Cider fermentation with three Williopsis saturnus yeast strains and volatile changes

Abstract

Williopsis saturnus var. subsufficiens NCYC 2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500 were used to carry out cider fermentation to assess their impact on the volatile composition of cider. The changes of yeast cell population, °Brix and pH were similar among the three yeasts. Strain NCYC 500 grew best, with the highest cell population of 1.14 × 10⁸ CFU ml⁻¹, followed by strains NCYC 2728 and NCYC 22 (8 × 10⁷ CFU ml⁻¹ and 3.19 × 10⁷ CFU ml⁻¹ respectively). Esters were the most abundant volatiles produced, followed by alcohols. Among the esters, ethyl acetate, 2-phenylethyl acetate, isoamyl acetate, cis-3-hexenyl acetate and hexyl acetate were the major volatiles. The major alcohols were ethanol, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol. The three Williopsis yeasts transformed volatile compounds during cider fermentation with significant variations in terms of volatile production and degradation. This study implied that fermentation with Williopsis yeasts could result in cider with a more complex yet fruity aroma.

Introduction

Cider is a fermented alcoholic beverage made from fresh or concentrated apple juice through a process usually in the presence of added yeasts, in which the consumption of sugars by yeasts yields ethanol, carbon dioxide and flavor compounds. The fermentation can also be carried out spontaneously with indigenous yeast species that originate from the surface of the fruit and the surfaces of juice processing equipment. In spontaneous fermentation, the succession of yeast species occurs. Species of Hanseniaspora, Kloeckera, Candida, Pichia, and Metschnikowia dominate initially, but they gradually die as the ethanol concentration increases, and give way to more ethanol-tolerant Saccharomyces cerevisiae and S. bayanus to complete alcoholic fermentation (Fleet 2003; Suarez et al. 2007; Ciani and Comitini 2011).

Saccharomyces cerevisiae wine yeast is well known to enable the rapid, efficient and complete conversion of sugars to alcohols to realize complete fermentation (Kunkee and Bisson 1993; Heard 1999; Fleet 2003), whereas non-Saccharomyces yeasts bring about the benefits of the distinct aromatic properties in wine from controlled fermentation, and the flavor profiles of the wines may be altered favorably (Dequin et al. 2003). The yeast genus Williopsis was defined by Zender in 1925 and accommodates the saturn-shaped ascospore-forming species Williopsis saturnus (formerly Hansenula saturnus) (Yilmaztekin et al. 2008). Non-Saccharomyces species tend to be weakly fermentative, consuming less sugar and producing less ethanol under normal wine making conditions, and thus, are sometimes used as adjunct cultures with S. cerevisiae in simultaneous or sequential inoculations; the latter is normally required to accomplish complete ethanol fermentation (Jolly et al. 2003a, Jolly et al. 2003b).

Esters make the greatest contribution to the bouquet of alcoholic beverages (Rapp and Mandery 1986). A mixture of ethyl caproate and ethyl caprylate (apple-like aroma) is a primary contributor to the characteristic fruity odors of fermentation bouquet, while isoamyl acetate imparts a banana-like aroma and 2-phenylethyl acetate contributes a fruity and flowery flavor (Rapp and Mandery 1986). Non-Saccharomyces wine yeasts are known as good producers of fruity acetate esters such as isoamyl acetate and 2-phenylethyl acetate (Rojas et al. 2001; Plata et al. 2003). Yeast species involved and a balance of ester formation by alcohol acetyl-transferases and ester hydrolysis by esterases determine the ester concentration (Suomalainen 1971; Inoue et al. 1997).

Strains of Williopsis saturnus are known to produce high levels of acetate esters that impart fruity aroma, and have been well studied in various fruit-based alcoholic beverages including grape wine, mango wine, longan wine and papaya wine (Erten and Campbell 2001; Lee et al. 2010; Trinh et al. 2011; Li et al. 2012; Tanguler 2012, 2013). However, these yeast strains have not been investigated in cider fermentation. Therefore, the objective of this study was to assess the volatile changes during cider fermentation inoculated with three Williopsis yeasts strains (W. saturnus var. subsufficiens NCYC2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500). The information gained would be useful for selection of non-Saccharomyces yeasts for modulation of cider flavor.

Material and methods

Yeasts and microbiological growth media

Three Williopsis yeast strains, including W. saturnus var. saturnus NCYC 22, the W. saturnus var. mrakii NCYC 500 and W. saturnus var. subsufficiens NCYC 2728, were used in cider fermentation. All these strains were obtained from the National Collection of Yeast Cultures (Norwich, UK). The yeasts were inoculated into the sterile nutrient broth with the composition of 2 % (w/v) glucose, 0.25 % (w/v) yeast extract, 0.25 % (w/v) bacteriological peptone and 0.25 % (w/v) malt extract (pH 5.0). Pure active dried yeasts were incubated statically at 25 °C for up to 48 h and then stored in 1-ml sterile tubes at −80 °C as stock cultures before use.

