The Biosynthesis of Strawberry Flavor (II ... - Wiley Online Library

Concise Reviews in Food Science

JFS:

Concise Reviews in Food Science.

The Biosynthesis of Strawberry Flavor (II): Biosynthetic and Molecular Biology Studies K.G. BOOD AND I. ZABETAKIS

ABSTRACT: The biogenesis of strawberry flavor is a popular area of current research bringing together diverse areas of science, including flavor chemistry, biochemistry, molecular biology, and genetics. This review concentrates on the biosynthesis of three major flavor classes of compounds in strawberry, namely carbohydrates, esters, and furanones. They are qualitatively discussed with respect to their importance in fruit flavor, their biochemical formation, and the biochemical relationships between each other and fruit ripening. Recent genetic studies are also critically evaluated. These can provide an insight into the expression of bioinformation controlling the biogenesis of these flavor compounds through identifying and studying the expression of the corresponding controlling enzymes. Keywords: strawberry, flavor, biosynthesis, esters, furanones

Introduction

I

N THIS PAPER, AN OVERVIEW INTO THE CURRENT RESEARCH ON

the biosynthesis of aroma compounds contributing to the flavor of strawberry is given. The three main classes of compounds that are reviewed are carbohydrates, esters, and furanones. These three classes are discussed with respect to their importance and their bioformation. Strawberry flavor is extremely popular worldwide as part of the fruit or as a flavoring added in many manufactured foodstuffs. Therefore, studying the origin of strawberry flavor is a worthwhile and challenging area of food research. Strawberry flavor, being a very complex mixture of nonvolatile and volatile compounds, is one of the most complicated fruit flavors with about 350 components (Latrasse 1991). Flavor research has progressed from the qualitative and quantitative analysis of volatile compounds towards the sensory evaluation of the most important ones. Biosynthetic studies have been carried out on these most sensory active compounds. Recently, genetic studies on these important flavor components have also been carried out. This evolution of the flavor research is described in this review.

Carbohydrates Sugars are the most important nonvolatile flavor compounds in strawberry fruit, in terms of both quality and quantity. It is the interactions of the sugar compounds that have the greatest effect on the properties of the strawberry in terms of ripening, flavor development, and color. The sugars are important in balancing out the increasing levels of fruit acids associated with ripening (Montero and others 1996). The general increase in soluble sugar content throughout ripening is the main reason why strawberries are so pleasant to eat. Sugars act as a carbon source for the precursors of the aroma compounds and, as ripening proceeds, the increase in these soluble sugars results in an increase in the availability of precursors able to produce aroma compounds. The strawberry fruit has an initial phase of growth and enlargement, followed by a maturation phase, during which the fruit acquires the capacity to ripen (Manning 1993). Throughout these stages the appearance and flavor of the strawberry both change, and it is these changes in the fruit characteristics that provide clues toward detailing the 2

JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 1, 2002

flavor compounds. Table 1 indicates the major sensory and analytical changes that the strawberry goes through during ripening. During fruit ripening, major changes occur to the strawberry. Some of these are degradative, for example starch hydrolysis and cell wall degradation, and some are constructive, such as the formation of flavor volatiles and color compounds (Biale and Young 1981). Ripening consists of a complex set of physicochemical reactions that are genetically controlled (Manning 1993). The fact that the strawberry fruit is nonclimacteric (Given and others 1988) provides difficulty in its study as comparisons cannot easily be made with research into flavor volatiles from climacteric fruits. Ogiwara and others (1999) carried out a study into the soluble sugar content sampled at different maturation stages. They concluded that sucrose, fructose, and glucose were the major sugars, and that inositol and sorbitol were the minor carbohydrates in strawberries. While the composition of the sugars varied between varieties, the ratio of each sugar to total sugars was almost constant. This study can be compared to earlier studies stating that glucose, fructose, and sucrose in strawberries accounted for 99% of the total sugars in the ripe fruit (Makinen and Soderling 1980).

Table 1—Changes in strawberry characteristics during maturation ( – : not detected, + : detected) (Yamashita and others 1977) Days after Weight bloomof ing Fruit (g)

