FORMATION OF AROMA COMPOUNDS DURING LONGAN JUICE FERMENTATION BY WILLIOPSIS SATURNUS VAR. SATURNUS WITH THE ADDITION OF SELECTED AMINO ACIDS

FORMATION OF AROMA COMPOUNDS DURING LONGAN JUICEFERMENTATION BY WILLIOPSIS SATURNUS VAR. SATURNUSWITH THE ADDITION OF SELECTED AMINO ACIDSjfpp_578 1..9

THI-THANH-TAM TRINH1, BIN YU2, PHILLIP CURRAN2 and SHAO-QUAN LIU1,3

1Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543,Singapore2Firmenich Asia Pte Ltd., Tuas, Singapore

3Corresponding author. TEL: +65-6516-2687;FAX: +65-6775-7895. EMAIL:[email protected]

Received for Publication August 9, 2010Accepted for Publication June 7, 2011

doi:10.1111/j.1745-4549.2011.00578.x

ABSTRACT

Longan (Dimocarpus longan Lour.) is a type of tropical fruit. This study investigatedthe impact of L-leucine and L-phenylalanine on the volatile profiles of longan winefermented with a non-Saccharomyces yeast Williopsis saturnus ssp. saturnus CBS254with a view to enhancing longan wine aroma. The results revealed the ability of thisyeast to enhance isoamyl alcohol and its ester isoamyl acetate (banana-like aroma),and 2-phenylethanol and its ester 2-phenylethyl acetate (rose-like aroma) with theaddition of L-leucine and L-phenylalanine, respectively. The increased productionof the targeted acetate esters appeared to be at the expense of other acetate estersexcept for methyl acetate, whereas the effects on the biotransformation of other vola-tiles were minimal. Therefore, these findings suggest that the combination(s) of anamino acid and yeast can be employed as a tool to manipulate the level of the targetedaroma compounds so as to impart a specific aroma or to add flavor complexity tolongan wine.

PRACTICAL APPLICATIONS

Tropical fruits such as longan are often in oversupply during the peak season andsome are wasted because of lack of adequate processing technologies. Thus, there is aneed to add value to longan fruit via processing. Although drying as a form of pro-cessing is commonly applied to longan fruit, fermentation is not widely practiced toprocess and preserve this fruit because of lack of research and information. Longanwine fermentation using the conventional grape wine fermentation technologyresults in longan wine with inferior flavor. The aim of this study is to investigate thefermentation of longan juice using a non-Saccharomyces yeast Williopsis saturnusvar. saturnus with addition of L-leucine and L-phenylalanine so as to modulate theformation of aroma compounds.

INTRODUCTION

Fruit wine refers to alcoholic beverages made from fruitsother than grapes. The popularity of fruit wine, especiallytropical fruit wine, is increasing in spite of the relatively infe-rior flavors due to lack of scientific knowledge. The additionof artificial flavors into fruit wine may not improve its orga-noleptic quality due to lack of flavor complexity and naturalcharacters; besides, consumers increasingly prefer foods and

beverages containing natural flavors because of perceivedhealth and environmental issues associated with syntheticchemicals and the production thereof (Vanderhaegen et al.2003). Furthermore, a simple adoption of grape winemakingtechnology for fruit wine production may not necessarily leadto quality fruit wine. The reasons can be attributed to thecompositional differences between grape fruits and tropicalfruits. As a consequence, tropical fruit wine production is afield that is yet to be fully explored.

Journal of Food Processing and Preservation ISSN 1745-4549

1Journal of Food Processing and Preservation •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

In the past, spontaneous fermentation was frequently prac-ticed and characterized by a succession of yeast growthwhereby non-Saccharomyces yeasts dominated during the earlystages while Saccharomyces yeasts made up higher percentagesat the later stages (Fleet and Heard 1993; Fleet 2003). However,with the application of selected wine yeast starter cultures ofSaccharomyces cerevisiae and Saccharomyces bayanus to ensureconsistency of the product and ease of control, wine flavorcomplexity is often compromised (Lambrechts and Pretorius2002; Romano et al. 2003).Consequently, a trend of using non-Saccharomyces yeasts is emerging in winemaking to take advan-tage of their positive role in imparting the organolepticcharacteristics back to wine (Fleet 2003; Viana et al. 2008).

