Postharvest Biology and Technology 50 (2008) 2530

Contents lists available at ScienceDirect

Postharvest Biology and Technology

journal homepage: www.elsevier.com/locate/postharvbio

Does freezing and thawing affect the volatile prole of strawberry fruit (Fragaria ananassa Duch.)?
D.M. Modise
College of Agriculture and Environmental Sciences, University of South Africa (UNISA), P.O. Box 392, Pretoria 0003, South Africa

a r t i c l e

i n f o

a b s t r a c t
This research was aimed at determining if there were any changes in the volatile prole of strawberries cv. Camarosa, when subjected to various freezing and thawing treatments. Strawberries were cut in half, one half of the berries were frozen directly at 20 C or 80 C or rapidly frozen in liquid nitrogen (N2 ) (196 C). They were then stored overnight or for a week. Berries were later left to thaw at room temperature (natural thawing) for about 1 h and some were forced-thawed in a 30 C water bath. Headspace volatile compounds were determined using an Atmospheric Pressure Chemical Ionisation-Mass Spectrometer (APCI-GPA) and validated with a Gas ChromatographMass Spectrometer (GCMS). In a comparison of thawed half berries and fresh berries, most esters such as hexyl acetate, ethyl methyl hexanoate, methyl acetate were increased signicantly by week-long and not by overnight freeze/thaw treatments. Ethyl butyrate was not affected by any treatment. The abundance of aldehydes such as the acetaldehyde compounds was increased signicantly when thawed naturally compared to when forced-thawed in all the cold storage treatments. 2008 Elsevier B.V. All rights reserved.

Article history: Received 29 October 2007 Accepted 15 March 2008 Keywords: Freezing Headspace Strawberries Thawing Volatile compounds Volatile prole

1. Introduction Due to the highly fragile structure of strawberry fruit and their high rates of respiration, their postharvest life is relatively short (Tucker, 1993; Richardson and Kosikattrun, 1995). This is because the fruit generally contains a high percentage of water as fresh weight, which may be as high as 98%, and also exhibits a high metabolic rate, thus making them highly perishable (Tucker, 1993; Pomper and Breen, 1997; Hancock, 1999). The characteristic avour of a fruit is due to the production of specic volatile avour compounds and a complex interaction of sugars, organic acids and phenolics (Kader, 1991; Moing et al., 2001). The perishability and inherent short life of the fruit can result in rapid changes in the volatile compound prole. There is little work on differences in the volatile prole of fresh strawberries compared to those stored frozen for different periods, before volatile compound analyses. The effect of different thawing regimes (slow versus rapid thawing) for volatile compound analysis is also not well documented. Previous experiments have tended to concentrate on comparing avour constituents of different cultivars subjected to the same freeze/thaw regime, e.g. Schreier (1980), Honkanen and Hirvi (1990) and Larsen and Poll (1992). Freezing of strawberries with

liquid nitrogen for individual quick freezing (IQF) is considered the most suitable method compared to conventional cold storage because the delicate texture and avour is maintained (Wang, 1991). This study was conducted to determine if there were any changes in the volatile prole of strawberries when subjected to various freezing and thawing treatments. Understanding whether freezing of strawberry fruit prior to volatile compound analysis, might change the volatile prole would enable researchers to decide whether to analyse the fruit immediately after harvesting or store them frozen for later analysis without fear of altering the volatile prole. The experiment was therefore set to test the hypothesis that the volatile prole of frozen and thawed strawberry fruit does not differ from that of fresh berries. 2. Materials and methods 2.1. Plant material The research was carried out at the Food Science Laboratory, University of Nottingham, in England. Strawberry (Fragaria ananassa Duchesne.), a hybrid of Fragaria chiloensis Fragaria virginiana cv. Camarosa was used in the experiment. Fresh strawberry fruit were purchased from a local supermarket in Loughborough and fruit of uniform size, shape and ripening stage (basing on the standard colour chart of KG Fruits Ltd., UK) were selected before the calyces were removed with a sharp knife. Fruit were thereafter cut in half