Color Intensity Absorbance, 520nm

Sweetness

5

0.37

0.038

–

10 20

0.91 2.50

0.038 0.040

– –

30

6.30

0.198

+

40

15.00

0.540

+

45

15.00

1.355

+

Flavor No characteristic strawberry flavor

Faint strawberry flavor Matured strawberry flavor

© 2002 Institute of Food Technologists

John and Yamaki (1994) discussed sugar distribution between cellular compartments at two maturation stages. This work provided an overall insight into the importance of sugar compartmentalization in the strawberry cells. This is important because the expansion of the strawberry fruit is determined by the turgor pressure that, in turn, is determined by solute concentration (for example, sugar content). If an understanding of sugar compartmentalization within the strawberry cells can be achieved, then it can be understood where other important flavor components are kept and how they interact with other flavor precursors or flavor compounds. A study of organic acids and soluble sugars was carried out by Montero and others (1996). This work provided more detail about the quantities of sugar compounds present in strawberry fruit and the changes that those sugar compounds go through during maturation. Figure 1 shows that the levels of soluble sugars (expressed as mg per gram of fresh weight) increase up until day 35 (day 28 in the case of sucrose) and then decrease. The increase in sugars can be attributable to the onset of ripening (Table 1). Glucose and fructose levels (reducing sugars) were found to increase until day 35 after fruit set, and then also start to decrease, which can be associated with the increase in weight due to ripening. Thereafter, the reduction in weight of the strawberry over time (in the later stages of ripening) may be accounted for by desiccation and fruit senescence.

Table 2—The free amino acids in strawberry var. Chandler during ripening in days after blooming (mg per 100g of fresh weight) (b stands for a value lower than 0.25mg/100g (Perez and others 1992) Amino Acid Asp Glu Asn Gln Ser Ala Pro Val Trp His

30 Days

36 Days

41 Days

46 Days

4.0 11.4 52.4 13.0 3.0 9.7 4.3 2.0 2.2 2.4

1.2 7.3 47.8 7.4 2.4 18.2 4.0 1.3 1.7 b

0.7 2.3 47.4 23.6 5.6 16.7 3.7 1.5 b 1.5

0.8 3.6 30.6 10.4 2.0 1.6 3.0 1.0 b 1.4

The action of ester production is through an enzyme called alcohol acyltransferase (AAT), which catalyzes the transfer of an acyl moiety of an acyl CoA onto a corresponding alcohol. Therefore, ester production is dependent on alcohol production. Many studies have been done that state the formation of volatile esters is a coenzyme-A-dependent reaction (Olias and others, 1995). Alcohol + Acyl CoA

Esters

AAT

—— Ɑ

Ester

Volatile esters are one of the largest and main groups of volatile compounds contributing to the overall flavor of strawberry. The most abundant esters identified by gas chromatography and mass spectrophotometry include methyl and ethyl butanoates, ethyl hexanoate, hexyl acetate, and trans-2-hexenyl acetate (Pyssalo and others 1979). There is a wide range of esters in the strawberry fruit that are found in cultivated strawberries; this range could be attributable to the lack of specificity of the enzymes involved in ester formation.

Esters are biosynthesized from branched chain amino acids (Wyllie and others 1996) (Figure 2). These branched chain amino acids are commonly studied because they can be identified with relative ease in later compounds using methods such as chiral analysis. The enzymes of importance in ester formation from branched chain amino acids are: ␣aminotransferase, ␣-ketoacid decarboxylase, ␣-keto dehydrogenase, alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT). However, the AAT and ADH enzymes are the most widely studied due to their high presence and activity in ripe fruit. The authors therefore concentrate here on the properties of the AAT and ADH enzymes. Evidence that amino acids are the precursors of esters can be provided by chiral analysis. For example, if the 2-methylbutyl moiety of the ester is derived stereospecifically from isoleucine, it will exhibit the S-configuration. Chiral analysis on 2-methylbutanoic acid from strawberries has shown an S:R ratio of 91.3:8.7; these data are consistent with the biosynthetic route of esters from specific amino acids. ADH is an enzyme responsible for the reduction of aldehydes to alcohols, and this enzyme has been measured in ripening fruit. ADH has been found to show marked selectivity towards aldehydes producing particular alcohols. In particular, crude enzyme extracts and tissue slices from strawberry fruits have shown that ADH reduces 3-methylbutanal more extensively than 2-methylbutanal. This can be explained if the biogenetic step where 2- and 3-methylbutyl skeletons are differentiated precedes the reduction step (Figure 2) (Wyllie and others 1996). Perez and others (1992) have stated that ethyl butanoate and ethyl hexanoate are the main two ester compounds in fully ripe fruit. The authors were concerned with the development of esters and the role that amino acids played as Figure 1—Changes in soluble sugars (sucrose, fructose, their precursors for the effects of flavor development and glucose) during the development and ripening of the strawberry fruit. FW: Fruit weight. (Montero and others through ripening. It was predicted that the free amino acid composition could explain the distribution of the different 1996). Vol. 67, Nr. 1, 2002—JOURNAL OF FOOD SCIENCE

3


Biosynthesis of strawberry flavor . . .


