Impact of Pure, Co, and Sequential Fermentations with Hanseniaspora sp. and Saccharomyces cerevisiae on the Volatile Compounds of Ciders

: Pure, co-, and sequential fermentations of Hanseniaspora uvarum , H. guilliermondii, and Saccharomyces cerevisiae strains were evaluated to improve the aromatic quality of ciders. In sequential fermentations, Hanseniaspora strains were used as starter, followed by S. cerevisiae inoculation suc-ceeding one, two, and three days of fermentation. Kinetics, physicochemical parameters, and volatile compounds were assessed during 10 days of fermentation. The headspace technique was used to capture the volatile compounds from the ciders obtained in each experiment and analyzed by gas chromatography. Fermentations with pure strains of Hansenisaspora sp. showed a high population (>10 10 CFU/mL) but had a low fermentation rate (2.3–3.8 CO 2 g/L/d), low consumption of amino acids (20–40 mg/L) with a high residual content, high sugar consumption (80–90 g/L), and low alcohol content (<2.0% v / v ). The H. uvarum strain produced a notably high ester content (245 mg/L). In the co-fermentations, H. guilliermondii with S. cerevisiae highlighted a significant production of higher alcohols, similar to that produced by S. cerevisiae alone (152–165 mg/L). In general, the maximum fermentation rate of the sequential inoculations was lower than co-fermentations but showed low residual nitrogen content (<69 mg/L) and good conversion of sugars into ethanol (4.3–5.7% v / v ). The highest concentrations of volatile compounds were observed in treatments involving the two non-conventional strains: H. uvarum with S. cerevisiae inoculation after three days (564 mg/L) and H. guilliermondii after just one day (531 mg/L) of fermentation. These differences stemmed from the metabolic activity of the strains. H. uvarum was influenced by the presence of Saccharomyces , whereas H. guilliermondii did not exhibit this effect. Thus, a pure H. uvarum inoculum has the potential to produce a demi-sec cider with low alcohol content and high content of esters, contributing to a fruity aroma. In addition, ciders with sequential inoculation were the most promising for dry cider processing concerning fermentation parameters and bioaroma enrichment.


Introduction
Cider, or hard cider, consists of sparkling beverages obtained by the total or partial alcoholic fermentation of apple musts from blends of dessert and industrial apples.Apple varieties and cider processing technology vary between countries, regions, and producers worldwide [1,2].This impacts its diversity of sensory attributes, such as color, turbidity, acidity, astringency, sweetness, carbonation, and foam.However, there is a consensus that the fruity aroma of ciders, mainly from esters and higher alcohols, is a significant indicator of quality [3].
Traditionally, the alcoholic fermentation of ciders made only with Saccharomyces sp.ensures a complete fermentation and avoids sensory deviations.However, pure S. cerevisiae inoculum, mainly in the presence of potassium metabisulphite, will result in no notable differences in the aromatic quality of ciders and can provide sensory notes such as a neutral or weak aroma [1,4].On the other hand, with some non-Saccharomyces strains as inoculum, it is possible to increase and improve the flavor of fermented beverages, including cider.Regarding ciders, sensory notes of "fruity, floral, and fruit aromas" may appear [2].The use of non-Saccharomyces in cider processing has been researched since the 1990s [5].However, recent studies with these strains were assessed, as single cultures or in co-inoculation with S. cerevisiae, for their capacity to increase the flavor complexity of cider [2,4,[6][7][8].
Among the non-Saccharomyces, Hanseniaspora uvarum, frequently isolated from ciders, has been one of the most studied [4,9].The ability of Hanseniaspora species to develop the aroma profile of cider, wine, and beer, even when in mixed-culture fermentations with S. cerevisiae, has been demonstrated in several studies [4,[9][10][11][12].Recently, Hanseniaspora guilliermondii was isolated from apple must [13].However, H. guilliermondii was not yet employed, to the best of our knowledge, in mixed and sequential fermentations with S. cerevisiae for cider bioflavoring.The positive impact of this species on ciders' aromatic characteristics could be promising.
Nevertheless, there are still limited data on flavor-active cider yeasts, and further research is needed to identify novel strains with interesting properties suitable to enrich cider flavor.Therefore, the present study aimed to evaluate the physicochemical properties, kinetics parameters, and aromatic profile of ciders, obtained by pure, co-, and sequential fermentation of H. uvarum, H. guilliermondii, and S. cerevisiae.

