mango wine aroma enhancement by pulp contact and beta-glucosidase

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Original article

Mango wine aroma enhancement by pulp contact and

b-glucosidase

Xiao Li,1 Sien Long Lim,1 Bin Yu,2 Philip Curran2 & Shao-Quan Liu1,3*

1 Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore City

117543, Singapore

2 Firmenich Asia Pte Ltd, Tuas 638377, Singapore

3 National University of Singapore (Suzhou) Research Institute, No. 377 Linquan Street, Suzhou Industrial Park, Suzhou, Jiangsu 215123,

China

(Received 6 March 2013; Accepted in revised form 11 May 2013)

Summary This study determined the influence of pulp maceration andb-glucosidase on mango wine physico-chemical

properties and volatile profiles. The distinction in pH, sugars and organic acids among different treatments

was not statistically significant. All wine samples reached around 8% (v/v) ethanol from about 16% (w/v)

of sugars. The wine with pulp contact contained about ten times highera-terpinolene and up to three timeshigher acetate esters than the wine without pulp contact, but mitigated the production of medium-chain

fatty acids and relative ethyl esters by up to six times.b-glucosidase enhanced terpenols by up to ten times

and acetate esters by up to three times. Furthermore, enzyme treatment mitigated, by up to five times, the

formation of medium-chain fatty acids and ethyl esters to moderate levels. Sensory evaluation showed pulp

contact, and b-glucosidase not only improved the intensity and complexity of wine aroma but balanced

odour attributes.

Keywords Flavour, juice, mango wine, pulp, volatiles,b-glucosidase.

Introduction

Many reports revealed that, besides free aromatic com-pounds, there are nonodorous and nonvolatile gluco-sides that represent an important source of fragrantcompounds in fruit juices and wines (Shoseyov et al.,1988; Gueguen et al., 1996). The aglycone moieties ofglycosides include monoterpene hydrocarbons, terpen-ols, C13-norisoprenoids, benzene derivatives andaliphatic alcohols, most of which possess pleasant flo-ral and fruity aromas with low perception thresholds(Palomo et al., 2005). Sarry & Gunata (2004) summar-ised that enzymatic and/or acid hydrolysis of glycosidi-cally bound volatiles could allow the liberation of freevolatiles (aglycones) in different fruit juices or winessuch as grape, apple and passion fruits.

Acid hydrolysis of glycosides occurs very slowly andmay induce terpenol rearrangements (Gunata et al.,1988). Enzymatic hydrolysis can be much more effec-tive and specific in liberating aromatic compoundsfrom glycosides and minimise terpenol rearrangements(Gunata et al., 1988). However, glycosidases fromfruits or Saccharomyces cerevisiae are not sufficiently

active because these enzymes show poor stability underwine conditions (Palomo et al., 2005). In contrast,

glycosidases from fungi such as Aspergillus niger haveshown very good stability and activity in wine (Sarry& Gunata, 2004).

The glycosidically bound volatiles such as terpenolsare frequently found in mango cultivars (Adedejiet al., 1992; Drider et al., 1994; Sakho et al., 1997;Lalel et al., 2003). These studies revealed an abun-dance of glycosidically bound volatiles, which includedmonoterpene hydrocarbons, terpenols, alcohols, alde-hydes, acids, esters, C13-norisoprenoids and benzenederivatives in mangoes. Furthermore, Lalel et al.(2003) demonstrated the role of b-glucosidase andhemicellulase in the release of glycosidically boundvolatiles during ripening process in both pulp and skin

of the Kensington Pride mango. They discovered thatmost glycosidically bound aroma compounds increasedin the pulp as maturity progressed. This suggests apossible method for enhancement of mango winearoma by fermenting both juice and pulp with the useof glycosidases. However, there is a lack of systematicinvestigations on the application of exogenous glyco-sidases in mango wine. Therefore, the aim of thisstudy was to determine potent aroma compounds in*Correspondent: Fax: +65 6775 7895; e-mail: [email protected]

International Journal of Food Science and Technology 2013, 48, 22582266

doi:10.1111/ijfs.12212

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mango wine on the basis of odour activity values(OAV) and the effect of application of exogenousb-glucosidase on wine aroma composition. For furtherenrichment of free aroma compounds and boundaroma precursors (glycosides), mango pulp was notdiscarded but included throughout the fermentation to

mimic the maceration step in grape wine fermentation.The outcome of this study would aid further researchon pulp contact and ascertain the effectiveness ofb-glucosidase application in mango wine flavour mod-ulation. The ultimate aim was to enable mango wineflavour intensification, diversification and/or differenti-ation so as to achieve unique mango wine style anddistinction.