Biosynthesis of strawberry flavor . . . esters in strawberry aroma. The amino acids were individually quantified to understand their role in flavor formation (Perez and others 1992). Amino acid metabolism generates aliphatic and branchedchain alcohols, acids, carbonyls, and esters. The 10 amino acids quantitatively determined by HPLC are shown in Table 2, with asparagine, glutamine, and alanine being the most abundant compounds, but alanine exhibiting the most significant changes during ripening. Isoleucine, leucine, phenylalanine, and lysine were also found, but at levels of less than 0.25 mg/100 g fruit. It was observed that the concentration of alanine sharply increased from 30 to 36 days, stayed constant until day 41, and then decreased at the fully ripe stage. Ester biosynthesis is directly proportional to the concentration of alanine, and therefore ester biosynthesis is enhanced when alanine reaches its highest level. This study proves the importance of amino acids in the development of the variety of ester compounds in strawberry aroma. A study into the enzymatic aspects of flavor biogenesis, and in particular the properties of the AAT enzyme involved in the biosynthesis of volatile esters in strawberry fruits, was carried out by the same group (Perez and others 1993). AAT is proposed to be a membrane-bound enzyme with a molecular weight of 70000 Da and having optimum activity at 35 °C and pH 8.0. The AAT was shown to act on all alcohols that were added to the standard assay mixture, including primary and secondary, straight chain and branched, saturated and unsaturated. Strawberry AAT showed maximum activity with hexyl alcohol as the substrate, and the enzyme had a higher activity against straight chain alcohols (for example, amyl alcohol) than against branched chain alcohols (for example, isoamyl alcohol) of the same carbon number. Unsaturated C6-alcohol showed a low esterification rate. All the alcohols tested were present as acetate esters, and among all of the acetate esters formed by enzymic reactions, hexyl acetate (4.5%) was the most abundant in strawberry aroma, followed by butyl acetate (1.9%), and amyl and isoamyl acetate (1%). There is a clear correlation between ester composition and the available alcohols. Alcohols with an even carbon number such as butanol and hexanol were the most important for flavor development (Perez and others 1993). AAT is therefore a key enzyme in aroma production because, while it may show specificity, it will still react with a large group of alcohols. An important study into the changes in AAT during fruit development in 4 different strawberry varieties found that, in all strawberry varieties, the AAT specific activity increases through maturation. It was therefore deduced that high AAT activity results in higher ester production and, subsequently, fruits with enhanced aroma (Perez and others 1996a). Olias and others (1995) have also studied the substrate specificity of AAT and demonstrated that acetyl CoA and hexyl alcohol are the preferred substrates of the enzyme. These results are in good agreement with the previously described work on AAT by Perez and others (1993). From Figure 2, it can be concluded that the two main factors determining the volatile ester composition in the fruit are the availability of the substrates (acyl CoAs and alcohols) and the inherited properties of the AAT enzyme. The NAD-dependent and NADP-dependent ADH enzymes are important components of the in vivo biosynthetic pathway of strawberry flavor volatiles, and they have been characterized (Mitchell and Jelenkovic 1995). The NAD- and NADP-dependent ADH activities of strawberries were found to have broad substrate specificities including alcohols and 4


aldehydes responsible for strawberry aroma and flavor either directly or through their ester products. So, through the actions of these enzymes a wide range of esters is produced. It has been found that NAD-dependent activities are greatest against short-chain alcohols and NADP-dependent activities are most active against aromatic and terpene alcohols (Mitchell and Jelenkovic 1995).

Furanones The compound 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF) is quantitatively a minor constituent of the fruit flavor but, because it has such a low threshold value, it is highly influential on the overall flavor. The low flavor threshold value of DMHF in water is 4 × 10 –5 mg/kg, and this value itself implies that this molecule has a great impact on the flavor (Latrasse 1991). DMHF is present in 4 forms, DMHF-glucoside, mesifuran, DMHF-malonyl-glucoside, and as the free aglycone, DMHF (Figure 3). Each of these has its own particular flavor attribute that, when complemented with other compounds in the strawberry fruit, provides the overall distinctive flavor of the fruit. It is DMHF and mesifuran, however, that have been quoted by many authors as being the most significant volatiles or character impact compounds (Sanz and others 1995; Zabetakis and others 1999a). DMHF-glucoside and DMHF-malonyl-glucoside are not volatile compounds, but still influence the overall flavor significantly (Zabetakis and Holden 1995; Roscher and others 1996) . The Oso

Figure 2—Pathways leading to ester formation involved in flavor production from branched chain amino acids (Wyllie and others 1996).