The genus Williopsis was first defined in 1925 by Zender(1925), since then, further species have been accommodatedwithin this genus (James et al. 1998) and demonstrated toproduce high levels of esters, e.g., isoamyl acetate (Iwase et al.1995; Yilmaztekin et al. 2008, 2009). Furthermore, it has beenreported that Williopsis saturnus var. saturnus strains wereknown to be able to convert higher alcohols into the corre-sponding acetate esters, e.g., isoamyl alcohol into isoamylacetate when isoamyl alcohol was added into the fermenta-tion medium (Vandamme and Soetaert 2002; Yilmaztekinet al. 2009). Other yeasts were also found to produce highlevels of 2-phenylethyl acetate (Kluyveromyces marxianus[Hansen] van der Walt., Fabre et al. 1998), acetate esters(Hanseniaspora guilliermondii 11027 and 11102, Hans-eniaspora osmophila 1471 and Pichia membranifaciens 10113and 10550, Viana et al. 2008).

Many amino acids are the precursors to volatile flavor com-pounds produced by yeasts. For example, L-tryptophan is theprecursor to undesirable tryptophol, L-phenylalanine is theprecursor to 2-phenylethanol (Webb and Ingraham 1963),L-leucine is the precursor to isoamyl alcohol (3-methyl-1-butanol) (Dickinson et al. 1997), L-valine is the precursor toisobutyl alcohol (2-methyl-1-propanol) (Dickinson et al.1998), L-isoleucine is the precursor to active amyl alcohol(2-methyl-1-butanol) (Dickinson et al. 2000). The alcoholsproduced react with acetic acid or other medium-chain fattyacids (e.g., butanoic acid, octanoic acid) to form aroma-activeester compounds.

Longan was chosen to make wine in this study because ofits rich amount of sugar, significant level of polyphenols,good source of ascorbic acid, potassium, copper and otherminerals (Rangkadilok et al. 2005; Wall 2006), thus longanwine can be considered to be a potential fruit wine for nichemarkets. Particularly, longan fruit flesh was found to containtotal sugar at 67.35 g/100 g dry weight with xylose (2.94%),fructose (2.3%), glucose (44.69%), maltose (15%) andsucrose (5.37%) in its composition (Chang et al. 1998).However, longan is low in those amino acids that play roles asflavor precursors in wine fermentation such as leucine, isoleu-cine and valine (2.39, 1.79 and 1.54 mg/100 g longan fruit

flesh, respectively) (Chang et al. 1998). The objective of thisresearch was to assess the impact of added selected aminoacids on the production of targeted aroma compounds inlongan wine fermented with W. saturnus var. saturnusCBS254 so as to enhance longan wine aroma.

MATERIALS AND METHODS

Yeasts and Chemicals

W. saturnus var. saturnus CBS254 from CBS Culture Collec-tions (The Netherlands), L-leucine and L-phenylalanine fromSigma-Aldrich (Oakville, ON, Canada) were used in longanjuice fermentation.

Preparation of Longan Juice andFermentation Conditions

Locally purchased longan fruits (Dimocarpus longan Lour.)were used in this research. The fruits were picked up from themarket and longan juice was obtained within 24 h.The storagetemperature of the fruits was maintained at 4C and the juice at-20C before fermentation. Longan juice (pH 6.9) wasadjusted to pH 3.6 with malic acid to hinder the growth of bac-teria, and then centrifuged at 21,000 rpm, 4C for 15 min. Thesupernatant was subsequently prefiltered through a 0.65-mm-pore-size prefilter (Sartorius, Göttingen, Germany) beforeaseptically filtering through a 0.45-mm-pore-size microfilter(Sartorius), which can retain the yeast cells, thus removingwild yeasts initially present in the juice (Chandler and Zydney2005); moreover, sterility check was done by plate counting.Aliquotsof 200 mLof sterile longanjuice inoculatedwithyeastpreculturegrowninthesamemediumat1 ¥ 105 cfu/mLinitialcell counts were fermented in a sterile 250 mL conical flaskcapped with a cotton wool followed by aluminum foil at 20Cfor 14 days. Three fermentations (each in replicate) were per-formed without shaking, including control (without aminoacid), and 0.05% (w/v) L-leucine-added and 0.05% (w/v)L-phenylalanine-added longan juices. All equipment incontact with the juices was autoclaved at 121C for 15 min.