Materials
Gala apples (100 kg) samples were obtained in Ponta Grossa, Paraná, Brazil.To check the ripeness of these apples, the starch-iodine test was employed [14].The iodine value fell between the range of 4-5, which indicates the ripe stage of the apples.The H. uvarum and H. guilliermondii strains were previously isolated from apple must [13] and identified by molecular profile analysis (PCR-fingerprinting) using the PCR mini/microsatellite (MSP-PCR) technique with synthetic oligonucleotide (GTC) 5 [15], along with DNA extraction [16].For sequencing, the D1/D2 domains of the larger subunit of the rRNA gene were amplified, and the sequences obtained were compared with those deposited in the GenBank database using the Basic Local Alignment Search Tool.The Hanseniaspora strains were stored at −80 • C with 20% sterile glycerol.The fermentative yeast used was S. cerevisiae Bouquet (Fermol Bouquet, ref.PB 2004, AEB Group, Brescia, Italy), chosen to produce high concentrations of volatile compounds, which contributed to a fruity and floral aroma [17].

Processing of Apple Must
The commercial apples were carefully selected, weighed, and sanitized.The fruits were crushed and pressed (AGM Máquinas, Bento Gonçalves, RS, Brazil) at 294 kPa for 5 min, resulting in the extraction of the integral apple must.The physical yield (η p ) averaged 72.5%.Apple must was blended and depectinized using a pectinolytic enzyme (Pectinex-Batch ® 1201371L, Novozymes Latin America LTDA, Araucária, Brazil) at 3.0 mL/hL at room temperature (25 ± 2 • C) for 4 h.The depectinized apple must was racked, filtered in a line filter with a 0.5 µm opening (Hidro Filtros do Brasil ® , Joinvile, Brazil) at 0.5 bar, and stored at −18 • C until cider processing.

Processing of Cider
The apple must was transferred to glass fermenters of 1450 mL (sterilized in an autoclave 121 • C/15 min, previously) with a working volume of 1200 mL, and kept in a climate room at a temperature of 20 • C. Antibiotic chloramphenicol was added to the apple must at 0.01% (Henrifarma Supplier) to control bacterial growth.The S. cerevisiae was inoculated with an initial population of approximately 4.0 × 10 6 CFU/mL and 2.0 × 10 6 CFU/mL for Hanseniaspora sp.To reach this population, one colony of each strain of Hanseniaspora sp., isolated in YMA (Merck) in 5 mL of GPBY broth-glucose peptone broth yeast (Merck), was inoculated, and after incubation for 24 h at 25 • C, the culture was transferred to 100 mL of the same broth, time, and temperature, reaching a population of approximately 10 12 CFU/mL [18].Then, a sufficient volume was collected to achieve the desired population in fermenters (about 2.0 × 10 6 CFU/mL).On the tenth day, fermentation was stopped (time required to keep residual sugar) and the cider was centrifuged at 10,200× g at 5 • C (Hitachi Himac CR21GII centrifuge, Tokyo, Japan) for 20 min, then racked, bottled, and stored at −18 • C before further analysis.
The strains H. uvarum, H. guilliermondii, and S. cerevisiae were inoculated as either pure or monoculture (Hu; Hg, and Sc, respectively), mixed or co-fermentation (Hu + Sc and Hg + Sc), and sequential, with the first inoculum being Hanseniaspora sp.The S. cerevisiae was inoculated after one (Hu + Sc 1d and Hg + Sc 1d), two (Hu + Sc 2d and Hg + Sc 2d), and three days of fermentation (Hu + Sc 3d and Hg + Sc 3d).The experiments were carried out in triplicate.