Grande variety of strawberry has the highest aroma value out of the 4 varieties studied at all 4 stages of ripening. At the fully ripe stage, the aroma value is very large (3720) and this is accounted for by the high concentration of DMHF at levels of 37 ␮g/g (Perez and others 1996b). A new in vitro method of studying DMHF and its methyl ether derivative has been used to study their biosynthetic pathways (Perez and others 1999). This method is especially convenient for studying the ripening physiology of fruits, where they are incubated in a growth medium for a period of few days. Sucrose was found to be important in terms of fruit development and ripening, but not for furanone biosynthesis, indicating that the reducing sugars discussed earlier are more influential in flavor development. When the strawberries were analyzed for DMHF content, the levels of DMHF-glucoside were found to be higher than for DMHF. This can be justified, as DMHF-glucoside is known to be the stable form of DMHF. However, this contradicts the findings of the group’s earlier work (Perez and others 1996b). These precursor studies were aimed to elucidate biochemical pathways in furanone development. D-fructose showed an increase of 28.4% in total furanones with an increase for DMHF (42.6%) and DMHF-glucoside (26.3%). Therefore, Dfructose could be a precursor of DMHF. D-fructose-6-phosphate feeding produced a 125% increase in DMHF and the derivatives, suggesting that the sugar phosphates are closer precursors of furanones (Perez and others 1999). The problem with adding precursors in this way is that their presence cannot be accounted for before the reaction has started, and this can lead to inaccuracies in the results achieved. While they can be compared to control levels of DMHF derivatives, it cannot be said how that compound was formed. The fact that DMHF and DMHF-glucoside readily interconvert needs to be taken into account. General trends can be obtained, but quantifying results can be inaccurate. The in vitro method uses strawberries in the pink stage, which is obviously where major transformation of compounds is taking place with the onset of ripening, but 4 d is a short development time compared to up to 4 wk used in other ripening studies. Nevertheless, the high rate of synthesis observed in this study proves that it is a very effective method. So, while there is an understanding of the production of compounds over one maturation phase, to achieve a truer picture into the development of flavor compounds over the lifetime of the fruit, several studies would have to be carried out over different maturation phases. While the work of Perez and others (1999) has shown the development of the DMHF and derivatives throughout ripening, the results are merely quantitative. It can be seen that DMHF is only detected in the stage of white fruit development, and this provides the evidence that it is the precursor to the other derivatives. Other precursor feeding studies have used strawberry callus cultures. The analysis of DMHF and derivatives in strawberries was carried out using HPLC-UV (Sanz and others 1994) or HPLC with Photo Diode Array (PDA) detection (Zabetakis and Holden 1996). Table 3 summarizes the results obtained from these studies. This plant tissue culturing method for the study of DMHF and its derivatives has the advantage that their endogenous amounts in the tissue are zero. Whereas in the case of feeding experiments with unripe fruits, the endogenous amounts of metabolites are rather high compared to the levels of exogenously supplied precursors. In callus cultures, the metabolic pool of furanones can therefore be quantified via

— Levels of DMHF, DMHF-glucoside and mesifuran Table 3— in control and precursor fed cultures Precursor Compound ConIdentified trola DMHF n.d. DMHFn.d. Glucoside Mesifuran n.d.

1,2propanediolb

6-deoxy D-fructosec

n.d. n.d. 0.94⫾0.03 1.03⫾0.22 n.d.

n.d.

Lactaldehyded

Methylobacterium Extorquens e

26.6⫾1.44 0.30⫾0.03

5.86⫾0.54 n.d.

n.d.

11.35⫾0.57

Levels mg per g of fresh weight callus; mean of 3 analyses + S.D. n.d. = not detected a,bZabetakis and Gramshaw 1997 c Zabetakis and Holden 1996 d Zabetakis and others 1999b e Zabetakis 1997

HPLC-UV or HPLC-PDA, and derived values are directly accountable to the exogenously applied precursors. In the studies of Roscher and others (1996), in vitro methods were used for analyzing the development of flavor compounds (Perkins-Veazie and Huber 1992) and radiolabeled precursors to identify biochemical pathways helping in the elucidation of compound formation. Indeed, this was the first group to study the malonyl glucoside derivative of DMHF in detail (Roscher and others 1996). Proof that DMHF-malonyl-glucoside is derived from malonic acid, glucose, and DMHF comes from their presence in an HPLC fractionation of a glycosidic extract of strawberry, as well as from the presence of DMHF-glucoside in this fraction (Roscher and others 1996). This is important in proving that the biosynthesis of this compound proceeds through esterification. This is a static study concerning the malonyl derivative, and it does not give any indication into its presence throughout ripening. However, it is known that DMHF and DMHF-glucoside levels increase throughout ripening (Perez and others 1996b) and also that sugars and acids are generated throughout fruit ripening (Montero and others 1996). So the production of this malonyl derivative is likely to occur in the later stages of fruit development. Radiotracer studies have proved that strawberries are able to assimilate and convert DMHF into mesifuran and DMHF-glucosides (Roscher and others 1997) and that Dfructose-6-phosphate is the closest precursor to DMHF. In support of the findings of Perez and others (1996b), DMHF would be converted to its derivatives with the onset of ripening and, in its later stages; the furanone would be converted into mesifuran and DMHF-glucoside (Roscher and others 1997). Studies with callus cultures have also shown that the preferred storage metabolite is indeed DMHF-glucoside and not DMHF. When 6-deoxy-D-fructose was supplemented to

