Impact of Hanseniaspora Vineae in Alcoholic Fermentation and Ageing on Lees of High-Quality White Wine

: Hanseniaspora vineae is an apiculate yeast that plays a signiﬁcant role at the beginning of fermentation, and it has been studied for its application in the improvement of the aromatic proﬁle of commercial wines. This work evaluates the use of H. vineae in alcoholic fermentation compared to Saccharomyces cerevisiae and in ageing on the lees process (AOL) compared to Saccharomyces and non- Saccharomyce s yeasts. The results indicated that there were not signiﬁcant di ﬀ erences in basic oenological parameters. H. vineae completed the fermentation until 11.9% v / v of ethanol and with a residual sugars content of less than 2 g / L. Di ﬀ erent aroma proﬁles were obtained in the wines, with esters concentration around 90 mg / L in H. vineae wines. Regarding the AOL assay, the hydroalcoholic solutions aged with H. vineae lees showed signiﬁcantly higher absorbance values at 260 (nucleic acids) and 280 nm (proteins) compared to the other strains. However, non-signiﬁcant di ﬀ erences were found in the polysaccharide content at the end of the ageing process were found compared to the other yeast species, with the exception of Schizosaccharomyces pombe that released around 23.5 mg / L of polysaccharides in hydroalcoholic solution. The use of H. vineae by the wineries may be a viable method in fermentation and AOL to improve the quality of white wines. These results allow us to differentiate the metabolism of both yeasts, even though these differences were not quantitatively observed. It is noted that we identified the same separation between the must


Introduction
The inoculation of commercial S. cerevisiae yeast strains is the most common practice in the industrial elaboration of commercial wines. However, nowadays, winemakers are trying to obtain quality wines with different organoleptic characteristics. In this regard, the use of different species of yeast could be interesting. Many studies have been done with respect to obtaining differentiated quality products and the use of non-Saccharomyces yeasts for this purpose [1][2][3]. The use of H. vineae in wineries could be a good alternative to the traditional Saccharomyces fermentations. This yeast and

Ageing on Lees Conditions
The AOL was done in hydroalcoholic solution (13.5% v/v) sulphited to 60 mg/L with K 2 S 2 O 5 and the pH was adjusted to 3.5 with phosphoric acid. The samples were prepared in triplicates, using ISO flasks of 0.5 L. The dosage of yeast lees was 6 g/L and the ageing process was done at 16 • C in a dark room for 156 days. The samples were mixed once a week to simulate a bâtonnage process.

NMR Spectroscopy
NMR spectra of a triplicate set of Albillo white wines fermented with H. vineae and S. cerevisiae yeast strains, were carried out on a Bruker 600 Avance III HD spectrometer, equipped with a 5-mm 1H/D TXI probehead equipped with a z-gradient at 298 ± 0.1 K of temperature. The following set of NMR experiments were conducted: (a) Standard 1H-one-dimensional NMR experiment was carried out as step for calibration of the water-to-ethanol multi-presaturation module: with 4 transients of 32,768 complex points, having recycling delays of 5 s and with acquisition times of 1700 milliseconds, produced an experimental time of 26 s. No apodization function was applied during Fourier Transform. (b) {1Hwater_presat NMR}: 1D single pulse NOESY experiment with a homemade shaped-pulse water-to-ethanol presaturation during both the relaxation delay (5 s) and mixing times (100 milliseconds), with a 8.18 × 10 −4 W power irradiation level for the solvent signals' elimination, centering the transmitter frequency at 4.7 ppm and shifting the decoupler frequency between 3.55 ppm (CH2-ethanol) and 1.08 ppm (CH3-ethanol) for accurate multi-presaturation of all signals [16,17] were acquired for each sample as follows: a total of 128 transients were collected into 32,768 complex data points, with a spectral width of 9615.4 Hz and acquisition times of 1700 ms, produce experimental times of 10 58 '. (c) NMR post-processing was carried out as follows: ppm calibration and manual phase corrections were conducted with the use of Bruker TopSpin 4.0.8 software. Global and soft baseline corrections, least-squares NMR alignments, variable size bucketing and data matrix normalization were carried out with NMRProcFlow [18]. Scaling and statistical analysis workflow for obtaining the Principal Component Analysis to determine relationships between H. vineae and S. cerevisiae wine samples, from the constant sum normalized NMR data matrix, were developed with the BioStatFlow 2.9.2 software. Identified metabolites were quantified (Table 1) through qNMR methods [19,20] routinely used in oenology [21,22].