The Growth Curves of Yeast
The total yeast counting was performed in culture medium potato dextrose agar (PDA, Acumedia ® , Lansing, MI, USA), then hydrated and sterilized in an autoclave (Phoenix ® VA 13811, Araraquara, São Paulo, Brazil) for 15 min (121 • C, 1.01 kgf/cm 2 ).Ten milliliters of culture medium and 50 µL of 10% tartaric acid (Biotec ® , Pinhais, Paraná, Brazil) were poured into 90 mm Petri dishes (J.Prolab ® , São José dos Pinhais, Paraná, Brazil).Subsequently, the solid medium received 100 µL of the sample with scattering and using a sterile Drigalski spatula.The plates were then incubated at 26 • C for 24 h, with subsequent counting of specific colonies.The results were expressed as CFU/mL.
To perform the plate count of Hanseniaspora sp., aliquots of 1.0 mL suspension obtained in the recovery were withdrawn.After serial dilutions, the plate surfaces were sown using a Drigalski handle in YMA.The plates were incubated at 25 • C for 48 h [19].

Monitoring of Fermentation and Characterization of Ciders
Fermentation was monitored by the loss of mass from the system caused by the release of CO 2 , and the weight was determined every two hours with a sensitivity of 0.01 g (BL3200h, Shimadzu-Brazil, São Paulo, Brazil) for 10 days [20].The fermentation rate was calculated by the variation in the loss of CO 2 vs time, Equation 1, where V is the fermentation rate (CO 2 g/L/day), ∆CO 2 is the variation in production of CO 2 (g/L), and ∆t is the variation in time (days): Samples of the apple juice and cider were analyzed for total reducing sugars (TRS) by the traditional method of Somogyi-Nelson [21].The ethanol content was determined by an ebulliometer (accuracy of 0.1%) and expressed as percentage by volume.Total titratable acidity was expressed as malic acid (g/L), volatile acidity was expressed as acetic acid (g/L), and total nitrogen was determined by the Kjedhal method [22].The pH was determined using a pH potentiometer (pH digital micro process Tecnal ® , model TEC3-MP, Piracicaba, São Paulo, Brazil).All the analyses were performed in triplicate.

Analysis of Volatile Compounds
The capture of compounds using headspace was performed according to the method of Saerens et al. [23] with modifications.Samples (6 mL) of apple juice and ciders were placed in glass vials with a capacity of 20 mL.A solution of internal standards containing 50 µL of heptanoic acid (Merck ® ) and 20 µL of 4-methyl-1-amyl alcohol (Merck ® ) was added.
Before analysis, the samples remained for 10 min at 60 • C under agitation in the oven of the automatic injector YL 6100 GC (Young Lin Instrument ® Gas Chromatograph, Anyang, Republic of Korea) equipped with a flame ionization detector (FID) and a 30 mlong capillary column (Phenomenex) with an internal diameter of 25 mm and 0.25 µm-thick ZB-WAX film.
The analysis of aromatic compounds was performed by gas chromatography according to the method of Pietrowski et al. [11].The injector temperature was 220 • C, the detector temperature was 230 • C, and the carrier gas was nitrogen at a flow rate of 2.5 mL/min.The injection technique was split 1:1.2 and the gas chromatography automatic injection mode was used, with a volume of 1500 µL.The analysis conditions were programmed with an initial temperature of 40 • C for 5 min and an increase of 10 • C/min to 150 • C, before remaining at this temperature for 10 min.This was followed by a rise of 10 • C/min to 200 • C, remaining there for 5 min, and a further rise of 10 • C/min to 220 • C, where it remained for 16 min.The compounds were identified by comparing retention times with those obtained in the reference solution.

Statistical Analysis
The data were shown as average and standard deviation.The homogeneity of variance was verified by Levene's test or the F-test (p ≥ 0.05).The differences between the samples were evaluated using one-way ANOVA, followed by Fisher's LSD test.The statistical analysis was performed using Statistica v. 13.3 software (TIBCO Software Inc., Palo Alto, CA, USA) and Origin ® 9.0 (OriginLab, Northampton, MA, USA).

Yeast Growth Kinetics
The growth curves of the pure strains showed significant differences (Figure 1A).This indicates specific metabolisms during cider fermentation.The H. uvarum and H. guilliermondii strains reached their maximum population (10 11 CFU/mL) on the first day of fermentation.In comparison, S. cerevisiae reached a lower population (10 8 CFU/mL) after three days of alcoholic fermentation and remained stable during the stationary phase (8 days).H. guilliermondii had the highest population, and it remained high during the stationary phase.However, H. uvarum showed a significant population reduction during this same phase (Figure 1A).The same behavior was observed in co-fermentation with S. cerevisiae (Figure 1B).The growth of H. guilliermondii was not affected by the inoculum in co-and sequential fermentation (Figure 1D), demonstrating better adaptation to competition with S. cerevisiae.The S. cerevisiae showed the same growth behavior observed with H. uvarum (Figure 1E).