Figure 3—The chemical structures of DMHF (1), mesifuran (2), DMHF-glucoside (3) and DMHF-malonyl glucoside (4). Vol. 67, Nr. 1, 2002—JOURNAL OF FOOD SCIENCE

5




Biosynthesis of strawberry flavor . . . strawberry callus cultures, DMHF-glucoside was formed (Zabetakis and Holden 1996). Mesifuran was proved to derive from DMHF (for the furanone structure) with S-adenosyl-L-methionine (SAM) providing the methoxy group (Roscher and others 1997). This was accounted for by a 0.4% radioactivity incorporated into mesifuran from applied radiolabelled SAM, and no incorporation into DMHF, proving that SAM supplied the methoxy group to mesifuran alone. Radiolabeled DMHF showed an incorporation rate into DMHF-glucoside of 0.7%, proving that DMHF is the precursor to the glucoside in accordance with the work of Perez and others (1996b). Further information regarding the precursors of DMHF was obtained in further radiolabeling studies which showed that the hexose sugars of radiolabeled fucose and rhamnose (isomers of 6-deoxy-fructose) were not incorporated into DMHF or one of its derivatives (Roscher and others 1998). Labeled lactaldehyde was not detected in any of the furanone compounds, therefore disproving the role of lactaldehyde condensing with DHAP to form 6-deoxy-D-fructose as a precursor to DMHF and derivatives (Zabetakis and others 1999b). However, it is not clear yet how the furanone ring is biosynthesized from either D-fructose or 6-deoxy-D-fructose or their phosphate derivatives and which enzymatic activities are involved in these steps. The radiolabeled C6-carbohydrates were incorporated into DMHF and derivatives, but phosphate homologues were incorporated at a higher rate. D-fructose 1,6-bisphosphate was found to show the highest incorporation into the DMHF derivatives, which suggests that it could be a closer precursor of DMHF and derivatives (Roscher and others 1998). This contradicts the results where unlabeled D-fructose 1,6-bisphosphate did not have any effect on DMHF production (Perez and others 1999). Interestingly, respiration inhibitors were found to increase the rate of furanone synthesis and, because respiration continues throughout the ripening of the fruit (Perkins-Veazie and Huber 1992), there may be competition from furanone biosynthesis pathways as against glycolysis and TCA cycles for these compounds. From the work of Roscher and others (1998), D-fructose 1,6 bisphosphate is claimed to be the closer precursor of DMHF, but transformation of a carbon chain of six 13C atoms directly into the furanone structure proves that D-fructose is not cleaved to two C3 units before incorporation into DMHF (Schwab 1998). Therefore, it is the whole bisphosphate ring that forms the structure of the furanone. While mass spectral data proves this biochemical transformation, it is a more favorable conformation as a bisphosphate molecule rather than as two C3 molecules following a biological Maillard reaction (Schwab 1998). The suggested biosynthetic pathway to DMHF and its derivatives is shown in Figure 4.

developmental changes in mRNA levels occurring throughout ripening. Differences in mRNA populations were examined between unripe and ripe fruit (Wilkinson and others 1995). The identification and initial characterization of 5 differentially expressed mRNAs was carried out. Of the 5 mRNAs expressed in this study, only 3 were related to actual proteins and, as they were all expressed at later stages of fruit development, they were believed to be influential in the later stages of fruit ripening. One was identified as a member of the annexin family of proteins. This could be important because the annexins, being calcium-dependent phospholipid-binding proteins, could account for changes to the cell wall; that is, the loss of firmness through disassembly of cytoskeletal arrangements. The presence of annexins could also account for the loss of pectic stabilization, therefore accounting for the degree of esterification contributing to fruit

Molecular biology studies General. Manning (1994) investigated the qualitative changes of mRNA throughout fruit ripening. This study provided evidence that mRNA changes throughout fruit ripening, some mRNA being only expressed at early stages in fruit development and some only expressed at late stages. The expression of more mRNAs throughout fruit development could be attributable to the greater number of reactions that occur as part of secondary metabolism later on in fruit development. In a development from the work of Manning (1994), strawberry fruits were used as model systems to study the 6


