EFFECT OF 1-MCP PRETREATMENT, CA STORAGE AND SUBSEQUENT MARKETING TEMPERATURE ON VOLATILE PROFILE OF GALA APPLE

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1 Saltviet, M. E. and F. Mencarelli Inhibition of ethylene synthesis and action in ripening tomato fruit by ethanol vapors. J. Amer. Soc. Hort. Sci. 113: SAS SAS/Stat User s Guide, Version 8. SAS Institute Inc., Cary, NC. Sisler, E. C. and M. Serek Inhibitors of ethylene responses in plants at the receptor level: Recent developments. Physiol. Plant. 100: Tovar, B., H. S. García, and M. Mata Physiology of pre-cut mango. I. ACC and ACC oxidase activity of slices subjected to osmotic dehydration. Food Res. Intl. 34: Winnicuk, P. P. and M. E. Parish Minimum inhibitory concentrations of antimicrobials against micro-organisms related to citrus juice. Food Micro. 14: Yanuriati, A., G. P. Savage, and R. N. Rowe The effect of ethanol treatment on the metabolism, shelf life and quality of stored tomatoes at different maturities and temperatures. J. Sci. Food Agric. 79: Zhang, S. and J. M. Farber The effects of various disinfectants against Listeria monocytogenes on fresh-cut vegetables. Food Micro. 13: Proc. Fla. State Hort. Soc. 116: A REFEREED PAPER EFFECT OF 1-MCP PRETREATMENT, CA STORAGE AND SUBSEQUENT MARKETING TEMPERATURE ON VOLATILE PROFILE OF GALA APPLE JINHE BAI AND ELIZABETH A. BALDWIN 1 USDA/ARS Citrus & Subtropical Products Lab. 600 Avenue S, N.W. Winter Haven, FL JAMES P. MATTHEIS USDA/ARS, Tree Fruit Research Lab. Wenatchee, WA Additional index words. 1-methycyclopropene, alcohol, aroma, controlled atmosphere, ester, Malus xdomestica Abstract. Gala apples pretreated or non-treated with 1-methylcyclopropene (1-MCP, µl L -1 ) were stored under controlled atmosphere (CA, 2 kpa O kpa CO 2 ) or regular air (RA) for 6 months at 1 C. Aroma compounds (measured by gas chromatograph and electronic nose) were analyed every month directly and after transferring to 20 C for one week to simulate marketing conditions. The effect of 1-MCP was stronger than CA after transfer of fruit to room temperature. For electronic nose data, canonical discriminant analysis separated the storage treatments (1-MCP + CA, 1-MCP, CA, RA), indicating that the aroma profile was different in apples from each treatment. This was confirmed by GC analysis. Fruit lost most of the esters but not alcohols after transferring apples from 1 C to 20 C for all treatments. 1-MCP + CA inhibition of volatile production was greater, and did not show any benefit for maintaining firmness and acidity, compared to 1-MCP alone. Therefore, 1-MCP alone was the best treatment for storing Gala apples with minimal loss of volatiles, while maintaining firmness and acidity. Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. The U.S. Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation, and marital or family status. 1 Corresponding author; ebaldwin@citrus.usda.gov. To whom reprint requests should be addressed. Controlled atmosphere (CA) storage has been used for apple commercial storage for decades, and had been recognied as the second most practical and effective method, in addition to proper temperature management, for extending the shelf life of intact and fresh-cut fruit and vegetables (Schlimme and Rooney, 1994). Recommended CA conditions were given as 0 C with 1-3 kpa O 2 and 1-5 kpa CO 2, with about 50% of U.S. apple production being stored under CA conditions (Kader, 1989). One of the major effects of CA storage for prolonging shelf life of commodities is inhibition of ethylene production and decreased sensitivity when products are exposed to ethylene. Although most of the ethylene inhibitors, such as aminooxyacetic acid (AOA) and aminoethoxyvinylglycine (AVG), have been found effective for prolonging storage life of commodities, but they can be toxic to fruit tissue and expensive. Sisler and coworkers (1996, 1999) have discovered a very effective ethylene action inhibitor 1-methylcyclopropene (1-MCP) during their searches for the ethylene receptor in plants. 1-MCP can be synthesied easily (Magid et al., 1971), and has been recently approved by Environmental Protection Agency (2002) for postharvest use on apples. Researches using 1-MCP on apples showed positive results in inhibiting ripening and senescence through decreased ethylene production, respiration, softening, loss of titratable acidity, color change and other senescence-related metabolic events (Baritelle et al., 2001; Fan and Mattheis, 1999; Rupasinghe et al., 2000; Saftner et al., 2003; Song et al., 1997; Watkins et al., 2000). In this study we report on the effect of 1-MCP on volatile profile of Gala apple in comparison to apples stored in air or CA. Materials and Methods Gala apples (Malus domestica Borkh.) were harvested from a commercial orchards located in Wenatchee, Wash. on 4 Sept Defect-free fruit were randomly divided into two groups, one for 1-MCP treatment and one for a non-treated control. Pretreatment with 1-MCP was immediately per- 400 Proc. Fla. State Hort. Soc. 116: 2003.