Characterization of Ciders
Table 1 shows the composition of apple must.The values correspond to the Gala apple must at the stage of ripeness used [1,3].This table also shows the composition values of ciders with different yeast inoculums after 10 days of fermentation.
The H. guilliermondii strain was able to consume larger quantities of sugar (89.6 g/L) Figure 1B,C correspond to the growth in co-and sequential fermentations of H. uvarum and S. cerevisiae, respectively.The growth of H. uvarum was affected by co-fermentation with S. cerevisiae (Figure 1B).On the other hand, the growth of S. cerevisiae, in co-and sequential fermentation with one day of inoculum, provided higher growth than that observed in the pure strain (Figure 1A,C).In these two treatments, there was no competition for nutrients, but H. uvarum probably released some growth factor into the medium.However, with the S. cerevisiae inoculum after 2 and 3 days, it was possible to observe a smaller population, though it was similar to the pure yeast (Figure 1A,C), indicating a possible depletion of nutrients essential to the growth of S. cerevisiae.
The growth of H. guilliermondii was not affected by the inoculum in co-and sequential fermentation (Figure 1D), demonstrating better adaptation to competition with S. cerevisiae.The S. cerevisiae showed the same growth behavior observed with H. uvarum (Figure 1E).

Characterization of Ciders
Table 1 shows the composition of apple must.The values correspond to the Gala apple must at the stage of ripeness used [1,3].This table also shows the composition values of ciders with different yeast inoculums after 10 days of fermentation.
The H. guilliermondii strain was able to consume larger quantities of sugar (89.6 g/L) than the H. uvarum strains (80.8 g/L).S. cerevisiae consumed only 10 to 20 g/L more sugar than the Hansenispora sp.strains, demonstrating the ability of the non-Saccharomyces strains to ferment apple sugars (glucose, fructose, and sucrose).According to Moreira et al. [24], during fermentation, H. uvarum metabolize glucose and fructose similarly, but H. guilliermondii has a different behavior (fructophilic), consuming fructose more quickly than glucose.
The ethanol content produced by the Hanseniaspora sp.strains was <2.0% vol.after 10 days of fermentation.This shows a low conversion of sugar into ethanol when compared to S. cerevisiae (>5.0%vol.).Moreira et al. [24] found higher ethanol concentration with longer fermentations, reaching 5.4% vol.for H. uvarum and 7.2% vol.for H. guilliermondii.However, these results indicate that pure Hanseniaspora sp.inoculum with 10 days of fermentation can be used to develop low-alcohol ciders.There is a growing tendency to reduce the concentration of alcohol in commercialized alcoholic beverages [25].
The ciders made with mixed inoculum showed the highest fermentation rates and high conversion of sugar into ethanol.However, when the inoculum was sequential, the reduction in fermentation rate, and consequently in ethanol production, was proportional to the inoculation time of S. cerevisiae (Table 1).In France, slow fermentation by reducing biomass is used to improve aromatic quality and facilitate the logistics of cider processing operations [1].
Total Kjeldahl nitrogen content classifies apple must as a high concentration (>120 mg/L) [17].Nitrogen is mostly found in apples in the form of amino acids, such as asparagine, glutamine, aspartate, glutamate, and serine, which together account for 86-95% of the nitrogen portion of the juice [17].In addition, these amino acids are the most relevant precursors and intermediates for the biosynthesis of many volatile compounds [11,26].In ciders elaborated with pure inoculum, nitrogen consumption by Hanseniaspora sp.strains were between 35-45 mg/L.However, S. cerevisiae consumed 87 mg/L.This difference in consumption is related to the size of the S. cerevisiae cell, which can be twice as large as that of Hanseniaspora sp.Nitrogen consumption was similar (around 80 mg/L) in ciders with mixed and sequential inoculum.This consumption can mostly be attributed to S. cerevisiae, which used similar amounts when inoculated in pure culture (Table 1).Therefore, this nutrient was not limiting for cell growth (protein synthesis) and fermentation activity during the initial stages of alcoholic fermentation.