Tel.: +27 12 352 4132; fax: +27 12 352 4117. E-mail address: modisd@unisa.ac.za. 0925-5214/$ see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.postharvbio.2008.03.009

26 Table 1 Freeze/thaw treatments Treatment 1 2 3 4 5 6 7 8 9 10 11 12 Freezing and storage None (fresh berry) 20 C overnight 20 C for 1 week 20 C for 1 week 80 C overnight 80 C overnight 80 C for 1 week 80 C for 1 week Liquid N2 /80 C overnight Liquid N2 /80 C overnight Liquid N2 /80 C for 1 week Liquid N2 /80 C for 1 week

D.M. Modise / Postharvest Biology and Technology 50 (2008) 2530

Thawing conditions None Ambient room temperature Ambient room temperature 30 C water bath Ambient room temperature 30 C water bath Ambient room temperature 30 C water bath Ambient room temperature 30 C water bath Ambient room temperature 30 C water bath

and each half was placed in a sample vial (Sterillin) and weighed before exposure to the different freeze/thaw regimes. 2.2. Treatments The half berries were frozen at 20 C in a conventional freezer, or at 80 C in a low temperature deep freezer, or rapidly frozen in liquid nitrogen (N2 ) (196 C), they were then stored overnight or for a week. Berries were later left to thaw at room temperature (natural thawing) for about 1 h and some were forced-thawed in a 30 C water bath (Table 1). There were 12 treatments and each treatment had 10 half berries, giving a total of 120 samples. 2.3. Volatile compound analysis

used for the freeze/thaw treatments was placed inside the mill and blender. The lid of the mill had a modied entry port that enabled entrance of the APCI-GPA interface sample line (Watson et al., 2002). The blender was activated for two 1-s pulses to disrupt the fruit cells in order to release volatile compounds and compartmentalised enzymes which produce additional volatiles (Grab and Gfeller, 1999; Baldwin et al., 2000; Forney et al., 2000). Headspace inside the mill was sampled via a heated deactivated heated (160 C) fused silica transfer line (electronic nose) at an air ow rate of 14 mL min1 . Ion mass (m/z) peaks recovered for 10 replicate samples on the APCI, were identied and quantied. Ethyl butyrate was used as a reference standard where 10 L was injected into the APCI using a syringe of 0.49 mm diameter set to inject 1.5 L min1 , at a ow rate of 14 mL min1 (Humonics Precision Flow, Veriow 500, USA). A preliminary full scan spectra of the headspace sampled from fresh strawberry fruit (cv. Camarosa) was thus obtained so as to validate the actual identity of the compounds using the Gas ChromatographMass Spectrometer (GCMS, GC 5890 Series II, Hewlett Packard and MS-Fisons Instruments, MD 800). Fruit were macerated with a pestle so as to release volatiles and a tenax trap was inserted in an airtight 100 mL bottle. A pressure pump was activated to blow the volatiles onto the tenax trap in the bottle for 10 min. The ow rate was set at 4050 mL s1 . The GCMS uses tenax to adsorb volatile compounds and sample injection is through re-concentration in a cooling trap. The GCMS was used to separate the ions via the Masslynx library (Masslynx V3.2, Micromass Ltd., Manchester, UK) and a quality t indicated the correlation of the volatile compound with the quality t, when the highest peak is selected. 2.4. Data analysis

Headspace proles (Hakala et al., 2002) of the volatile compounds of the rst half of fresh/thawed strawberries were analysed immediately using an Atmospheric Pressure Chemical Ionisation-Gas Phase Analysis (APCI-GPA, Platform LC2 Micromass, Manchester, UK) as described by Brauss et al. (1998) and utilised by Modise et al. (2004). The APCI-GPA was tted with a custombuilt sampling interface that allowed the introduction of gas phase samples. It is characterised by a soft ionisation technique (operating in the API positive ion mode) with real time monitoring, and high sensitivity, enabling it to measure very low headspace concentrations of volatile compounds among its other characteristics. Headspace proles were obtained from macerated strawberry samples prepared using a mill and blender (BL 350 Blender, Kenwood Ltd., UK). A half berry corresponding to the other half that was