Cider fermentation

Commercial apple juice (produced from a concentrate by the juice manufacturer) was purchased from a local supermarket in Singapore (Marigold® Apple 100 % juice, Malaysia Dairy Industries Pte Ltd.) with a composition of 12 g of total carbohydrate, 10.4 g of sugar, 0.8 g of dietary fiber and 1.2 mg of sodium per 100 ml, and there was no added sugar, preservative or flavoring according to the label.

Two-hundred and twenty ml of sterile apple juice were fermented in sterile 250-ml conical flasks after inoculation with 2 % (v/v) of 10⁷–10⁸ CFU ml⁻¹ of respective yeast strain. Two independent fermentations for each strain were carried out for 14 days at 20 °C. During fermentation, samples were taken at intervals to measure total soluble solids (°Brix), cell count and pH by using a refractometer (ATAGO, Japan) and a pH meter (Metrolim, Switzerland). Yeast enumeration was conducted at the beginning and at the end of fermentation by spread plating method on potato dextrose agar (PDA).

Volatile analysis

Volatile compounds in apple juice and cider were analyzed by using headspace (HS) solid-phase microextraction (SPME) using a carboxen/polydimethylsiloxane fiber (85 μm) (Supelco, Inc., Bellefonte, PA) coupled with Agilent 7890A gas chromatography (GC)-5975 series mass spectrometry and flame ionization detector (FID) (Agilent Technology, USA). Five ml of each sample (adjusted to pH 2.5 using 1 M HCl) was tightly capped into a 20-ml glass vial and sealed with a Teflon/Silicone septum and was extracted by HS-SPME at 60 °C for 30 min with stirring at 250 rpm. After extraction, the fiber was inserted into the injection port of the GC set at 250 °C for 3 min to desorb the analytes from the fiber. A capillary column (DB-FFAP, Agilent Technology, USA) of 60 m × 0.25 mm I.D., coated with 0.25 μm film thickness of polyethylene glycol modified with nitroterephthalic acid, was used for separation and analysis. Oven temperature was initially programmed at 50 °C for 5 min, then was increased to 230 °C at a rate of 5 °C min⁻¹ and held at this temperature for 20 min. Carrier gas was helium at 19.37 psi and a total flow rate of 42.7 ml min⁻¹ was used. Mass spectra of unknown compounds were compared with those in the Wiley database (Agilent Technologies Inc.), and identification was checked and confirmed with the linear retention index values. GC-MSD ChemStation software G1701EA was used for data acquisition and analyses were carried out in duplicate. The results shown represent the mean values from two independent fermentations.

Statistical analysis

An analysis of variance (ANOVA) using Microsoft Office Excel (version 2003) was applied to day 14 experimental data (end of the fermentation period) to determine the significant differences in volatile compounds produced by three Williopsis yeasts. The results were significantly different if the associated p value was below 0.05 at the 95 % confidence level. The mean values and standard deviations were calculated from the data obtained from two independent fermentations. It must be pointed out that standard deviations in this context referred to system errors (two replicate fermentations and duplicate analyses).

Result and discussion

Growth of yeasts, changes of total soluble solids, and pH during cider fermentation

The viable cell counts of all three yeast cultures reached the maximum cell populations at the end of the fermentation (day 14), where strain NCYC 500 showed the highest growth at 1.14 × 10⁸ CFU ml⁻¹, followed by strain NCYC 2728 at 8 × 10⁷ CFU ml⁻¹ and by strain NCYC 22 at 3.19 × 10⁷ CFU ml⁻¹ (Table 1). The total soluble solids content decreased from about 12 °Brix to 7.14–8.9 °Brix depending on yeasts. Strain NCYC 500 with the highest cell population had a maximum residual soluble solid content of 8.9 °Brix, followed by strain NCYC 22 with a final °Brix of 7.6 and strain NCYC 2728 with a final °Brix of 7.15. These data indicate that there was no correlation between soluble solids reduction and cell count among different Williopsis yeasts, which could be attributed to strain variations in their ability to utilize sugars, as demonstrated elsewhere (Trinh et al. 2011). Overall, sugar consumption by three yeasts was weak, such that the ethanol content would be low (potential alcohol of approximately 1.8 to 3.0 % v/v). The pH increased from its initial value of 3.53 to a final value of 3.58–3.63.