Longan Wine Analysis and YeastEnumeration

A 20-mL sample was taken aseptically after swirling gently theconical flasks for homogenization on days 0, 3, 6, 10 and 14 offermentation. The samples were then used to measure pH,Brix values and optical density using UV-vis spectrophotom-eter (Shimadzu, Kyoto, Japan) at 600 nm. One mL of sampleswas aseptically taken for yeast enumeration by plating usingpotato dextrose agar (Oxoid Ltd., Hampshire, England) ondays 0 and 14. The remaining samples were centrifuged at5,000 rpm, 4C for 15 min in capped tubes to remove yeast

LONGAN WINE AROMA FERMENTED WITH WILLIOPSIS SATURNUS T.-T.-T. TRINH ET AL.

2 Journal of Food Processing and Preservation •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

cells. The cell-free supernatants were stored at -20C beforeanalysis. All determinations were conducted in duplicate.

Analysis of Volatile Compounds inLongan Wine

Volatile compounds were analyzed by headspace-solid phasemicroextraction sampling combined with gas chromatography6890-flame ionization detector/mass spectrometry detector5975 (Agilent Technologies, Santa Clara, CA) (HS-SPME-GC-FID/MSD).Five mL of samples was placed in a 15-mL glass vialtightly capped with a poly-tetra-fluoro-ethylene/siliconeseptum and subjected to automatic HS-SPME exposure for30 min at 60C to get the required headspace–liquid equilib-rium.The SPME fused silica fiber coated with 85 mm carboxen/polydimethylsiloxane (Supelco Co., Bellefonte, PA) wasapplied for the extraction of volatiles.After extraction, the vola-tiles were thermally desorbed by inserting the fiber into the GCinjector set at 250C and operated in splitless mode for 150 s.AnAgilent code of stationary phase of column which is composedof Nitroterephthalic acid modified polyethylene glycol,highly polar and applied to analyze volatile fatty acids andphenols (Agilent Technologies) capillary column (0.25 mmI.D. ¥ 60 mL ¥ 0.25 mm film thickness) was performed forchromatographic separation. The GC oven temperature wasprogrammed to operate from 50 to 230C, first maintained at50C for 4 min and then increased to 230C with a rate of5C/min and kept for 20 min. FID temperature was set at 250C,and MSD was operated in the electron impact mode at 70 eV.Identification of the eluted compounds (in duplicate) wasachieved by matching their mass spectra against NIST 8.0 MSlibrary (National Institute of Standards and Technology,Gaith-ersburg, MD) and confirmed with linear retention index (LRI)values. LRI values on the FFAP column were determined usinga series of alkanes (C5–C25) run under identical conditions. Theresults shown (in duplicate analysis) represent the means fortwo independent fermentations with their standard deviations.

Statistical Analysis

An analysis of variance was applied to the experimental dataobtained at day 14 of fermentation. The significant differ-ences were determined by Tukey’s honestly significant differ-ence test. All statistical analyses were performed using thesoftware R for Windows, version 2.9.1. The mean values andstandard deviations were calculated from the data obtainedfrom two independent fermentations.

RESULTS

Yeast Growth, Total Soluble Solids and pHChanges during Longan Juice Fermentation

The growth kinetics of the yeast W. saturnus CBS254 weresimilar irrespective of leucine and phenylalanine addition,

except that the yeast grew at a slower rate with a lower OD600 nm

(Fig. 1) when phenylalanine was added. There were essen-tially no differences between Brix or pH changes with andwithout added amino acids. The Williopsis yeast utilizedsugars weakly (approximately 36% initial sugar wasconsumed).

Kinetic Changes in Volatile Compoundsduring Longan Juice Fermentation

Figure 2 shows the kinetic changes in acetate esters in thepresence and absence of added leucine and phenylalanine.Most of the acetate esters increased initially then decreased,except for 2-phenylethyl acetate that increased steadily. The

FIG. 1. GROWTH OF WILLIOPSIS SATURNUS VAR. SATURNUS CBS254(AS OPTICAL DENSITY [OD] AT 600 nm), BRIX AND PH CHANGESDURING LONGAN JUICE FERMENTATION WITH AND WITHOUT ADDEDAMINO ACIDSLongan juice without added amino acid (control) (�). Longan juice withadded L-leucine (�). Longan juice with added L-phenylalanine ( ).