A multi-factorial design was used for statistical data analysis where factors under consideration were storage environment, period of storage and the repetition of the intensity of volatile compounds (for freeze/thaw experiments), using Genstat 5 Release 4.1 (Rothamstead Institute), and means were separated using Standard Error Differences (S.E.D.) at (P < 0.05). 3. Results Ions with prominent peaks were chosen (Fig. 1) from the scan for selected ion recording (SIR) of the headspace of 10 replicates of each sample. The identities of the selected ions are shown in Table 2. Generic terms rather than the International Union of Pure

Fig. 1. A full spectral prole of the strawberry fruit (cv. Camarosa).

D.M. Modise / Postharvest Biology and Technology 50 (2008) 2530 Table 2 Ions observed in the headspace of fresh macerated strawberry fruits cv. Camarosa, their probable and actual identities Ion mass 45.3 59.2 61.2 75.2 89.3 99.3 101.2 103.2 115.2 117.2 131.2 143.2 145 159.2 Probable compound Acetaldehyde Acetone Acetic acid Methyl acetate Ethyl acetate (E)-Hexenal Hexanal Methyl butyrate Heptanone Ethyl butyrate Ethyl methyl butyrate Furanone Ethyl hexanoate Ethyl methyl hexanoate Actual identity after validation Acetaldehyde Acetone Ethyl acetate Methyl acetate Ethyl acetate 3-Hexenal (Z) Hexanal Methyl butyrate Methyl propyl acetate Ethyl butyrate Methyl hexanoate Hexyl acetate Methyl propyl butyrate Ethyl methyl hexanoate

27

and Applied Chemistry (IUPAC) nomenclature, were used in the results. The prole of some of the strawberry avour compounds was altered signicantly by the freeze/thaw treatments, e.g. acetaldehyde (Fig. 2). The intensity (abundance) of the volatile content was particularly increased by 80 C and liquid nitrogen (IQF) treatments and frozen storage for one week and later thawing under ambient temperature.

Compounds 3-hexenal (Z) and hexanal (Figs. 3 and 4) were not altered by overnight storage in a refrigerator and thawing regimes but were altered signicantly by the freeze/thaw treatments over one week. Thawing of these fruit at ambient temperature caused an increase in the intensity of these two compounds. Most esters such as hexyl acetate (Fig. 5), ethyl methyl hexanoate (Fig. 6), methyl butyrate ethyl acetate, methyl acetate, methyl propyl acetate and methyl hexanoate were all not altered by overnight freeze/thaw treatments but were altered signicantly by freeze/storage for one week and subsequent thawing. Ethyl butyrate was not altered by any of the treatments. On the whole, week-long freeze/thaw treatments resulted in increased volatile content levels, much greater than the overnight freeze/thaw treatments. Strawberry fruit that were thawed at ambient temperature mostly had increased intensity levels of volatile compounds, e.g. acetaldehyde, 3-hexenal (Z), hexanal, ethyl acetate and methyl acetate compared to the rapid thawing treatment, but some, e.g. methyl hexanoate and hexyl acetate, were increased by thawing of fruit using the water bath. 4. Discussion The APCI-GPA allows simultaneous measurement of all avour volatile compounds within a short time on the basis of mass only. It is unable to differentiate between stereoisomers and positional isomers or compounds producing ions of the same mass (Brauss et al., 1998). Validation of the actual identities of the compounds by

Fig. 2. Intensity of acetaldehyde due to freeze/thaw treatments.

Fig. 3. Intensity of 3-hexenal (Z) due to freeze/thaw treatments.