Table 1 Cell counts of Williopsis saturnus before and after cider fermentation

Volatile compounds in apple juice

The volatile compounds in apple juice contained three acids, four alcohols, six aldehydes, nine esters and two ketones (Table 2). Among the volatiles, esters were the most abundant constituents in apple juice. C6 compounds (alcohol, aldehyde and corresponding acetate esters) are character-impact apple aroma compounds. However, not all volatiles detected were naturally present in apple juice. For example, furfural and 5-methyl-2-furfural are known thermal and/or light-induced compounds (Blanco-Gomis et al. 1991), whereas cis-3-hexenyl lactate and menthyl acetate have not been reported in apple juice and are considered to be added flavorings.

Table 2 Major volatile compounds (GC-FID peak area; Mean ×10⁶ ± SD) found in apple juice and cider fermented with three Williopsis saturnus strains at day 14

Volatile compound variations during cider fermentation and in final cider

During cider fermentation (day 0–14), several classes of volatile compounds, such as fatty acids, alcohols, aldehydes, esters and ketones, were transformed (Figs. 1–6) by the three strains of W. saturnus. Some volatile compounds in the apple juice including alcohols (hexanol, cis-3-hexanol, trans-2-hexenol), aldehydes (hexanal, 5-methyl-2-furfural), acetate esters (cis-3-hexenyl acetate, hexyl acetate, menthyl acetate), propanoate ester (cis-3-hexenyl lactate), ethyl esters (ethyl butanoate, ethyl 2-methylbutanoate) and ketones (beta-damascenone, furyl methyl ketone) were transformed (decreased or disappeared) during cider fermentation. Examples included disappearance of ethyl 2-methylbutanoate and trans-2-hexenol (Figs. 3 and 5), as well as depletion of hexanal and initial increases in hexanol and hexyl acetate (Table 2, Figs. 2 and 4). It is likely that esters initially present were hydrolyzed by yeast esterases and aldehydes were reduced to alcohols, followed by the conversion of alcohols to esters.

Fig. 1

Changes of acids during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCY C22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

Fig. 2

Changes of alcohols during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCYC 22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

The changes of fatty acids among three W. saturnus strains showed similar trends, exemplified by a continuous increment of acetic and octanoic acids (Fig. 1). Strain NCYC 500 produced the least amount of fatty acids compared to strain NCYC 22 and strain NCYC 2728. There were statistical differences in the final concentrations of fatty acids at day 14 among or between the yeasts (Table 2).

Ethanol, isobutyl alcohol, isoamyl alcohol and 2-phenethyl alcohol were the major alcohols synthesized by the three yeasts, and these alcohols continuously increased during cider fermentation (Figs. 2 and 3). Ethanol is the most abundant product resulting from the fermentation of sugars by fermenting yeasts, but was found to be weakly produced by Williopsis saturnus yeasts (Erten and Campbell 2001). In this study, it was also observed that low levels of ethanol were produced, being in agreement with low sugar consumption. The lower production of ethanol would affect the overall smoothing effect on taste characteristics, losing the sensory characteristics of ethanol, i.e., body, sweetness, fullness and mouth-warming effect (Williams 1972).

Fig. 3

Changes of other alcohols during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCYC 22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

Hexanol, cis-3-hexenol and trans-2-hexenol that were indigenous to the apple juice decreased or disappeared during cider fermentation. Interestingly, hexanol and cis-3-hexenol were initially produced by strain NCYC 500, possibly from the reduction of aldehydes and/or trans-cis isomerization (Figs. 2 and 3), resulting in their higher residual levels. Unlike the results reported by Viana et al. (2008), there were no significant differences for isoamyl alcohol between the three strains, whereas the amount of 2-phenylethyl alcohol produced by strains NCYC 500 and NCYC 2728 were approximately twofold higher than that of strain NCYC 22 (Table 2). Aromatic eugenol has been found in cider before (Xu et al. 2007; Fan et al. 2011), but its origin in cider is still not clear. Figure 3 shows that eugenol increased consistently during cider fermentation, indicating its biological origin. Eugenol was probably glycosidically bound and was released by glycosidases from the yeasts. Williopsis and other non-Saccharomyces cider yeasts are reported to contain glycosidase activities (Li et al. 2012; Pando et al. 2012).

The most abundant volatile compounds in cider were esters that could be categorized into acetate esters, ethyl esters and propanoate esters (Table 2, Figs. 4–5). All these esters had similar dynamic patterns of formation and degradation during cider fermentation amongst the three strains of W. saturnus yeasts. cis-3-hexenyl acetate, hexyl acetate, ethyl 2-methylbutanoate, menthyl acetate and trans-2-hexenyl acetate initially present in the apple juice were degraded, whereas butyl acetate, 2-phenylethyl acetate and isoamyl acetate were formed. Strains NCYC 22 and 2728 initially produced cis-3-hexenyl acetate and hexyl acetate, followed by almost complete degradation. The indigenous volatile compounds that impart the distinct characteristic apple aroma were mostly degraded by W. saturnus yeasts and were retained to some content, such as hexyl acetate and cis-3-hexenyl acetate (a major ester compound in the final cider).