Materials and methods

Enzymes

The commercial enzyme Novarom Blanc (Novozymes,

Bagsvaerd, Denmark) used in this study was chosen outof three other enzyme preparations: Lallzyme Beta andLallzyme Cuvee Blanc (Lallemand SA, Blagnac,France) and Macer8 W (Biocatalysts, Parc Nantgarw,United Kingdom). This selection was based on anenzyme activity test where Novarom Blanc (a commer-cial enzyme prepared from Aspergillus niger) exhibited aconsistently higher b-glucosidase activity under pH andtemperature ranges of pH 27 and 2070 C, respec-tively (data not shown). The Novarom Blanc enzymewas mainly composed of maltodextrin-encapsulatedb-glucosidase. The dosage of the enzyme used in thisexperiment was 0.4 g L1 to accelerate the enzymatic

process (vs. the recommended dosage of 0.1 g L

1

forabout 23 weeks).

Reagents and chemicals

Food-grade DL-malic acid (>97%) was purchasedfrom Suntop Ltd. (Singapore). Potassium metabisulfite(>97%) was bought from Goodlife Homebrew centre(Norfolk, England). Standards of glucose, fructose,sucrose, tartaric, citric, malic, succinic, lactic and pyru-vic acids (>99%) were purchased from Sigma-Aldrich(St. Louis, MO, USA); glycerol (>99%) was boughtfrom Merck (Darmstadt, Germany). Firmenich Asia(Singapore) provided the following standards: hexa-

noic, octanoic, decanoic and dodecanoic acids; etha-nol; isobutyl alcohol; active amyl alcohol; isoamylalcohol; (Z)-3-hexenol; 2-phenylethyl alcohol; linalool;a-terpineol; b-citronellol; nerol; geraniol; ethyl acetate;isobutyl acetate isoamyl acetate; (Z)-3-hexenyl acetate;citronellyl acetate; 2-phenylethyl acetate; ethyl hexano-ate; ethyl octanoate; ethyl decanoate; ethyl dodecano-ate; and a-terpinolene (>98%). Ethanol (>99%) wasbought from Fisher Chemical, Singapore.

Preparation of mango juice

Chok Anan mangoes from Malaysia were purchasedfrom a local market in Singapore and kept at 25 Cuntil fully ripened before use. The ripened mangoeswere juiced and divided into two lots. The first lot was

centrifuged at 41 415 g and 4 C for 10 min to removethe pulp (Beckman Coulter Allegra 64R Centrifuge,Brea, CA, USA). The second lot was not centrifuged.The sugar concentration of centrifuged and uncentri-fuged juice was 158 and 165 g L1, respectively.

Preculture media prepared from the mango juice weresterilised at 100 C for 3 min and cooled to room tem-perature. The media were inoculated with 10% (v/v) ofS. cerevisiae MERIT.ferm (Chr.-Han., Horsholm, Den-mark, maintained in nutrient broth) and incubated for48 h at 25 C until yeasts grew to at least 107 cfu mL1.The mango juice used for fermentation was adjusted topH = 3.5 with food-grade 50% (w/v) DL-malic acidand was treated with 100 ppm (w/v) potassium metab-

isulphite and then heated at 60 C for 15 min andcooled to room temperature.

Fermentation and enzyme treatment

Triplicate mango juice fermentations were carried outin 500-mL sterile Erlenmeyer conical flasks (pluggedwith cotton wool and then wrapped with aluminiumfoil), and each flask contained 450 mL of centrifugedmango juice or uncentrifuged pulpy mango juice. Allsamples were inoculated with 1% (v/v) preculture ofS. cerevisiae MERIT.ferm, and the fermentation wasconducted at 20 C statically for 10 days. The samples

were divided into two 225-mL portions at the end offermentation. One portion was subjected to enzymetreatment (enzyme dosage of 0.4 g L1) and incubatedat 20 C for another 4 days. The other portion waskept as control and incubated at 20 C. All enzymetreatments and controls were carried out in triplicate.Sampling was done at the end of enzyme treatment.