28

D.M. Modise / Postharvest Biology and Technology 50 (2008) 2530

Fig. 4. Intensity of hexanal due to freeze/thaw treatments.

Fig. 5. Intensity of hexyl acetate due to freeze/thaw treatments.

a GCMS library enables conrmation of ions by correlation of t (Modise et al., 2004). This is because the GCMS library is able to separate and identify volatile compounds by structural data. The GCMS may, however, not be relied upon to differentiate between E and Z isomers. The major problem with this analytical method is that it is slow and may result in the degradation of avour compounds by other enzymes during the analysis (Buttery et al., 1988). Therefore, once actual identity of the compound has been deter-

mined using GCMS, APCI-GPA can be used since it allows rapid analysis of avour compounds. This procedure was followed in the present study prior to the measurement of changes in the volatile prole of the berries due to the various freeze/thaw treatments that were imposed. There was no loss of any identied volatile compounds in the berries that were stored overnight or over one week. The freeze/thaw treatments however did alter the abundance of some

Fig. 6. Intensity of ethyl methyl hexanoate due to freeze/thaw treatments.

D.M. Modise / Postharvest Biology and Technology 50 (2008) 2530

29

individual volatile compounds making up the headspace prole. The intensities of compounds such as 3-hexenal (Z), hexyl acetate, ethyl methyl hexanoate, methyl butyrate, ethyl acetate, methyl acetate, methyl propyl acetate and methyl hexanoate were altered signicantly by the week-long freeze/thaw treatments and not by overnight freeze/thaw treatments. This suggests that overnight freeze storage of strawberry fruit, i.e. in a conventional freezer, does not alter the volatile prole of the fruit but freeze/storage of the fruit over one week will cause a signicant change of the volatile prole of the fruit. Acetaldehyde was the only volatile compound that was altered by all treatments. This would suggest that this compound will be altered irrespective of any freeze/thaw treatment. Since acetaldehyde is associated with off-odours (Larsen and Watkins, 1995) and low temperature injury (Smagula and Bramlage, 1977), changes in this compound by treatments should be expected. Ueda and Iwata (1982) reported that aroma off-odours often accumulate and develop immediately following freezing and frozen storage. Deng et al. (1996) attributed off-avour development in frozen strawberries to the production of H2 S. They further hypothesised that H2 S evolution could be due to the decrease in the pH of the cytosol caused by the disruption of cells by freezing, resulting in the release of sulphide ions as H2 S. Hirvi (1983) determined that secondary volatile compounds formed by enzymaticoxidative cleavage of linoleic and linolenic acids tend to increase during freezing and thawing, resulting in off-avours. Week-long freezing generally increased levels of volatile compounds after thawing and the effect was less at 20 C. This could be attributed to possible formation of large ice crystals in a longer freeze period which in turn may cause more tissue disruption during thawing (Jeremiah, 1996). Ethyl butyrate was the only compound that was not affected by any of the treatments. The unknown compounds were identied as fragments of other compounds that are mostly esters that have broken up during the ionisation process. This shows that the freeze/thaw protocol described in this experiment would affect the volatile prole of these compounds. Some of the compounds perceived to be key volatile compounds of strawberries were therefore altered. Fischer and Hammerschmidt (1992) regarded 3-hexenal (Z) and furanone as some of the key strawberry components using Aroma Extraction Dilution Analysis (AEDA). In this investigation, it has been shown that hexanal is altered by all treatments except treatments 2 and 4. Furanone was not found in cv. Camarosa. Zabetakis and Holden (1997), Fischer and Hammerschmidt (1992) and several other researchers found that furanone was a major volatile compound that affects strawberry avour. Furaneol (2,5-dimethyl4-hydroxy-3(2H)-furanone) is not stable due to its water-soluble nature, therefore its extraction and recovery is very low (Larsen and Poll, 1992). Jetti et al. (2007) conceded that furaneol recovery and detection is affected by isolation and detection techniques since it is pH and temperature dependent. They were also unable to recover it in cv. Camarosa even though they used a different technique, SolidPhase Microextraction (SPME) gas chromatography. Nevertheless, other ndings, e.g. Dirinck et al. (1981) have shown that compounds formed from organic acids such as acetic, butanoic and hexanoic acids were the most important ones in cultivated strawberries. Interestingly, the intensity of volatile compounds in almost all the treatments was much greater after the one-week freeze/thaw treatments. Similarly, in a study of six strawberry cultivars, Larsen and Watkins (1995) showed that the contents of volatile compounds, linalool and methyl hexanoate were doubled after freezing and thawing. Signicant increases in the content of ethyl butanoate and ethyl hexanoate were also observed. Volatile compound values as expressed by odour threshold values (Rothe, 1975) using methods such as Charm Analysis or