Figure 4—Suggested biosynthetic pathway of DMHF and derivatives (Roscher and others 1998).

softening. A second mRNA was determined to encode a ribosomal protein that was already present in ripening fruit but increased to its highest levels in fully ripe fruits. This could suggest that developing berries contain a very metabolically active tissue compared to the vegetative tissue. As it is known that flavor and color production are confined to the fruit receptacle (Perez and others 1992), this provides evidence that ripening is controlled primarily by reactions taking place in the receptacle. Protein synthesis continues throughout fruit ripening (Manning 1993); therefore a greater demand for ribosomal activity could be associated with the late increase in cellular respiration observed at the full to overripe stage of berry ripening. Medina-Escobar and others (1997) used a powerful technique combining the previous differential screening technique with Southern blot screening by means of Polymerase Chain Reaction (PCR). The method of the modified MagnetAssisted Subtraction Technique (MAST) was used to search for cDNAs differentially expressed during the process. This specific method could elucidate the molecular mechanisms underlying strawberry fruit ripening. But, while a very good, quick, and sensitive technique is discussed, there is little information regarding the specific genes identified and how these genes control the biosynthesis of flavor in strawberries. A second study by Manning (1998) into ripening related genes aimed to give more detail into the possible function of isolated genes in relation to fruit metabolism and quality traits. A high quality cDNA library from ripe strawberries was produced and 26 families of cDNAs were identified through Northern analysis. All of the clones were identified as ripening enhanced, and were involved with key metabolic processes such as cell wall degradation. From 26 ripeningrelated cDNAs described, the only one of flavor relevance was that of the Acyl Carrier Protein (ACP). The presence of this enzyme is important in the production of esters from alcohols and aldehydes in the fruit; it has therefore important implications in flavor development. The expression of protein at later stages of fruit ripening can be linked to the higher levels of esters in developed fruit. Nam and others (1999) specifically isolated 9 cDNA clones from wild strawberry that were all related to the accumulation of pigments and sugars. Eight out of the 9 clones that hybridized to mRNAs were all induced over ripening of the fruit. However, only 1 clone was predicted to encode the Acyl Carrier Protein (ACP). This proves that the onset of ripening induces specific proteins responsible for flavor formation (Manning 1998). These proteins produce flavors through secondary metabolism later on in the development of the fruit. These genetic studies are important as they attempt to deal with the complex problem of fruit ripening. Indeed, a cDNA library constructed from isolated DNA will identify cDNA clones that can be studied as homologues to actual proteins. These cDNA clones will help in the understanding of how specific proteins are expressed throughout the ripening of the fruit. If cDNA clones can be identified that are homologues to proteins involved in flavor formation, then there is the possibility for genetic engineering to overexpress those particular genes in order to obtain a product with optimum flavor. While the authors discuss specific clones identified, there are only few clones relevant to discussions regarding the ripening of fruit and even less regarding the production of flavor compounds. However, the work of Nam and others (1999) is important as it provides an understanding into some of the developments that occur during ripen-

ing on a molecular level, and how those techniques could be manipulated for specific genes, especially those specific for flavor. Esters. A molecular biology approach to studying ester compounds, taking the form of a genetic study, investigated the Strawberry AAT gene (SAAT) (Aharoni and others 2000). This study supports the chemical studies of Perez and others (1996a). A cDNA clone isolated from strawberry fruit has shown sequence similarity to genes expressing the acyltransferase activity, and is therefore used in this study to test the enzyme against a variety of different substrates determining its expression. SAAT was exclusively expressed in the receptacle tissue, and there was a 16-fold greater degree of expression during the red (ripe) stage of the fruit development than during the green (unripe) stage. GC-MS analysis of the strawberry fruits showed that the first detectable sign of release of volatile esters is during the pink stage and the greatest amounts are in the red stage. Substrate specificity of the SAAT gene was assessed using a range of alcohols and acyl CoAs, and the volatiles were analyzed using radioactivity detection-gas chromatography and GC-MS. In support of earlier work on acyl CoA which is the preferred precursor (Perez and others 1996a), the enzyme was found to accept a broad range of alcohols as substrates. Enzyme activity was seen to increase with an increase in carbon chain length, up to octanol, after which the activity decreased (Aharoni and others 2000). The above results correlate well with the work of Perez and others (1996a) and provide supporting evidence for the absence of esters at early stages of fruit ripening due to a lack of ester-producing enzymes. This work also provides more evidence that major volatiles emitted in the later stages of fruit ripening are genuine products of the recombinant SAAT enzyme. The presence and activity of this enzyme could explain how such a large number of different compounds are produced in a fruit with a relatively low metabolic energy. It is more energetically efficient to have a few enzymes responsible for producing a large number of different volatiles through a pathway such as this, rather than a larger number of different pathways all leading to different compounds. However, this wide range of esters originated from the same pathway could be regarded as an “accident” of nature that we happen to find attractive. Because of rather wide substrate specificity of this enzyme, a range of esters with attractive flavor is biosynthesized.