Analysis of sugars and organic acids

Cell-free samples were obtained by centrifugation andfiltration through a 0.2-lm regenerated cellulose filtermembrane (Sartorius Stedim Biotech, Goettingen, Ger-many). Sugars were measured by Shimadzu ultra-fast

liquid chromatography (UFLC, Shimadzu Asia Paci-fic, Singapore) according to the method of Li et al.(2012) using Agilent Zorbax carbohydrate column(150 9 4.6 mm, 5 lm, Santa Clara, CA, USA) con-nected to an ELSD-LT detector. External standards ofglucose, fructose, sucrose and glycerol were used foridentification and quantification. Organic acids werealso determined by Shimadzu UFLC according toLi et al. (2012) using a Supelcogel C-610H column

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(7.8 9 300 mm, 9 lm, Bellefonte, PA, USA) con-nected to a photodiode array detector. Identificationand quantification were done by comparing the reten-tion time and peak area with those obtained fromexternal standards of tartaric, citric, malic, succinic,lactic and pyruvic acids at 210 nm. All samples were

analysed in duplicate.

Analysis of volatiles

Volatiles were analysed using a headspace (HS) solid-phase microextraction (SPME) method coupled withgas chromatography (GC)mass spectrometer (MS)and flame ionisation detector (FID). The system wasAgilent (Palo Alto, CA, USA) 6890 N network gaschromatography system equipped with a DB-FFAPcapillary column (60 m 9 0.25 mm 9 0.25 lm, Agi-lent, Woodbridge, USA), 5975 inert mass selectivedetector (MSD) and FID. Adsorption of volatiles wascarried out with an 85 lm carboxenpolydimethylsilox-

ane fibre (Supelco, Bellefonte, PA, USA). Details ofanalysis were described in Li et al. (2012). The volatileswere identified using Chemstation integrated with Wi-ley mass spectral library. The linear retention indices(LRI) of the compounds were used to express theirretention times on the gas chromatographic column rel-ative to a homologous series of n-alkanes. For semi-quantification of the volatiles, FID peak area was usedto represent the amount of each volatile. A total oftwenty-six major volatiles were quantified by compari-son with available external standards. The method wasbased on Chen et al. (2006). All standards were dis-solved in 10% v/v diluted mango juice with water,

except for ethanol that was dissolved in 100% v/vmango juice. The same HS-SPME-GC-MS/FID condi-tion was used for quantitative analysis, and good line-arity was obtained for all standard curves (R2 > 0.98).All samples were analysed in duplicate. Thereafter,odour activity values (OAVs) of quantified volatileswere calculated according to their known threshold lev-els in the literature (Guth, 1997; Lambrechts & Pretori-us, 2000; Yamamoto et al., 2004; Zemni et al., 2007;Bartowsky & Pretorius, 2008).

Sensory analysis

Four wine samples were assessed by a panel of seven

trained flavourists from Firmenich Asia Pte. Ltd., Sin-gapore. The four wine samples were assessed in thefollowing order: (i) non-enzyme-treated wine from cen-trifuged juice; (ii) non-enzyme-treated wine from non-centrifuged pulpy juice; (iii) enzyme-treated wine fromcentrifuged juice; and (iv) enzyme-treated wine fromnoncentrifuged pulpy juice. A set of descriptive termsfor nine attributes were rated on a five-point scale forthe intensity perceived, where zero indicated that the

descriptor was not perceived and five indicated a veryhigh intensity.

Statistical analysis

The statistical differences of the effect of enzyme treat-

ment on the volatiles of mango wine fermented withand without pulp were evaluated using analysis ofvariance (ANOVA). All tests of significance were con-ducted at a probability level of P < 0.05. Means andstandard deviations were obtained from triplicate fer-mentation samples. The volatile and aroma profiles forenzyme-treated mango wines and control were furtheranalysed using principal component analysis (PCA) tocharacterise the multidimensional data.