Synthesis of Volatile Compounds
Table 2 shows the profile of volatile compounds formed in ciders with different inoculums after 10 days of alcoholic fermentation.Some classes of volatiles, such as esters (78-92% of the total), higher alcohols (6-16% of the total), carboxylic acids, ketones, and aldehydes (2-6%), are formed by yeasts during cider fermentation and contribute to the production of fruity aromas [1].The ester content of the cider made with the pure culture of H. uvarum was four and six times higher than that produced with S. cerevisiae and H. guillermondii, respectively.In addition, ciders with sequential fermentation containing H. uvarum had a high ester concentration (Table 2); however, the cider made with mixed culture had the lowest ester content.This indicates a possible interference in the biosynthesis of these compounds in the presence of S. cerevisiae.Esters are directly related to the formation of fruity aromas in fermented beverages; the concentration of these volatiles after the fermentation process depends on several factors, including the yeast strain and the amount of initial amino acids [17].In cider fermentation, the amino acids aspartate, asparagine, and glutamate correlate with the production of 3-methyl-1-butanol, 2-phenylethanol, 2-hexanol, ethyl ethanoate, 3-methyl-ethanoate, ethyl octanoate, ethyl butanoate, ethyl decanoate, and hexyl ethanoate [2,17].
The formation of higher alcohols was much higher in the cider with pure S. cerevisiae, reaching 14 times more than the cider with H. guilliermondii.However, cider with a mixed culture of H. guilliermondii and S. cerevisiae showed similar results to cider with only S. cerevisiae, so there was no interference in the production of these compounds.In the sequential cultures with H. guilliermondii and S. cerevisiae, the same effect was confirmed, with a higher concentration of higher alcohols compared to the mixed and sequential H. uvarum inoculums (Table 2).Studies have demonstrated that more than 90% of acids and higher alcohols (and their acetate esters derivatives) in wine are produced via central carbon metabolism (CCM) [25,27].
The compounds hexyl ethanoate, ethyl lactate, and 2-octanone showed lower values than the threshold odor, playing little or no role in the aroma of the ciders.Regardless of this, more than 99% of the volatile compounds identified were above the threshold value (Table 2), demonstrating the importance of these compounds in the formation of cider aroma.Consequently, the H. uvarum strain showed a greater propensity to produce esters, and the S. cerevisiae strain was greater in the formation of higher alcohols.Both the mixed culture and the sequential culture of H. guilliermondii with S. cerevisiae maintained a high formation of higher alcohols.
Figure 2A shows the sum of the volatile compounds, giving an overview of the production of each treatment.Considering that the production of volatiles in cider inoculated only with S. cerevisiae corresponds to the usual content, only pure H. guilliermondi and mixed H. uvarum and S. cerevisiae fermentations had lower contents.The other treatments showed higher values, with between 1.25 and 2.00 times more volatiles (Figure 2A).
The residual amino acids after 10 days of alcoholic fermentation can be observed in Figure 2B.Consumption by the Hanseniaspora sp.strains was between 20 and 50 mg/L and still resulted in high populations and production of volatiles in the pure cultures.High levels of residual nitrogen, usually in the form of amino acids, can cause microbiological instability in cider [17].Nitrogen consumption was similar in all other treatments with the presence of S. cerevisiae at about 88%.Therefore, the use of pure H. uvarum fermentations requires further studies to maintain stability in the final product.  (7  Note: Concentration of volatile compounds in mg/L: data shows the average of three replicates for headspace; Different letters in the same row indicate significant differences existing between the quantities in headspace samples (p < 0.05); nd = not detected; LOD: limit of detection.Pure inoculum (Hu: H. uvarum; Hg: H. guilliermondii; Sc: S. cerevisiae), mixed inoculum (Hu + Sc and Hg + Sc), and sequential inoculum (Hu + Sc 1d; 2d, and 3d; and Hg + Sc 1d, 2d and 3d), 1d, 2d, and 3d represent days of alcoholic fermentation of Hanseniaspora strains in which the S. cerevisiae inoculum was performed. (1)Yu et al. [28]; (2) Li et al. [29]; (3) Wei et al. [8]; (4) Karl et al. [30]; (5) Arcari et al. [31]; (6) Baiano et al. [32]; (7) Wang et al. [33].It is clear that the H. uvarum strain, as a pure and sequential inoculum, showed higher volatile compound content.The inoculum in a mixed culture of H. uvarum and S. cerevisiae had a low volatile content, making it an uninteresting inoculum.The same was observed with H. guilliermondii as a pure inoculum, which also showed low production of volatile compounds.However, in the fermentation with the mixed and sequential cultures of H. guilliermondii, the values were interesting and cannot be neglected.
(A) (B) The residual amino acids after 10 days of alcoholic fermentation can be observed in Figure 2B.Consumption by the Hanseniaspora sp.strains was between 20 and 50 mg/L and still resulted in high populations and production of volatiles in the pure cultures.High levels of residual nitrogen, usually in the form of amino acids, can cause microbiological instability in cider [17].Nitrogen consumption was similar in all other treatments with the presence of S. cerevisiae at about 88%.Therefore, the use of pure H. uvarum fermentations requires further studies to maintain stability in the final product.