Volatile Compounds from the Alcoholic Fermentation Analysis
The volatile compounds of the wines obtained in fermentation assay were measured using an Agilent Technologies 6850 gas chromatograph, equipped with an integrated flame ionization detector (GC-FID) and DB-624 column (60 m × 250 µm × 1.40 µm). Analyses were performed according to the method described by [23]. The injector temperature was 250 • C, and the detector temperature was 300 • C. The column temperature was 40 • C for the first 5 min, rising linearly by a 10 • C/min until reaching 250 • C; this temperature was maintained for 5 min. Hydrogen was used as the carrier gas. The flow rate was 22.1 L/min. The injection split ratio was 1:10. The detection limit was 0.1 mg/L.

Proteins and Nucleic Acids Estimation by Absorbance at 260 and 280 nm
The absorbance measurements were done through the ageing after centrifugation (1200 rcf for 3 min) using a 1-cm path-length quartz cuvette. All spectrometric measurements were obtained using an 8453 spectrophotometer from Agilent Technologies™ (Palo Alto, CA, USA).

Statistical Analysis
Statgraphics v.5 software (Graphics Software Systems, Rockville, MD, USA) was used to calculate means, standard deviations, analysis of variance (ANOVA), least-significant difference (LSD) test and principal component analysis (PCA). The LSD test was used to detect significant differences between means. Significance was set at p < 0.05.

Basic Oenological Parameters
In general, no significant differences were found in the wines fermented by H. vineae compared to conventional wines fermented by S. cerevisiae, with the exception of the total acidity parameter. The S. cerevisiae wines showed 0.5 g/L more total acidity than the H. vineae wines (Table 2). However, no differences in lactic acid, malic acid and volatile acidity content were found, therefore, the decrease of total acidity may be due to the precipitation of tartaric acid during the alcoholic fermentation. It is important to mention that these differences were not reflected in the pH values, since the pH was similar in all the wines studied. Table 2. Ethanol content (% v/v), pH, total acidity (g/L) as tartaric acid, volatile acidity as acetic acid (g/L), malic acid (g/L), lactic acid (g/L) and glucose and fructose (g/L) after fermentation process. Mean ± SD for three replicates.
Regarding the residual sugar content, both yeasts have been able to ferment all the sugar, with final concentrations in the wine below 2 g of residual sugar per litre. These results are in line with those obtained by other authors that compared both yeast species in Macabeo and Merlot grape wines [25]; nevertheless, [26] found 0.5 g/L of glucose and frutose more in H. vineae wines than in S. cerevisiae wines before the malolactic fermentation. This fact is linked to the glycolytic power-all wines showed similar ethanol contents around 11.9% v/v. These results indicate that both yeast species may produce wines with similar basic oenological parameters.
Targeted NMR analysis allowed the identification and quantification (Table 1) Figure 1A). The distribution was better explained with the first three components (PC1 = 43.1%, PC2 = 24.23% and PC3 = 13.59%). Even though the results were not statistically significant between the two yeasts studied (Table 1), the PCA made it possible to differentiate the wines studied into two independent clusters corresponding with the two target yeasts ( Figure 1). Chemical shift loading plots ( Figure 1B  . These results allow us to differentiate the metabolism of both yeasts, even though these differences were not quantitatively observed. It is noted that we identified the same separation between the must fermented by H. vineae and S. cerevisiae when the PCA was done on fermentative volatile compounds ( Figure 2). . These results allow us to differentiate the metabolism of both yeasts, even though these differences were not quantitatively observed. It is noted that we identified the same separation between the must fermented by H. vineae and S. cerevisiae when the PCA was done on fermentative volatile compounds ( Figure 2).

Volatile Compounds from the Alcoholic Fermentation
Considering the total volatile compounds identified, S. cerevisiae produced a larger amount of volatile compounds (Table 3) with around 1200 mg/L. In this regard the concentration of acetaldehyde and 2,3-butanediol have a special importance. The amount of these compounds was significantly higher in the wines from S. cerevisiae. Similar results were obtained after the fermentation of artificial red must [27].
Both yeast species did not show significant differences in the sum of higher alcohols. It interesting to point out that other authors reported a decrease in higher alcohols after the fermentation of the Chardonnay grapes must have with H. vineae compared with that of S. cerevisiae [5].
The fermentation with H. vineae resulted in increases in acetate esters and some ethyl esters, like ethyl acetate with concentrations around 79 mg/L. These results are similar to the results obtained by [5]. process [14,20,21]. During the entire wide-gap brazing process, the additive powder with high melting point remains largely unmelted, thereby providing the necessary capillary force to retain the molten braze powder that would otherwise be too fluid to bridge the gap faying surfaces [16,17]. However, formation of hard and brittle eutectic structures with uneven distribution cannot be avoided, due to their sensitivity to the chemical composition of the filler metal, brazing temperature and brazing time [22][23][24][25]. We previously reported [26] the successful design of a new type of Ni-based mixed filler powder without eutectic transformation, which can be used in the repair of K417G alloy with a wide gap of 20 mm by introducing in situ precipitated borides. In the process, the high-melting point additive powder with a supporting function can split the large gap into tiny virtual gaps, while the lowmelting point braze powder with a filler function can be fully melted, for wetting and filling the numerous tiny gaps within the large gaps. However, the effect of the brazing time on the properties of the wide-gap brazing joints and the strengthening mechanisms are not clear. In this study, we analyzed the microstructure and mechanical properties of the K417G alloy brazed repairs by vacuum hot-pressing for different brazing time. In particular, the precise regulation of the shape and distribution of the M3B2 boride phase inside the wide-gap brazed (WGB) region were investigated, ultimately achieving a high-quality brazing repair of the wide gap of the K417G alloy.