Dilution Analysis (Fischer and Hammerschmidt, 1992; Larsen and Poll, 1992) and AEDA (Schieberle and Hoffman, 1997) would be invaluable in describing the odour units of the volatile compounds. However, these methods fall short of addressing the concept of synergism, antagonism and interactions of the volatile compounds. The altering of the volatile compounds in strawberries by freeze/thaw treatments when compared to fresh berries suggests that strawberries would best be analysed fresh and not frozen for later analysis, and that validation of compound identity by GCMS is necessary. Acknowledgements The author is indebted to Robert S. Linforth (University of Nottingham) and Richard Watson (Complete Medical Communications) for the technical support they provided for this study. References
Baldwin, A.E., Scott, J.W., Shewmaker, C.K., Schuch, W., 2000. Flavor trivia and tomato aroma: biochemistry and possible mechanisms for control of important aroma components. HortScience 35, 10131031. Brauss, M.S., Linforth, R.S.T., Taylor, A.J., 1998. Effect of variety, time of eating, and fruit-to-fruit variation on volatile release during eating of tomato fruits (Lycopersicon esculentum). J. Agric. Food Chem. 46, 22872292. Buttery, R.G., Teranishi, R., Ling, L.C., 1988. Fresh tomato aroma volatiles: a quantitative study. J. Agric. Food Chem. 35, 540544. Deng, H., Ueda, Y., Chachin, K., Yamanaka, H., 1996. Off-avour production in frozen strawberries. Postharvest Biol. Technol. 9, 3139. Dirinck, P.J., De Pooter, H.L., Willaert, G.A., Schamp, N.M., 1981. Flavor quality of cultivated strawberries: the role of the sulphur compounds. J. Agric. Food Chem. 29, 316321. Fischer, N., Hammerschmidt, F.J., 1992. A contribution to the analysis of fresh strawberry avour. Chem. Mikrobiol. Technol. Lebensm 14, 141148. Forney, C.F., Kalt, W., Jordan, M.A., 2000. The composition of strawberry aroma is inuenced by cultivar, maturity and storage. HortScience 35, 1022 1025. Grab, W., Gfeller, H., 1999. Flavorspace a technology for the measurement of fast dynamic changes of avor release during eating. In: Proceedings of the 9th Weurman Symposium, Freising, Germany. Hakala, M.A., Lapvetelainen, A.T., Kallio, H.P., 2002. Volatile compounds of selected strawberry varieties analyzed by purge-and-trap headspace GCMS. J. Agric. Food Chem. 50, 11331142. Hancock, J.F., 1999. Strawberries. CABI Publishing, London, UK. Hirvi, T., 1983. Mass fragmentographic and sensory analyses in the evaluation of the aroma of some strawberry varieties. Lebensm. Wiss. Technol. 16, 157161. Honkanen, E., Hirvi, T., 1990. The avour of berries. In: Morto, I.D., Macleods, A.J. (Eds.), Food Flavours. Part C. The Flavour of Fruits. Elsevier Science Publishers, The Netherlands, pp. 125193. Jeremiah, E.L. (Ed.), 1996. Freezing Effects on Food Quality. Marcel Dekker Inc., New York, pp. 183237. Jetti, R.R., Yang, E., Kurnianta, C., Finn, C., Qian, M.C., 2007. Quantication of selected aroma-active compounds in strawberrrys by headspace solid-phase microextraction gas chromatography and correlation with sensory descriptive analysis. J. Food Sci. 72, 487497. Kader, A.A., 1991. Quality and its maintenance in relation to the postharvest physiology of strawberry. In: Dale, A., Luby, J.J. (Eds.), The Strawberry into the 21st Century. Proceedings of the Third North American Strawberry Conference. Houston, Texas, 1416 February 1990. Timber Press, Oregon, pp. 145152. Larsen, M., Poll, L., 1992. Odour thresholds of some of some important aroma compounds in strawberries. Z. Lebensm Unters For. 195, 120123. Larsen, M., Watkins, C.B., 1995. Firmness and concentration of acetaldehyde, ethyl acetate and ethanol in strawberries stored in controlled and modied atmospheres. Postharvest Biol. Technol. 5, 3950. Modise, D.M., Wright, C.J., Watson, R., Linforth, R., Taylor, A.J., 2004. Flavour volatile compound analysis in strawberry (Fragaria ananassa Duch.) fruits: comparison of two mass spectrometer techniques for identifying volatile compounds. South Afr. J. Bot. 70, 306309. Moing, A., Renaud, C., Gaudillere, M., Raymond, P., Roudeillac, P., Denoyes-Rothan, B., 2001. Biochemical changes during fruit development of four strawberry cultivars. J. Am. Soc. Hort. Sci. 126, 394403. Pomper, K.W., Breen, P.J., 1997. Expansion and osmotic adjustment of strawberry fruit during water stress. J. Am. Soc. Hort. Sci. 122, 183189. Richardson, D.G., Kosikattrun, M., 1995. Off-avour development of apples, pears, berries, and plums under anaerobiosis and partial reversal in air. In: Fruit Flavors. Am. Chem. Soc, pp. 211222. Rothe, M., 1975. Aroma valuesa useful concept? In: Aroma Symposium of the Central Institute for Nutrition and Food Research. Zeist, The Netherlands.