T.-T.-T. TRINH ET AL. LONGAN WINE AROMA FERMENTED WITH WILLIOPSIS SATURNUS


addition of leucine increased the production of isoamylacetate, while decreasing the formation of butyl, isobutyland hexyl acetates, and 2-phenylethyl acetate remainedunchanged, relative to the control. Similarly, the addition ofphenylalanine enhanced the production of 2-phenylethylacetate, while decreasing the formation of butyl, isobutyl,isoamyl and hexyl acetates, compared with the control.

The kinetics of ethyl esters in the presence of added leucineand phenylalanine are shown in Fig. 3. Most of the ethyl esters(ethyl butyrate, ethyl 2-butenoate, ethyl 3-hydroxybutyrateand ethyl benzoate) that were naturally present in the longanjuice decreased consistently without being affected by theaddition of leucine or phenylalanine, relative to the control.Ethyl acetate and ethyl octanoate increased consistently and

FIG. 2. CHANGES IN ACETATE ESTERS DURINGLONGAN JUICE FERMENTATION BY WILLIOPSISSATURNUS VAR. SATURNUS CBS254Longan juice without added amino acid(control) (�). Longan juice with addedL-leucine (�). Longan juice with addedL-phenylalanine ( ). GC-FID, gaschromatography 6890-flame ionizationdetector.

FIG. 3. CHANGES IN ETHYL ESTERS DURINGLONGAN JUICE FERMENTATION BY WILLIOPSISSATURNUS VAR. SATURNUS CBS254Longan juice without added amino acid(control) (�). Longan juice with addedL-leucine (�). Longan juice with addedL-phenylalanine ( ). GC-FID, gaschromatography 6890-flame ionizationdetector.



their synthesis was decreased to a small extent by the additionof leucine or phenylalanine, compared with the control.

Figure 4 shows that most of the alcohols increased consis-tently with the exception of linalool that decreased. The addi-tion of leucine and phenylalanine markedly increased theproduction of isoamyl alcohol and 2-phenyethanol, respec-tively. The production of ethanol and isobutyl alcohol was notmarkedly influenced by the presence of added amino acids,nor was linalool reduction.

The changes in fatty acids including acetic, hexanoic,octanoic and decanoic acids are presented in Fig. 5. Aceticacid is a common product of yeast metabolism, thus account-ing for its continuous increase during fermentation. More-over, acetic acid is considered to be unpleasant at

concentrations near its flavor threshold because of its highvolatility. The addition of leucine and phenylalanineappeared to slightly decrease acetic acid production. Whilehexanoic acid decreased initially then stabilized, octanoic anddecanoic acids increased initially then decreased, neither wasaffected by the addition of leucine or phenylalanine.

Figure 6 shows the changes in aldehydes (acetaldehyde,2-butenal and benzaldehyde) that occur naturally in longanjuice. Short-chain volatile aldehydes are important to theflavor of a number of foods and beverages, contributingflavor characteristics ranging from “apple-like” to “citrus-like” to “nutty” depending on the chemical structure (Zoeck-lein et al. 1995). Both acetaldehyde and benzaldehydedecreased consistently, whereas 2-butenal decreased initially

FIG. 4. CHANGES IN ALCOHOLS DURINGLONGAN JUICE FERMENTATION BY WILLIOPSISSATURNUS VAR. SATURNUS CBS254Longan juice without added amino acid(control) (�). Longan juice with addedL-leucine (�). Longan juice with addedL-phenylalanine ( ). GC-FID, gaschromatography 6890-flame ionizationdetector.

FIG. 5. CHANGES IN ACIDS DURING LONGANJUICE FERMENTATION BY WILLIOPSISSATURNUS VAR. SATURNUS CBS254Longan juice without added amino acid(control) (�). Longan juice with addedL-leucine (�). Longan juice with addedL-phenylalanine ( ). GC-FID, gaschromatography 6890-flame ionizationdetector.



then increased, neither was impacted by the addition ofleucine or phenylalanine.

DISCUSSION

A majority of volatile flavor compounds identified in longanwine (Table 1) are known to convey sensory properties togrape and other fruit wines such as orange wine, pineapplewine, etc. (Rapp and Mandery 1986; Selli et al. 2003; Pino andQueris 2010). Moreover, yeasts belonging to the Williopsisgenus have the ability to utilize sugar oxidatively for cellgrowth with the production of desirable fruity flavors (Iwaseet al. 1995; Yilmaztekin et al. 2008, 2009). Particularly, W. sat-urnus strains are known to be able to convert higher alcoholsinto the corresponding acetate esters (Janssens 1991). Anexample is the conversion of isoamyl alcohol into isoamylacetate (Yilmaztekin et al. 2009). The present study has dem-onstrated that this yeast can also convert 2-phenylethanolinto 2-phenylethyl acetate that is more stable than isoamylacetate and other acetate esters (Fig. 2).