Results and discussion

Physico-chemical properties of mango wine with

maceration and enzyme treatmentThe pH increased slightly in macerated (with pulpcontact) wines and viable cell count reached about7 9 107 8 9 107 cfu mL1 (Table 1) for all fourwines. The difference of total soluble solids was notsignificant in all four treatments (Table 1), but theBrix value was slightly higher for the macerated winecompared with the nonmacerated for both enzyme andnonenzyme treatments, which is logically due to ahigher content of pectins and other soluble solids(Rouse et al., 1974). In addition, there was no signifi-cant difference for the organic acids between the con-trol and enzyme-treated wine (Table 1), and lactic acid

was not detected in all samples. The residual sucroseand fructose content was 0.080.19 g L1 higher in the

enzyme-treated wines compared with the controls. Thehigher sugar content could be due to b-glucosidaseand pectinase activity from the enzyme treatment,resulting in the hydrolysis and release of glycosidicallybound saccharides into the solution (Joshi et al., 2011;Pei et al., 2012). The higher sugar content could alsolead to significantly higher glycerol level in theenzyme-treated wine, which was shown in Table 1,and this is probably because high osmolar concentra-tions of solutes (e.g. sugar) would enhance glycerolsynthesis to counteract cell dehydration (Lopes et al.,2000). Glycerol does not contribute to the aroma of

the wine due to its nonvolatile nature but does con-tribute to sweetness, smoothness and viscosity of wine(Eustace & Thornton, 1987). The typical concentrationof glycerol in wine is from 1 to 15 g L1 (Scanes et al.,1998). The threshold taste level of glycerol is observedat 5.2 g L1 in wine (Noble & Bursick, 1984). Glycerolin macerated mango wine with enzyme addition wasremarkably improved, and its concentration washigher than the reported threshold level (Table 1). In

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addition, the final ethanol reached 7.878.49% (v/v)for four wines and was slightly higher in the enzyme-treated wines (Table 1); however, this difference wasnot significant. The slight difference could be due tomore sugars being released from glycoside hydrolysisin enzyme-treated wine, or its higher fermentationactivity suggested by higher viability of cells (Table 1).

Volatiles of mango wine with pulp contact and enzymetreatment

Terpenols and monoterpene hydrocarbonsFew terpenols in the control wine were at levels abovetheir odour thresholds and thus scarcely contribute tothe overall aroma (Table 2). Enzyme treatment signifi-cantly enhanced the concentrations of free terpenols(Table 2), but pulp contact only facilitated enrichmentof b-citronellol and nerol but not all terpenols. Thisresult was slightly different from the report of Cabaro-glu et al. (2003). This was probably due to some extentof interconversion of terpenols catalysed by Saccharo-

myces cerevisiae as summarised by King & Dickinson(2000). The concentration of geraniol increased signifi-cantly in both nonmacerated and macerated wine withenzyme addition, achieving an OAV of 14 and 8,respectively. In addition, enzyme treatment increasedb-citronellol to 0.1 and 0.23 mg L1 in the nonmacer-ated and macerated wines, respectively, which were atthe level of or higher than its odour threshold(Table 2). On the other hand, linalool was found to

have an OAV of 12 and 7.3 in both nonmacerated andmacerated wines, respectively, with enzyme treatment.The concentrations of a-terpineol and nerol were alsoenhanced by enzyme treatment but were still belowtheir threshold levels. The result was consistent withthe results obtained in fruit juices (Gueguen et al.,1996) and grape wine (Rogerson et al., 1999; Cabaro-

glu et al., 2003). Terpenols such as geraniol, linalooland citronellol have been found to be important in thecontribution of floral and citrus notes to wine espe-cially those from aromatic grape varieties such asMuscat and Riesling (Marais, 1984; Swiegers et al.,2005).