Conclusions

T

ECHNIQUES USING RADIOLABELED COMPOUNDS AND PRE-

cursor studies are important tools in providing information regarding potential biosynthetic pathways leading to flavor formation. In the next steps, biochemical techniques have provided information on the enzymes involved in these pathways. Once these enzymes were characterized, molecular biological techniques have been used to clone these enzymes. These studies have provided valuable information on how the genes involved in the biosynthesis of flavor are expressed during ripening, and whether it is feasible to overexpress these genes in order to maximize flavor production.

References Aharoni A, Keizer LCP, Bouwmeester HJ, Sun Z, Alvvarez-Huerta M, Verhoeven HA, Blass J, Van Houwelingen AMML, De Vos RCH, Van der Voet H, Jansen RC, Guis M, Mol J, Davis RW, Schena M, Van Tunen AJ, O’Connell AP. 2000. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 12:647-661. Biale JB, Young RA. 1981. Respiration and ripening in fruit: Retrospect and prospect. In: Friend J, Rhodes MJC, editors. Recent advances in the biochemistry of

Vol. 67, Nr. 1, 2002—JOURNAL OF FOOD SCIENCE

7




Biosynthesis of strawberry flavor . . . fruits and vegetable. London: Academic Press. P 1-39. Given NK, Venis MA, Grierson D. 1988. Hormonal regulation of ripening in the strawberry, a nonclimacteric fruit. Planta 174:402-406. John OA, Yamaki S. 1994. Sugar content, compartmentation, and efflux in strawberry tissue. J Amer Soc Hort Sci 119:1024-1028. Latrasse A. 1991. Fruits III. In: Maarse H, editor. Volatile compounds in food and beverages. New York: Marcel Dekker. P 329-387. Makinen KK, Soderling E. 1980. A quantitative study of mannitol, sorbitol, xylitol, and xylose in wild berries and commercial fruits. J Food Sci 45:367-371. Manning K. 1993. Soft fruit. In: Seymour GB, Taylor JE, Tucker GA, editors. Biochemistry of fruit ripening. London: Chapman and Hall. P 347-377. Manning K. 1994. Changes in gene expression during strawberry fruit ripening and their regulation by auxin. Planta 194:62-68. Manning K. 1998. Isolation of a set of ripening-related genes from strawberry: Their identification and possible relationship to fruit quality traits. Planta 205:622-631. Medina-Escobar N, Cardenas J, Valpuesta V, Munoz-Blanco J, Caballero JL. 1997. Cloning and characterization of cDNAs from genes differentially expressed during the strawberry fruit ripening process by a MAST-PCR-SBDS method. Anal Biochem 48:288-296. Mitchell WC, Jelenkovic G. 1995. Characterizing NAD- and NADP- dependent alcohol dehydrogenase enzymes of strawberries. J Amer Soc Hort Sci 120:798801. Montero TL, Molla EM, Esteban RM, Lopez-Andreu FJ. 1996. Quality attributes of strawberry during ripening. Sci Hort 65:239-250. Nam YW, Tichit L, Leperlier M, Cuerq B, Marty I, Lelievre JM. 1999. Isolation and characterization of mRNAs differentially expressed during ripening of wild strawberry (Fragaria vesca L.) fruits. Plant Mol Biol 39:629-636. Ogiwara I, Miyamoto R, Babutsu S, Suzuki M, Hakoda N, Shimura PY. 1999. Variation in sugar content in fruit of four strawberry cultivars grown in the field and under forced culture, harvest years, and maturation stages. J Jap Soc Hort Sci 67:400-405. Olias JM, Sanz C, Rios JJ, Perez AG. 1995. Substrate specificity of alcohol acyltransferase from strawberry and banana fruit. In: Rouseff RL, editor. Fruit flavors. Washington: American Chemical Society. P 134-139. Perez AG, Rios JJ, Sanz C, Olias JM. 1992. Aroma components and free amino acids in strawberry variety Chandler during ripening. J Agric Food Chem 40:2232-2235. Perez AG, Sanz C, Olias JM. 1993. Partial purification and some properties of alcohol acyltransferase from strawberry fruits. J Agric Food Chem 41:14621466. Perez AG, Sanz C, Olias R, Rios JJ, Olias JM. 1996a. Evolution of strawberry alcohol acyltransferase activity during fruit development and storage. J Agric Food Chem 44:3286-3290. Perez AG, Olias R, Sanz C, Olias JM. 1996b. Furanones in strawberries: Evolution during ripening and postharvest shelf life. J Agric Food Chem 44:3620-3624. Perez AG, Olias R, Olias JM, Sanz C. 1999. Biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone and derivatives in vitro-grown strawberries. J Agric Food Chem 47:655-658. Perkins-Veazie PM, Huber DJ. 1992. Development and evaluation of an in vitro system to study strawberry fruit development. J Exp Bot 43:495-501. Pyssalo T, Honkanen E, Hirvi T. 1979. Volatiles of wild strawberries, Fragaria vesca L., compared to those of cultivated berries, Fragaria x ananassa cv. Senga Sengana. J Agric Food Chem 27:19-22. Roscher R, Steffen JP, Herderich M, Schreier P, Schwab W. 1996. 2,5-Dimethyl-4-