Materials
The K417G alloy with a size of Φ80 × H20 mm, and a chemical composition as shown in Table 1, was used as the cast base metal (BM). A wide gap with dimensions of L20 mm × W20 mm × H20 mm was machined into the core of the K417G alloy. In order to perform bonding by wide-gap brazing, the high-melting point additive powder similar in composition to the BM and low-melting point braze powder are mixed at the weight ratio of 95:5, to obtain the mixed filler powder. The chemical compositions of the base metal, additive powder, braze powder and mixed filler powder as shown in Table 1.

Volatile Compounds from the Alcoholic Fermentation
Considering the total volatile compounds identified, S. cerevisiae produced a larger amount of volatile compounds (Table 3) with around 1200 mg/L. In this regard the concentration of acetaldehyde and 2,3-butanediol have a special importance. The amount of these compounds was significantly higher in the wines from S. cerevisiae. Similar results were obtained after the fermentation of artificial red must [27].
Both yeast species did not show significant differences in the sum of higher alcohols. It interesting to point out that other authors reported a decrease in higher alcohols after the fermentation of the Chardonnay grapes must have with H. vineae compared with that of S. cerevisiae [5].
The fermentation with H. vineae resulted in increases in acetate esters and some ethyl esters, like ethyl acetate with concentrations around 79 mg/L. These results are similar to the results obtained by [5].
2-Phenylethyl acetate is an ester with strong aromatic power and its perception threshold reported is 250 µg/L [28]. This compound is associated with fruity, floral and honey aromas [25]. The 2-phenylethyl acetate concentration was significantly higher in H. vineae wines than in S. cerevisiae wines (Table 3). This fact has been reported by several authors [25,29] who identified up to 50 times more abundance of this compound in wines fermented by H. vineae. However, no significant differences in 2-phenylethanol content were found. This can be due to the fact that there are significant differences between these two yeast species in the acetylation step due to an increase in the copy number of the acetyl transferases genes in H. vineae [29].
In addition, the "odour activity values" (OAV) were calculated (see Table 3). It allows us to estimate the contribution of a specific compound to the aroma of the wine [30]. Among the compounds that have been identified, only ethyl acetate, 2-methyl-1-butanol, 2.3-butanediol, isoamyl acetate, hexanol and 2-phenylethyl acetate have obtained an OAV greater than one. It must again be emphasized the importance of the 2-phenylethyl acetate. This compound had 31.84 OAV and statistically higher concentrations in H. vineae than in S. cerevisiae wines. In this regard, the concentration identified as 2-phenylethyl acetate had an important organoleptic repercussion in the wines obtained by the fermentation of H. vineae, providing fruity, floral and honey aromas to these wines.
A principal component analysis (PCA) was done for the 15 volatile compounds identified after the fermentation process ( Figure 2) and it allowed to differentiate the aromatic profile between the yeasts studied. The distribution was explained with the first two components. The compounds 2-phenylethyl acetate, ethyl acetate, 3-methyl-1-butanol, 1-propanol, hexanol, isoamyl acetate and methanol are associated positively with the PC1. A cluster including the wines fermented by H. vineae was found in the positive values of the PC1 with the highest concentration of these volatiles. It is noteworthy the contribution of the 2-phenylethyl acetate produced by the metabolism of this yeast species; on the contrary, on the negative values of the principal component PC1, a cluster composed of the wines fermented by S. cerevisiae was identified, including the contribution of 2-phenyl ethanol and indicating the difference between the two yeast species in the acetylation of this compound.  [32].

