J Sci Food Agric 1995,68,257-260

Characterisation of Bound Flavour Components 0 --. . A

Harry Young* and Vivienne J Paterson

The Horticulture and Food Research Institute of New Zealand Ltd, Private Bag 92169, Auckland, New Zealand

(Received 9 August 1994; revised version received 6 December 1994; accepted 13 February 1995)

Abstract: Glycosidically bound volatiles in kiwifruit have been studied. The com- pounds were isolated from kiwifruit juice by absorption onto a column of Amberlite XAD-2 followed by washing with pentane and elution with methanol. Volatiles were released by enzymic hydrolysis with 8-glucosidase. Major com- ponents found and identified by GC-MS were E-hex-2-enal and benzaldehyde. Compounds not previously identified in kiwifruit include octan-3-01, camphor, 4-methylbenzaldehyde, 2-hydroxybenzaldehyde, neral, geranial, methyl 2- hydroxybenzoate, nerol, geraniol and 2-phenylethanol.

Key words : kiwifruit, bound flavour components, glycoside.

INTRODUCTION EXPERIMENTAL

Kiwifruit (Actinidia deliciosa (A Chev) Liang et Fergu- son var deliciosa cv Hayward) flavour volatiles have been the subject of several studies (Cossa et a1 1988; Bartley and Schwede 1989; Young and Paterson 1990; Paterson et a1 1991). Some sensory attributes have been shown to be associated with flavour compounds but the causal factors for many of the attributes are still unknown (McMath et a1 1992; Young et a1 19929 1993)* Recent studies have shown that an important portion of the flavour compounds in fruit and vegetables occurs as non-volatile glycosides (Koulibaly et a1 1992; Marlatt et a1 1992; Zhou et a1 1992; Sefton et 1994). These non- volatile compounds release volatile flavour components, often by enzyme-mediated reactions, which could be

ducts. With kiwifruit the marked change in flavour with processing is a major problem. The resulting flavour is considered as inferior to that of the fresh product. This

flavour compounds in kiwifruit.

Preparation of non-volatile precursors

A column of Amberlite XAD-2 resin (240 mm 20 mm id) was washed sequentially with water (100 ml), acetone (150 ml), water (100 ml), 3 M HCl(100 ml), and finally water until C1--free.

The fruit used were stored at 0°C for 3 months. The flesh from fruit with mean soluble solids concentration (sSc) of 16.00Brix and mean firmness of 0.67 kgf (kgf = 9.8 N) was scooped from 4 kg of fruit and blended in a Wasng blender for 15 s. The juice was extracted using a laboratory cloth and rack press (900 ml) and filtered through a pad of Celite 535 before passing it down the Amberlite XAD-2 column. The

water (200 ml), followed by pentane (250 to non-glycosidic compounds. The non-volatile flavour precursors were then eluted from the column with methanol (150 ml). The methanol eluate was evaporated to dryness on the rotary evaporator and further dried under vacuum (<0-3 kPa) at ambient temperature overnight. The dried methanol eluate was kept in the freezer at - 20°C until required.

important contributors to the flavour of processed pro- column was eluted with reversed osmosis purified (RO)

demonstrates the presence Of bound

* To whom correspondence should be addressed.

J Sci Food Agric 0022-5142/95/$09.00 0 1995 SCI. Printed in Great Britain 257

258 H Young, V J Paterson

Release of the aglycone

The buffer for the hydrolysis was prepared as follows : citric acid (4.2 g) and Na,HPO, (2.84 g) was dissolved in RO water (100 ml) and adjusted to pH 5 with 4 M NaOH.

The residue from the dried methanol eluate was taken up in 30 ml of pH 5 buffer and extracted with purified pentane (3 x 10 ml). A portion (75%) of the non-volatile pentane-insoluble fraction was treated with 8- glucosidase from almond (50 mg, Sigma Chemical Co product no. G-0395) dissolved in pH 5 buffer (1 ml). The enzyme was used as received from Sigma and was not checked for contaminating activities. After stirring for 42 h at 37°C in a closed flask, the mixture was extracted with purified pentane (3 x 1 ml). The pentane extracts were combined and concentrated to c 600 pl with a micro-Dufton column followed by slow static distillation (Blomberg and Roeraade 1988) to 50 PI. The non-enzyme treated portion (control) was extracted in the same way.

Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS)

GC was carried out on a J and W scientific DBWax (30 m x 0.32 mm id) column using H, carrier gas at 30 an s-' and a flame ionisation detector was used. The split/splitless injector temperature was held at 150°C. Column temperature programme was 30"C, hold 6 min, 3°C min-' to 190"C, hold 20 min. Quantitation was based on an average response factor calculated from some of the quantitatively more important, and chemically stable, components found in kiwifruit

0.4

3 5 0.2 >

0

5

2

flavour volatiles (Young and Paterson 1990), namely methyl and ethyl butanoate, butanol, pent-1-en-3-01, methyl and ethyl hexanoate, hexanol, methyl and ethyl benzoate.

GC-MS was carried out on a VG70-SE spectrometer operated in the electron impact mode at 70 eV fitted with a directly coupled GC. The carrier gas was replaced with He.