Conclusions
This research evaluated the physicochemical properties, kinetic parameters, and aromatic profile of ciders obtained by pure, co-fermentative, and sequential fermentation by H. uvarum, H. guilliermondii, and S. cerevisiae.In yeast growth, it was possible to observe a different metabolic behavior of volatile production in H. uvarum compared to H. guilliermondii in the presence of S. cerevisiae in co-or sequential fermentations.The ciders produced with pure Hansenisaspora sp.inoculum showed a high population but low

Conclusions
This research evaluated the physicochemical properties, kinetic parameters, and aromatic profile of ciders obtained by pure, co-fermentative, and sequential fermentation by H. uvarum, H. guilliermondii, and S. cerevisiae.In yeast growth, it was possible to observe a different metabolic behavior of volatile production in H. uvarum compared to H. guilliermondii in the presence of S. cerevisiae in co-or sequential fermentations.The ciders produced with pure Hansenisaspora sp.inoculum showed a high population but low fermentation rate, low amino acid consumption with high residual content, high sugar consumption, and low alcohol content (<2.0%v/v).However, the H. uvarum strain was the one that produced a high ester content (245 mg/L).In the co-fermentations, the highlight was H. guilliermondii with S. cerevisiae with a high production of higher alcohol, though similar to that produced by S. cerevisiae alone (152-165 mg/L).The presence of S. cerevisiae in both co-and sequential fermentations improved the fermentation parameters of the Hanseniaspora sp.strains.
In general, the maximum fermentation rate of the sequential inoculations was lower than that of the co-fermentations.In addition, they showed low residual nitrogen content and good conversion of sugars to ethanol.The highest concentrations of volatile compounds in these treatments were obtained for the two non-conventional strains, H. uvarum with S. cerevisiae inoculum, after three days, and H. guilliermondii after only one day.These differences were due to the metabolic activity of the strains.H. uvarum was influenced by the presence of Saccharomyces; however, H. guilliermondii did not show this effect.
Thus, pure H. uvarum inoculum has the potential to develop a demi-sec cider with a low alcohol content and a high content of esters responsible for a fruity aroma.In addition, the ciders with sequential inoculum were the most promising for industrial cider processing, in terms of fermentation parameters and bioaroma enrichment.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Table 1 .
The chemical composition and kinetics parameters of ciders (10 days of fermentation) elaborated with different yeast inoculums (pure, mixed, and sequential).
Note: N: total nitrogen; TA: titratable acidity; VA: volatile acidity; RS: reducing sugars; TS: total sugars; Vmax: Maximum fermentation rate in CO 2 g/L/d; Time Vmax: Time to maximum fermentation rate; Hu: H. uvarum; Hg: H. guilliermondii; Sc: S. cerevisiae.1d, 2d, and 3d represent days of alcoholic fermentation of Hanseniaspora strains in which the S. cerevisiae inoculum was performed; Different letters on the same line indicate that there is a significant difference between the quantities in the samples (p ≤ 0.05).

Table 2 .
Values of volatile compounds (mg/L) in ciders elaborated with different yeast inoculums (pure, mixed, and sequential).