Intracellular Components and Polysaccharides Content Measured in the Ageing on Lees
The relative measurement of the intracellular components release has been done by the UV absorbance at 260 and 280 nm [40,41]. These measurements correspond to the relative amount of nucleic acids and proteins, respectively [42].
Regarding the monitoring at 260 nm, the samples with HV yeast lees showed the highest values during the entire ageing period. However, the SCG37 samples showed the lowest absorbance values without significant differences with SP938 through the AOL stage. It is also interesting to note the difference between the two Saccharomyces strains studied, the SC7VA samples showed absorbance values around 0.4-1 AU, while the lees of the yeast SCG37 resulted in lower values, around 0.1-0.2 AU. These results may indicate that the same yeast species can show different capacities for releasing cellular compounds depending on the strain used.
Similar results were obtained in the monitoring of 280 nm absorbance, but in this case no significant differences were obtained between the HV and SC7VA samples during the 91 days of ageing. These values indicated that both yeasts could be used to accelerate the release of cellular compounds. Therefore, the use of HV and SCVA yeast strains could be indicated to perform an AOL process.
The polysaccharides released after the action of glucanases are a good indicator of the autolysis process, being the parietal mannoproteins the majority of these polysaccharides [12]. After 156 days of ageing, the samples on SP938 lees have shown the highest content of polysaccharides with values around 23.5 mg/L. This quick releasing of compounds from the Schizosaccharomyces cell wall has already been observed by other authors [12]. It is interesting to stress the fact that the SP938 samples did not show the greatest absorbance values at 260 and 280 nm (Figure 3). This is possibly due to the fact that the high molecular weight polysaccharides do not have absorbance at these wavelengths as nucleic acids and proteins.
The HV samples showed a polysaccharides content of around 11 mg/L; this concentration was not statistically significant with respect to samples aged on the lees of the two Saccharomyces yeast strains ( Figure 4). In the same way, it was not significantly different from the results obtained in L31 samples. The results obtained in the hydroalcoholic solution of these three yeast species were similar to the result of other assays with Saccharomyces previously done [13]. In other words, the yeast H. vineae could be an alternative to replace S. cerevisiae yeast in an AOL process after the alcoholic fermentation. Fermentation 2020, 6, x FOR PEER REVIEW 11 of 15 The HV samples showed a polysaccharides content of around 11 mg/L; this concentration was not statistically significant with respect to samples aged on the lees of the two Saccharomyces yeast strains (Figure 4). In the same way, it was not significantly different from the results obtained in L31 samples. The results obtained in the hydroalcoholic solution of these three yeast species were similar to the result of other assays with Saccharomyces previously done [13]. In other words, the yeast H. vineae could be an alternative to replace S. cerevisiae yeast in an AOL process after the alcoholic fermentation.

Conclusions
The use of H. vineae yeast in alcoholic fermentation resulted in wines with similar basic oenological parameters like the wines obtained by the S. cerevisiae fermentation. However, different aromatic profiles were identified by the PCA. Two clusters were shown with more production of acetate esters and ethyl esters by H. vineae. This yeast stands out for its higher production of 2phenylethyl acetate, thus enhancing the fruity character of the wines.
The monitoring of the absorbance at 260 and 280 nm allowed to obtain a relative amount of nucleic acids and proteins released during the AOL process. In this context, the H. vineae yeast lees resulted in higher values of absorbance at these wavelengths throughout the ageing process. Nevertheless, the measurement of polysaccharides concentration by HPLC-RI after 156 days of ageing showed that there were no significant differences between the use of H. vineae yeast lees and the rest of yeast species studied, with the exception of the S. pombe samples.
H. vineae is an interesting yeast species to be used in alcoholic fermentation that can provide wines with more esters. In the same way, this yeast could be used in AOL processes because it is apparently quick to transfer certain cellular compounds. Nevertheless, further studies are necessary to obtain information on the cell wall polysaccharides released by this yeast and their sensory repercussion on aged wines.

Conclusions
The use of H. vineae yeast in alcoholic fermentation resulted in wines with similar basic oenological parameters like the wines obtained by the S. cerevisiae fermentation. However, different aromatic profiles were identified by the PCA. Two clusters were shown with more production of acetate esters and ethyl esters by H. vineae. This yeast stands out for its higher production of 2-phenylethyl acetate, thus enhancing the fruity character of the wines.
The monitoring of the absorbance at 260 and 280 nm allowed to obtain a relative amount of nucleic acids and proteins released during the AOL process. In this context, the H. vineae yeast lees resulted in higher values of absorbance at these wavelengths throughout the ageing process. Nevertheless, the measurement of polysaccharides concentration by HPLC-RI after 156 days of ageing showed that there were no significant differences between the use of H. vineae yeast lees and the rest of yeast species studied, with the exception of the S. pombe samples.
H. vineae is an interesting yeast species to be used in alcoholic fermentation that can provide wines with more esters. In the same way, this yeast could be used in AOL processes because it is apparently quick to transfer certain cellular compounds. Nevertheless, further studies are necessary to obtain information on the cell wall polysaccharides released by this yeast and their sensory repercussion on aged wines.