RESULTS AND DISCUSSION

Care was taken to remove free volatile compounds from the methanol eluate from the XAD-2 column by thor- ough washing with pentane. Figure 1 shows a typical chromatogram of the volatile compounds recovered from the enzyme hydrolysate of the methanol eluate while Table 1 gives a list of the compounds identified. The major components were E-hex-2-enal and benz- aldehyde. The identifications were made by comparing the mass spectra and retention indices with either a proprietary HortResearch database or with data found in the literature. Many of the compounds have been reported in previous studies of the free volatile flavour components of kiwifruit. The aroma of the hydrolysate and the subsequent pentane extracted material had an aroma similar to that of heat-treated kiwifruit juice.

Several of the components released by enzyme- mediated hydrolysis were also found in an experiment in which the methanol eluate from the XAD-2 column was dissolved in the buffer only. These compounds, which are important aroma components of the free volatiles, included hexanal, 2- and E-hex-2-enal. These compounds are probably the result of non-enzymic

12

11

I I I I 1 1 0 10 20 30 40 50 60

I 20

Mi n Utes Fig 1. Gas chromatogram of volatiles recovered by pentane extraction from enzyme hydrolysate of bound kiwifruit flavour precur-

sors. See Methods for details of GC conditions. Numbering corresponds to Table 1.

Bound flavour components in kiwifruit 259

TABLE 1 Compounds identified in the /I-glucosidase-treated glycoside-containing fraction from

kiwifruit"

Number Compounds RI WfW ID

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

2-Methylbutanal* Hexanal* 1 -Butanol Z-Hex-2-enal* E-Hex-2-enal* 1-Hexanol Z-Hex-3-en- 1-01 octan-3-ol Z-Linalool oxide A, furanoid Z-Linalool oxide B, furanoid Camphor Benzaldehyde Linalool QMetb ylbenzaldeb yde 2Hydroxybenzaldebyde Neral a-Terpineol Geranial Methyl 2bydroxybenmate Nerol /I-Damascenone Geraniol 2-Phyletbaaol Unknown Unknown Unknown Unknown Iridomyrmecia 1-(2Hydroxy-5-metbylphenyI~t~no~

984 1087 1138 1210 1227 1371 1402 141 1 1458 1488 1528 1538 1566 1639 1697 1700 1719 1788 1795 1824 1841 1872 1939 2018

2091 2126 2199 2226

8.6 20.7 2.3 1.2

138.5 5.4 0-7

11.3 0.3 1-3 0.8

142.2 1.4 0.8 1.5 1.6 1 .o 1.6 0.7 0.2 2.1 2.9 4.9 1 *6 0.5 0.9 0.7 1 *4 2.3

MS MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS, RI MS MS MS, RI MS, RI MS, RI MS MS MS, RI MS, RI MS, R I MS, RI MS, RI MS, RI MS, RI MS

MS, t MS, t

a Identification by Retention Index (RI) and/or Library Mass Spectra (MS). (*) indi- cates that the compound was also found in untreated control sample. Compounds not previously reported for kiwifruit are in bold print. t = tentative identification.

Semi-quantitative data assuming 100% juice recovery.

hydrolysis as the vacuum drying and the pentane washing are expected to completely remove any free volatile aroma components.

New compounds identified were octan-3-01, camphor, 4-methylbenzaldehyde, 2-hydroxybenzaldehyde, neral, geranial, methyl 2-hydroxybenzoate, nerol, geraniol and 2-phen yle t hanol.

The most abundant compound found in the enzyme hydrolysate was benzaldehyde, a compound which is found in a large variety of foods (Maarse and Visscher 1987). Benzaldehyde has been postulated as arising from enzymatic cleavage of the glycoside in high pres- sure treated peaches (Sumitani et al 1994).

The identification of /I-damascenone is of interest. Pasteurized kiwifruit juice has been often described as having an 'apple-like aroma'. /I-Damascenone is known to exist as several glycosides in apple and has been reported as the key component in heated apple juice, contributing to 32% of the total aroma potency (Zhou

et al 1992). It has also been found in the free flavour volatiles from kiwifruit when elevated temperature tech- niques, such as simultaneous distillation extraction (Shiota 1982), were used for isolation of the flavour volatiles.

Other new compounds identified were mainly mono- terpene and aromatic alcohols and aldehydes. Linalool, Z-linalool oxide, and a-terpineol have been previously found in the free volatiles of kiwifruit, but not consis- tently. It is possible that these compounds arise as arti- facts from the bound precursors during isolation of the free volatiles at the low pH of kiwifruit juice (c 3.3). Monoterpene glycosides are susceptible to acid hydro- lysis. Engel and Tressl (1983) reported that under distillation-extraction conditions at pH 3-0, passionfruit monoterpene glycosides gave a complex mixture of ter- penoid products. The occurrence of glycosidically bound monoterpenes in fruit and vegetables is well established (Engel and Tressl 1983; Wu et al 1990;

H Young, V J Paterson

Marlatt et a1 1992; Sefton et a1 1994). The existence of monoterpene flavour compounds in a fruit in bound form only, has precedence in passionfruit (Engel and Tressl 1983).

In conclusion several new flavour components of kiwifruit have been identified from non-volatile precur- sors. It is most likely that these precursors are glycosidi- cally bound but until the individual components have been isolated and characterized the nature of the bonding cannot be confirmed. Preliminary sensory data suggest that these compounds may be important con- tributors to the aroma of processed kiwifruit products.

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Engel K-H, Tressl R 1983 Formation of aroma components from nonvolatile precursors in passion fruit. J Agric Food Chem 998-1002.

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