30

D.M. Modise / Postharvest Biology and Technology 50 (2008) 2530 Ueda, Y., Iwata, T., 1982. Off-odor of strawberry by freezing. J. Jpn. Soc. Hort. Sci. 51, 219223. Wang, S., 1991. Freezing and thawing characteristics of cryogenically processed IQF strawberries. In: Dale, A., Luby, J.J. (Eds.), Strawberry into the 21st Century. Timber Press Inc., Portland, Oregon, pp. 177178. Watson, R., Wright, C.J., McBurney, T., Taylor, A.J., Linforth, R.S.T., 2002. Inuence of harvest date and light integral on the development of strawberry avour compounds. J. Exp. Bot. 53, 21212129. Zabetakis, I., Holden, M.A., 1997. Strawberry avour: analysis and biosynthesis. J. Sci. Food Agric. 74, 421434.

Schieberle, P., Hoffman, T., 1997. Evaluation of the character impact odorants in fresh strawberry juice by quantitative measurements and sensory studies on model mixtures. J. Agric. Food Chem. 45, 227232. Schreier, P., 1980. Quantitative composition of volatile constituents in cultivated strawberries, Fragaria ananassa cv. Senga sengana, Senga litessa and Senga gourmella. J. Sci. Food Agric. 31, 487494. Smagula, J.M., Bramlage, W.J., 1977. Acetaldehyde accumulation: is it a cause of physiological deterioration of fruits? HortScience 12, 200203. Tucker, G.A., 1993. Fruit ripening. In: Seymour, G., Taylor, J., Tucker, G. (Eds.), Biochemistry of Fruit Ripening. Chapman and Hall, London, pp. 120.