Monoterpene hydrocarbons (C10H16, most impor-tantly a-terpinolene in Chok Anan mango), the keyvolatile constituents of the fresh mango aroma, are lar-gely formed in the mango during ripening. Thesecompounds significantly contributed to typical freshmango aroma. Unfortunately, monoterpene hydrocar-bons were significantly lost throughout the fermenta-tion (Li et al., 2011, 2012) possibly due to their

volatility. In this experiment, pulp contact stronglyretained the level of a-terpinolene (Table 2), butsurprisingly, its concentration decreased with enzymetreatment. Although other important monoterpenehydrocarbons such as d-3-carene, a-phellandrene,a-terpinene, limonene and sabinene were not quantifieddue to a lack of standard compounds, they also showedhighest FID peak areas in the macerated control sam-ple relative to the enzyme-treated sample. The decrease

Table 1 Physico-chemical properties, alcohol, organic acid and sugar concentrations of nonmacerated and macerated mango wine, with and

without enzyme treatment

Fermentation medium

Control Enzyme

Nonmacerated Macerated Nonmacerated Macerated

Physico-chemical properties

Ph 3.57 0.00ab 3.70 0.06c 3.50 0.01a 3.64 0.01bc

Total soluble solids (Brix) 5.58 0.14ab 5.98 0.13a 5.09 0.16b 5.87 0.13a

Cell count (106 cfu mL1) 89.75 28.64a 73.88 24.57a 79.75 28.64a 85.13 6.19a

Alcohol

Ethanol (% v/v) 7.87 0.83a 8.08 0.41a 8.49 0.31a 8.14 0.47a

Glycerol (g L1) 4.62 0.11a 5.33 0.30ab 5.94 0.21b 8.46 0.52c

Organic acids (g L1)

Citric acid 2.21 0.30a 1.72 0.02b 1.95 0.04ab 1.93 0.03ab

Tartaric acid 0.28 0.07a 0.25 0.01a 0.26 0.01a 0.27 0.01a

Malic acid 3.81 0.41a 3.13 0.45b 3.62 0.35ab 3.27 0.01ab

Pyruvic acid 0.10 0.01a 0.05 0.00b 0.08 0.00c 0.03 0.00d

Succinic acid 1.21 0.07a 1.33 0.08a 1.11 0.05a 1.19 0.04a

Reducing sugars (g L1)

Fructose N. D. N. D. 0.17 0.00a 0.19 0.00b

Glucose 0.36 0.00a 0.48 0.01a 0.45 0.02a 0.49 0.01a

Sucrose 0.39 0.01a 0.39 0.05ab 0.47 0.02ab 0.47 0.03b

abcdANOVA(n = 6) at 95% confidence level with same letter indicating no significant difference.

N.D.: not detected, standard deviation.

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in monoterpene hydrocarbons as a result of enzymetreatment was possibly due to monoterpene hydrocar-bons being bound by the enzyme protein via hydropho-bic interactions or being trapped by maltodextrin (thecarrier substance for b-glucosidase in the NovaromBlanc enzyme preparation). The nonpolar monoterpene

hydrocarbons became less volatile when interactingwith protein and/or maltodextrin (Misharina, 2010).

Alcohols(Z)-3-Hexenol was considered as an important agly-cone in fresh mango juice, and it could be efficientlyreleased by either exogenous glycosidase or glycosidasefrom fruits (Sakho et al., 1997; Lalel et al., 2003).(Z)-3-Hexenol has also been reported to be presentabove their odour threshold values in some mangocultivars, contributing to the floralcitrus odour andgreen grassy odour (Pino & Mesa, 2006). In this study,(Z)-3-Hexenol, which was already present above itsodour threshold in both wines, was significantly

increased in the nonmacerated wine after the enzymetreatment. This was consistent with the result in otherreports (Sakho et al., 1997; Lalel et al., 2003). How-ever, there was no statistically significant differencebetween the concentrations of other alcohols (exceptactive amyl alcohol) after enzyme treatment, but themacerated wine was higher in fusel alcohols, whichcould be related to the higher availability of relativeamino acids in pulpy juice.