A variety of volatile metabolites can be formed via themetabolism of yeasts. The most typical volatile product in

wine is ethanol. Moreover, other alcohols are produced fromamino acids such as branched-chain alcohols from branched-chain amino acids (e.g., isoamyl alcohol from leucine, Dickin-son et al. 1997) and aromatic alcohols from aromatic aminoacids (e.g., 2-phenylethanol from phenylalanine, Webb andIngraham 1963). Higher alcohols, characterized by theirstrong and pungent smell and taste,can significantly influencewine taste and character (Lambrechts and Pretorius 2002).They are also precursors for ester formation (Soles et al.1982).

In this study, W. saturnus CBS254 was able to significantlyenhance the production of aroma-active compounds isoamylalcohol and its ester isoamyl acetate (banana-like aroma), and2-phenylethanolanditsester2-phenylethylacetate(rose-petalaroma), when longan juice was fortified with L-leucine orL-phenylalanine, respectively. The catabolism of leucine andphenylalanine by yeast resulted in the production of isoamylalcohol and 2-phenylethanol, respectively. These two alcoholstogether with acetyl-CoA were used for the biosynthesis ofisoamyl acetate and 2-phenylethyl acetate by the action ofalcohol acetyltransferase (Yoshioka and Hashimoto 1981),which may account for the enhanced formation of isoamylacetate and 2-phenylethyl acetate with the addition of leucineand phenylalanine described above (Fig. 2). The reduction inthe formation of butyl, isobutyl, isoamyl and hexyl acetatesmay be explained by the diversion of acetyl-CoA to the biosyn-thesis of isoamyl acetate and 2-phenylethyl acetate becauseacetyl-CoA is one of the precursors to acetate ester productioncatalyzed by alcohol acetyltransferases (Yoshioka and Hash-imoto 1981). On the other hand, the slightly decreased forma-tion of acetic acid induced by the addition of L-leucine andL-phenylalanine may also be due to diversion of acetyl-CoA toacetate ester biosynthesis instead of being used to synthesizefatty acids (Lambrechts and Pretorius 2002).

The addition of leucine and phenylalanine to longan juicewas expected to increase the formation of isoamyl alcohol and2-phenylethanol and the corresponding isoamyl acetate and2-phenylethyl acetate in longan wine, respectively. Indeed,isoamyl acetate and 2-phenylethyl acetate together withisoamyl alcohol and 2-phenylethanol were produced ingreater amounts in longan wine with added leucine and phe-nylalanine and differed significantly at the statistical levelrelative to the control, whereas most other volatiles were notstatistically different from the control (Table 1). Moreover,ethanol, ethyl acetate, isoamyl acetate and 2-phenylethylacetate were the major aroma compounds. Together withisoamyl alcohol and 2-phenylethanol, their correspondingacetate esters play positive roles in wine aroma, impartingfruity and flowery flavor (Rapp and Mandery 1986), and wereamong the target esters to be produced by non-Saccharomycesyeasts (Viana et al. 2009; Lee et al. 2010; Trinh et al. 2010,2011).

Therefore, the addition of a specific amino acid into longanjuice can be exploited to raise the level of the targeted aroma

FIG. 6. CHANGES IN ALDEHYDES DURING LONGAN JUICEFERMENTATION BY WILLIOPSIS SATURNUS VAR. SATURNUS CBS254Longan juice without added amino acid (control) (�). Longan juice withadded L-leucine (�). Longan juice with added L-phenylalanine( ). GC-FID, gas chromatography 6890-flame ionization detector.



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compounds in longan wine, making longan wine productfavorable to consumers. This application has been employedfor making other fruit wines such as apple and lychee wine inChina (Gai et al. 2005; Zhang et al. 2008). Sensory analysis isrequired to evaluate the relative contribution of each volatilecompound to the organoleptic characteristics of longan wine.

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FORMATION OF AROMA COMPOUNDS DURING LONGAN JUICE FERMENTATION BY WILLIOPSIS SATURNUS VAR. SATURNUS WITH THE ADDITION OF SELECTED AMINO ACIDS