8


hydroxy-3(2H)-furanone 6’O-malonyl-b-D glucopyranoside in strawberry fruits. Phytochem 43:155-159. Roscher R, Schreier P, Schwab W. 1997. Metabolism of 2,5-dimethyl-4-hydroxy3(2H)furanone in detached ripening strawberry fruits. J Agric Food Chem 45:3202-3205. Roscher R, Bringmann G, Schreier P, Schwab W. 1998. Radiotracer studies on the formation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in detached ripening strawberry fruits. J Agric Food Chem 46:1488-1493. Sanz C, Perez AG, Richardson DG. 1994. Simultaneous HPLC determination of 2,5 dimethyl-4-hydroxy-3(2H)-furanone and related flavor compounds in strawberries. J Food Sci 59:139-141. Sanz C, Richardson DG, Perez AG. 1995. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone and derivatives in strawberries during ripening. In: Rouseff RL, editor. Fruit flavors. Washington: American Chemical Society. P 268-275. Schwab W. 1998. Application of stable isotope ratio analysis explaining the bioinformation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone in plants by a biological maillard reaction. J Agric Food Chem 46:2266-2269. Wilkinson J, Lanahan MB, Conner TW, Klee HJ. 1995. Identification of mRNAs with enhanced expression in ripening strawberry fruit using polymerase chain reaction differential display. Plant Mol Biol 27:1097-1108. Wyllie SG, Leach DN, Nonhebel HN, Lusunzi I. 1996. Biochemical pathways for the formation of esters in ripening fruits. In: Taylor AJ, Motrram DS, editors. Flavor science: Recent developments. Cambridge: Royal Society of Chemistry. P 52-57. Yamashita I, Lino K, Nemoto Y, Yoshikawa S. 1977. Studies on flavor development in Strawberries: Biosynthesis of volatile alcohol and esters from aldehyde during ripening. J Agric Food Chem 25:1165-1167. Zabetakis I, Holden MA. 1995. A study of strawberry flavor biosynthesis. In: Etievant P, Schreier P, editors. Bioflavour 95:Analysis-precursor studies-biotechnology. Paris: INRA Editions. P 211-216. Zabetakis I, Holden MA. 1996. The effect of 6-deoxy-D-fructose on flavor bioinformation from strawberry (Fragaria x ananassa, cv. Elsanta) callus cultures. Plant Cell Tiss Org Cult 45:25-29. Zabetakis I. 1997. Enhancement of flavor biosynthesis from strawberry (Fragaria x ananassa) callus cultures by Methylobacterium species. Plant Cell Tiss Org Cult 50: 179-183. Zabetakis I, Gramshaw JW. 1997. 1,2-Propanediol in strawberries and its role as a flavor precursor. Food Chem 61:351-354. Zabetakis I, Holden MA. 1997. Strawberry flavor: analysis and biosynthesis. J Sci Food Agric 74:421-434. Zabetakis I, Gramshaw J W, Robinson D S. 1999a. 2,5-Dimethyl-4-hydroxy-2Hfurano-3 one and its derivatives: Analysis, synthesis and biosynthesis¾A review. Food Chem 65:139-151. Zabetakis I, Moutevilis-Minakakis P, Gramshaw JW.1999b. The role of 2 hydroxypropanol in the biosynthesis of 2,5-dimethyl-4-hydroxy-2H-furan-3-one in strawberry (Fragaria x ananassa, cv Elsanta) callus cultures. Food Chem 64:311314. MS 20010298 Submitted 6/12/01, Revised 8/24/01, Accepted 8/27/01, Received 8/28/01

Author Bood is with the Procter Dept. of Food Science, Univ. of Leeds, Leeds LS2 9JT, U.K., and author Zabetakis is located at 6, Stratigou Makriyanni St., GR-14342 Nea Filadelfia, Athens, Greece. Direct inquiries to author Zabetakis (E-mail: [email protected]).