Esters and fatty acidsEsters are the principal odourants in mango wine dueto their high OAVs (Table 2). The esters are mainly of

two types: ethyl esters of medium-chain fatty acidsand acetate esters of higher alcohols, and both typesare formed enzymatically or chemically during fermen-tation, but chemical formation is very slow and can beignored (Lambrechts & Pretorius, 2000). Major acetateesters such as isoamyl acetate and 2-phenylethyl ace-tate give banana-like and rose-like aromas, respec-tively. Major ethyl esters such as ethyl hexanoate andethyl octanoate engender an apple-like character(Rojas et al., 2003). However, an overproduction ofacetate esters and ethyl esters may lead to nail polisherand soapy off-flavours (Lambrechts & Pretorius,2000).

Compared with non-Saccharomyces yeasts such as

Hanseniaspora and Pichia (Rojas et al., 2001), the pro-duction of acetate esters is generally moderate inSaccharomyces yeasts. However, Saccharomyces yeastsare able to vigorously produce ethyl esters (Li et al.,2011), which is also shown in Table 2. According toSaerens et al. (2008), production of ethyl esters is posi-tively correlated with the production of respective fattyacids, and this is consistent with our results. Enzymeaddition (except for ethyl hexanoate) or pulp contact

seemed to induce decreased production of fatty acids,which correspondingly led to more moderate produc-tion of ethyl esters (Table 2). Nonetheless, the detailedmechanism involved remains to be elucidated.

Minor volatile compounds

Some quantitatively minor volatile compounds wereidentified, such as (E)-b-damascenone, p-cymen-8-ol,nerol oxide, geranyl ether, and 1,8-menthadien-4-ol.They could not be quantified because the referencecompounds are hardly commercially available, but theFID peak area of these compounds showed that theywere significantly enhanced by b-glucosidase treatment[FID chromatogram of (E)-b-damascenone was shownin Fig. 1]. More importantly, some of these volatilesare potent odour-active compounds such as(E)-b-damascenone, which has a flowery, quince-likeodour, and its odour threshold is 2 ng L1 in waterand 50 ng L1 in 10% alcoholic solution (Cabarogluet al., 2003).

Sensory characteristics of mango wine

The sensory profiles of the mango wines were repre-sented in a spider web plot as shown in Fig. 2. Fromthe analysis of variance (ANOVA), no significant differ-ences were found for all sensory attributes except foryeasty attribute. This was likely due to high standarddeviations, which were probably caused by variationsof panellists sensitivity to different aroma attributes,small panellist size of seven and a small five-pointscale.

The control samples for nonmacerated wine were

significantly more yeasty than the rest. This was prob-ably related to its higher concentration of ethyl estersand fatty acids. Yeasty note has been used to cha-racterise the complexity of sparkling wine (Torrenset al., 2010), but this aroma attribute has been deter-mined to be unfavourable in other alcoholic drinkssuch as Tannat grape wines (Varela & Gambaro,2006), and it may affect the fruitiness of wine. Also,the sensory result concomitantly showed that pulpcontact and enzyme treatment mitigated the protrud-ing yeasty note in control sample. In addition, theterpenic or woody aroma here referred to the charac-teristic aromas associated with fresh mango. The mac-erated wines were perceived as more terpenic

compared with the nonmacerated wines, where themacerated wine without enzyme treatment achievedthe highest score, and this was consistent with its high-est concentrations of a-terpinolene (Table 2). Macer-ated wines were also considered as winey and thiscorrelated with their higher concentration of higheralcohols. The enzyme-treated wines had higher scoresfor rosy (floral) and fruity attributes compared withthose without enzyme treatment (Fig. 2). The rosy and

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fruity notes were probably ascribed to the higher levelof short-chain acetate esters such as isobutyl acetateand isoamyl acetate, as well as terpenols such as linal-

ool, geraniol and b-citronellol (Table 2).

Principal component analysis of volatiles in mango wine

Principal component analysis (PCA) was applied todiscriminate the volatile profiles of the mango wines(Fig. 3). The first principal component (PC1)accounted for 62.7% of the total variance in the dataset, while the second principal component (PC2)

accounted for 23.2% of the total variance. The PCAbiplot separated the twenty-six different volatile com-pounds and the four different samples.

0

1

2

3

4

5Green

Fruity

Waxy

Terpenic/Woody

Rosy/FloralWiney

Sweet

Yeasty

Acidic

Figure 2 Aroma profile of mango wines: nonmacerated control

(), macerated wine control (), nonmacerated with enzyme (),

and macerated wine with enzyme (9).