Fig. 4

Changes of acetate esters during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCYC 22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

Fig. 5

Changes of ethyl esters during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCYC 22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

In line with reports by Rojas et al. (2001, 2003), our findings also showed that acetate esters such as isoamyl acetate and 2-phenylethyl acetate were formed in relatively high concentrations by non-Saccharomyces yeasts. Moreover, non-Saccharomyces yeasts proved to be good producers of isoamyl acetate and 2-phenylethyl acetate and the genera Hanseniaspora and Pichia were better producers of acetate esters (Viana et al. 2008). Similar results were obtained in our study (Table 2), where the three strains of W. saturnus produced most of the acetate esters such as isoamyl acetate, 2-phenylethyl acetate, cis-3-hexenyl acetate, hexyl acetate, furfuryl acetate and butyl acetate. It was possible that furfural (a thermal product in the juice) was reduced to furfuryl alcohol by the three yeast strains, and furfuryl acetate was derived from the alcoholysis reaction between acetyl Co-A and furfuryl alcohol catalyzed by the alcohol acetyl Co-A transferease from the yeasts.

Among the ethyl esters, ethyl butanoate and ethyl 2-methylbutanoate that were naturally present in the apple juice declined continuously during cider fermentation, whereas ethyl acetate and ethyl octanoate were generated by the three strains of W. saturnus, but the ethyl esters differed statistically in their final levels between or among the yeasts (Fig. 5). Ethyl acetate was the main ester, produced in the greatest amount (39.9–292.8 mg L⁻¹) in wine that was synthesized by selected non-Saccharomyces yeast strains (Viana et al. 2008). Ethyl and acetate esters are the key contributors of fruity aroma to wine (Rapp and Mandery 1986). Non-Saccharomyces yeasts are generally low producers of ethyl esters relative to Saccharomyces cerevisiae yeasts.

The aldehyde compounds such as hexanal, furfural and 5-methyl-2-furfural in the apple juice (the latter two were likely the products of juice heat treatment) decreased significantly or were completely catabolized throughout the fermentation process, while benzaldehyde increased (Table 2, Fig. 6). In addition, ketones like beta-damascenone and furyl methyl ketone were degraded continuously during cider fermentation (Fig. 6). Furfural, 5-methyl-2-furfural and furyl methyl ketone reportedly pose potential health risks (Stich et al. 1981) and their decrease after fermentation would be beneficial for consumer health. It is likely that aldehydes and ketones acted as electron acceptors and were reduced to their corresponding alcohols that can be converted into esters such as furfuryl acetate and hexyl acetate (Fig. 4).

Fig. 6

Changes of carbonyls during cider fermentation by three Williopsis yeast strains: W. saturnus var. saturnus NCYC 22 (◆), W. saturnus var. mrakii NCYC 500 (■), and W. saturnus var. subsufficiens NCYC 2728 (▲)

The three strains of W. saturnus played an essential role in transforming the vast array of volatile compounds in apple juice and cider. The kinetic changes of formation and utilization of the volatile compounds had similar characteristics, yet with some variations, with statistical differences at day 14 between or among the yeasts. Non-Saccharomyces wine yeasts are good producers of acetate esters (Rojas et al. 2001; Plata et al. 2003). We also showed that the volatile compounds like ethyl acetate, butyl acetate, hexyl acetate, cis-3-hexenyl acetate, furfuryl acetate and 2-phenylethyl acetate, which contributed fruity and sweet-aroma to cider, were formed in the greatest amounts by strain NCYC 22, followed by strains NCYC 2728 and NCYC 500. However, isoamyl acetate, which gave the fruity, sweet and banana-like aroma to cider, was produced in the highest concentrations by strain NCYC 2728 , while strain NCYC 22 produced the least amount of it.

Williopsis saturnus strains are metabolically active and their metabolites are expected to improve the wine quality and flavor via ester-synthesizing activities, although they are weak ethanol producers. Based on their sugar consumption, W. saturnus strains can form lower levels of ethanol and higher alcohols, such as propanol, isobutyl alcohol, isoamyl alcohol and active amyl alcohols, than Saccharomyces cerevisiae (Romano et al. 1992; Zironi et al. 1993). Non-Saccharomyces yeasts are capable of producing wine with a more complex aroma, which may improve wine quality and have a significant and favorable effect on the sensory characteristics (Ciani and Maccarelli 1998; Egli et al. 1998; Jolly et al. 2003a; Romano et al. 1992), but are not able to complete fermentation due to their low ethanol tolerance. Therefore, further research has to be carried out using non-Saccharomyces wine yeasts as part of mixed starters together with Saccharomyces cerevisiae to complete alcoholic fermentation (Jolly et al. 2003b; Rojas et al. 2003; Romano et al. 1992).