultrasound (US) has been used to improve different food industry processes; it could be interesting to accelerate the yeast autolysis during AOL. This work evaluates the use of the US technique together with AOL and oak chips for this purpose studying the effects of different oenological parameters of red wines. The results obtained indicate an increase of polysaccharides content when US is applied in wine AOL. In addition, total polyphenol index (TPI) and volatile acidity were not affected. However, this treatment increases the dissolved oxygen affecting the volatile compounds and total anthocyanins.

Formation of polymeric pigments in red wines through sequential fermentation of flavanol-enriched musts with non-Saccharomyces yeasts.

Non-Saccharomyces yeasts may contribute to enrich wine aroma while promoting the formation of stable pigments. Yeast metabolites such as acetaldehyde and pyruvate participate in the formation of stable pigments during fermentation and wine aging. This work evaluated the formation of polymeric pigments in red musts added with (+)-Catechin, ProcyanidinB2 and ProcyanidinC1. The non-Saccharomyces yeasts used were Lachancea thermotolerans, Metschnikowia pulcherrima and Torulaspora delbrueckii in sequential fermentation with Saccharomyces cerevisiae and Schizosaccharomyces pombe. Use of Lachancea thermotolerans led to larger amounts of polymeric pigments in sequential fermentation. (+)-Catechin is the flavanol prone to forming such pigments. The species Metschnikowia pulcherrima produced higher concentration of esters and total volatile compounds. The sensory analysis pointed out differences in fruitiness and aroma quality. The results obtained strengthen the fact that metabolites from non-Saccharomyces yeasts may contribute to form stable polymeric pigments while also influencing wine complexity.

Study of the Interaction of Anthocyanins with Phenolic Aldehydes in a Model Wine Solution.

Aldehydes may be present in wines as a result of metabolic processes during wine fermentation or through oxidation and extraction from wood during wine aging in oak barrels. Apart from acetaldehyde, the most abundant aldehyde in wine, other aldehydes such as furfural and more recently vanillin have shown to contribute to the formation of more stable pigments. The copigmentation effect of phenolic molecules, including flavanols and anthocyanins themselves, has been previously evaluated in wine and model solutions, and even the effect of aldehydes related to wine aging has been documented at different pHs and molar ratios. The copigmentation phenomenon is observed by hyperchromic effects and bathochromic shifts of λmax, and, in the same time, the presence of larger molecular weight pigments, potentially less susceptible to degradation, was followed up. This experimental work intended to evaluate the potential of five different aldehydes, all of which are safe for human consumption and are used in the food industry, to the formation of pyranoanthocyanin-like and polymeric pigments in the model solution.

Use of non-Saccharomyces yeasts and oenological tannin in red winemaking: Influence on colour, aroma and sensorial properties of young wines.

Today, many non-Saccharomyces strains have been verified can be positive for the development of wine anthocyanin and aroma in different fermentation scenarios. Moreover, oenological tannins are widely used in wine industry to improve the colour profile and aroma complexity. The aim of this work is to analyze the fermentation characters of non-Saccharomyces strains and investigate the effects of pre-fermentative addition of oenological tannins on the wine components as well as sensory properties. For this purpose, five selected non-Saccharomyces strains and grape seed tannin were used to carry out the different fermentation trials. As a result, the grape seed tannin were less likely to influence growth kinetics of non-Saccharomyces strains. Schizosaccharomyces pombe has been proved can be effective to reduce the malic acid content while increase the level of vinylphenolic pyranoanthocyanin, which is positive for wine colour stability. Pre-fermentative use of oenological tannin was verified could be beneficial for the wines fermented with non-Saccharomyces regarding the improvement of wine colour, anthocyanin composition and the complexity of volatile compounds. Nevertheless, sensory analysis showed that oenological tannin could be less effective to modify the aroma impression of non-Saccharomyces wines.

The Effects of Pre-Fermentative Addition of Oenological Tannins on Wine Components and Sensorial Qualities of Red Wine.

Today in the wine industry, oenological tannins are widely used to improve wine quality and prevent oxidation in wine aging. With the development of tannin products, new oenological tannins are developed with many specific functions, such as modifying antioxidant effect, colour stabilization and aroma modifications. The aim of this work is to investigate effects of pre-fermentative addition of oenological tannins on wine colour, anthocyanins, volatile compounds and sensorial properties. In this case, Syrah juice was extracted with classic flash thermovinification from fresh must in order to release more colour and tannins. Three types of oenological tannins, which are, respectively, derived from grape skin, seed (Vitis vinifera) and French oak (Quercus robur and Querrus petraea), were selected to carry out the experiments with seven treatments. Results indicated that tannin treatments significantly improved wine aroma complexity and sensorial properties. However, the concentration of some stable pigments such as Vitisin A, Vitisin A-Ac and Vitisin B was negatively affected by tannin additions. Nevertheless, by means of cluster analysis and principal component analysis, it was observed that higher alcohols were significantly promoted by grape seed tannin while most anthocyanins can be improved by addition of grape tannins. In conclusion, low amount of oenological tannin derived from grape seed is a promising method to be applied especially for young red wine making.