0.4 0.3 0.2 0.1 0 0.1 0.2 0.3 0.40.4

0.3

0.2

0.1

0

0.1

0.2

0.3

0.4

1

234

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

PC 1 (64%)

PC2(25%)

PCtrl

JCtrl

PEnz

JEnz

Figure 3 Biplot of principal component analysis of mango wines:

nonmacerated control (JCtrl, ), macerated wine control (PCtrl, ),

enzyme-treated nonmacerated wine (JEnz, ) and enzyme-treated

macerated wine (PEnz, 9): (1) acetic acid; (2) hexanoic acid; (3)

octanoic acid; (4) decanoic acid; (5) dodecanoic acid; (6) ethanol; (7)

isobutyl alcohol; (8) active amyl alcohol; (9) isoamyl alcohol; (10)

(Z)-3-hexenol; (11) linalool; (12) a-terpineol; (13)b-citronellol; (14)

nerol; (15) geraniol; (16) 2-phenylethyl alcohol; (17) ethyl acetate;

(18) isobutyl acetate; (19) isoamyl acetate; (20) (Z)-3-hexenyl acetate;

(21) ethyl hexanoate; (22) ethyl octanoate; (23) ethyl decanoate; (24)

citronellyl acetate; (25) 2-phenylethyl acetate; (26) ethyl dodecanoate;

(27) a-terpinolene.

31.20 31.21 31.22 31.23 31.24 31.25 31.26 31.27

130 000135 000140 000145 000150 000

155 000160 000165 000170 000175 000180 000185 000190 000

195 000200 000205 000210 000215 000

Time-->

Abundance

Signal: Juice A Enz.D\FID1A.chSignal: Juice A Ctrl.D\FID1A.chSignal: Pulp A Ctrl.D\FID1A.chSignal: Pulp A Enz.D\FID1A.ch

Macerated with Enzyme

Non-macerated with Enzyme

Macerated control

Non-macerated control

(E)--damascenone

Figure 1 Flame ionisation detector (FID) chromatogram of (E)-b-damascenone in nonmacerated control, macerated wine control, nonmacer-

ated with enzyme and macerated wine with enzyme.

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The first principal component (PC1) separated thenonmacerated control wine from other samples basedon the higher concentrations of medium-chain fattyacids (octanoic, decanoic and dodecanoic acids), med-ium-chain ethyl esters (ethyl esters of octanoic, decan-oate and dodecanoate) 2-phenylethyl acetate. PC1 also

separated the macerated wine with enzyme additionfrom other samples due to higher concentrations ofacetic acid, most of higher alcohols, short-chain ace-tate esters such as isoamyl acetate as well as someterpenols and their acetate esters such as b-citronelloland citronellyl acetate. Furthermore, PC2 separatedthe nonmacerated wine with enzyme addition andmacerated wine without enzyme addition based onhigher concentrations of ethanol and isoamyl alcoholin the former and higher a-terpinolene in the latter.

The compounds that correlated with the nonmacer-ated wine were those imparting waxy, soapy, fatty, flo-ral, green and fruity notes. The macerated wines wereassociated with compounds with winey, fruity, terpen-

ic, citrus and floral odours, but also with the acidicodour. Moreover, the enzyme addition generallyfurther intensified the aroma difference between non-macerated and macerated wines.

Conclusion

This work assessed the use of exogenous b-glucosidaseand pulp contact in mango wine for enhancing flavour.Pulp contact could enhance monoterpene hydrocar-bons, higher alcohols and acetate esters. The additionofb-glucosidase could accelerate the release of odour-active volatiles such as terpenols, acetate esters, ben-

zene derivatives and C13-norisoprenoids. Furthermore,both b-glucosidase addition and pulp contact couldmitigate production of excess fatty acids and theirethyl esters (yeasty note in sensory test). The applica-tion of b-glucosidase with mango pulp contact waseffective in intensification, diversification and alsobalancing of mango wine aroma profile.

Acknowledgment

This work was supported, in part, by an ARF grantfrom Ministry of Education of Singapore (WBS No.R-143-000-507-112).

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