2 formed after harvest at µl L -1 1-MCP (EthylBloc, Floralife, Inc., Waltersboro, SC) at 20 C for 18 h at the USDA Tree Fruit Research Laboratory in Wenatchee, Wash. The initial concentration of 1-MCP was determined by gas chromatography (GC) (Gong et al., 2002). Both the 1-MCP-treated and non-treated groups were further divided into two batches, one for CA storage and another for regular air (RA) storage at 1 C and 95% RH. The CA conditions were 2 kpa CO 2 and 2 kpa O 2. Immediately after 1-MCP treatment, and also after each month of storage, fruit were shipped by refrigerated truck to the USDA Citrus and Subtropical Products Laboratory in Winter Haven, Fla., overnight with separate boxes for the 1- MCP-treated and non-treated fruit. Every shipment included 40 fruit for each treatment, 20 for immediate analysis of quality attributes, and another 20 fruit were transferred to 20 C and held for a week at that temperature, to simulate the marketing conditions. Flesh firmness was measured on 20 individual fruit per treatment, with a penetrometer (FT 327, McCormick, Facchini, Alfonsine, Italy), equipped with a cylindrical plunger 11 mm in diameter. The measurement was obtained from equatorial area with the peel removed. Titratable acidity (TA), headspace GC volatile and electronic nose analysis were determined using 5 composite replicates of 4 fruit each. For TA content, 50 g of fruit tissue was blended with 50 ml deionied water at speed 4 for 45 s with a homogenier (Model PT 10/35, Brinkmann Instruments, Co., Switerland). The homogenate was vacuum-filtered through µm Büchner funnel. The filtrates were titrated to ph 8.1 with 0.1N NaOH, and the acidity was calculated as malic acid on weight basis. For headspace GC volatile and electronic nose analysis, 50-g apple slices were homogenied with 25 ml deionied water and 25 ml of a saturated NaCl solution. The homogenate was transferred to glass vials sealed with a crimp-topped Teflon-silicone septum, flash froen in liquid nitrogen and stored at -80 C prior to analysis. The sample prepared for headspace volatile analysis by GC and electronic nose consisted of 2 ml homogenate in a 6-mL vial, and 3 ml in a 10-mL vial, respectively. Sample vials were thawed under running tap water immediately prior to analysis. For headspace GC analysis, sample vials were incubated at 80 C for 15 min by a heating block (Perkin Elmer HS-6 headspace sampler, Norwalk, Conn.) before the headspace sample was injected into the GC. The analysis was carried out using a gas chromatograph (Perkin Elmer Model 8500, Norwalk, Conn.) equipped with a 0.53 mm 30 m polar Stabilwax capillary column (1.0-µm film thickness, Restek, Bellefonte, Pa.) and a flame ioniation detector (FID). Oven temperature was held 40 C for 6 min, then raised to 180 C at a rate of 6 C min -1. The compounds were identified by comparison of retention times with those of authenticated standards and by enrichment of apple homogenate with authentic compounds. For electronic nose analysis, a Fox 4000 system (Alpha MOS, Toulouse, France) was used, fitted with 18 metal oxide sensors, some with coated surfaces (SY/LG, SY/G, SY/AA, SY/Gh, SY/gCTl, SY/gCT, T30/1, P10/1, P10/2, P40/1, T70/2, PA2, P30/1, P40/2, P30/2, T40/2, T40/1, and TA2). The electrical output from the sensors was measured at 0.5 s intervals. Samples were incubated in an agitator at 500 rpm and 40 C for 2 min before the headspace sample (500 µl) was taken from the vial and injected into the electronic nose. The carrier gas was pure air with a flow rate of 150 ml min -1. The electronic nose data acquisition program was set as 2 min sampling time followed by an 18 min delay between samples for sensor recovery. Data were analyed using the general linear model (PROC GLM) and the canonical discriminant (PROC CAN- DISC) program, and mean separation was determined by the Scheffe s test (SAS Version 8, SAS Institute, Cary, N.C.). Results and Discussion Total volatile abundance in GC FID response in RA control fruit increased slightly in the first two months and then declined consistently throughout storage at 1 C (Fig. 1). The increase of volatiles indicates that the fruit continued ripening during storage, and the subsequent decrease indicates that the fruit was undergoing senescence. Mattheis et al. (1998) reported similar pattern of Gala volatile productions during storage. However, there were significant drops of total volatile abundance after transferring fruit from 1 C to 20 C for one week (Fig. 1). The average decrease in total volatiles, caused by the increased storage temperature compared to volatile levels averaged over 6 months storage at 1 C was 50% (Table 1) for RA control. The shorter the storage of fruit at 1 C, the greater decrease of total volatile abundance after transferring to 20 C (Fig. 1). Volatile accumulation, as measured in this research, depends on the production of volatiles by the fruit in relation to loss of volatiles due to off-gassing from the fruit. Since transferring apples from 1 C cold storage to 20 C room temperature usually increases metabolism and the production of volatiles, the decreased volatile levels at 20 C may have been due to increased evaporation. There was much less total volatile abundance in the fruit from treatments of 1-MCP and CA compared to that of control (RA-stored fruit, Fig. 1). The magnitude of inhibition of 1-MCP was similar to that of CA during storage at 1 C. Average total volatile abundance in apples from CA, and 1-MCP Fig. 1. Total volatile abundance (FID response peak height) of Gala apples pretreated or not with 1-MCP and stored in CA or RA at 1 C (Solid line), sub-samples were subsequently transferred to 20 C for an additional week (Dotted line). Proc. Fla. State Hort. Soc. 116:

3 Table 1. Relative amount (%) of major esters, alcohols and total volatile abundance of Gala apples after transferring from 1 C to 20 C for 1 wk compared to levels exhibited immediately after removal from cold (1 C) storage (as 100%). Volatiles were analyed every month directly from cold storage and after transferring to 20 C for 1 wk, within storage period of 6 months at 1 C. Averaged data over the 6 months were used.,y Component RA CA 1-MCP 1-MCP+CA Butyl acetate 27 ab** 48 a** 15 b** 31 ab** 2-methylbutyl acetate 57 a* 55 a** 14 b** 14 b** Hexyl acetate 19 b** 55 a* 10 c** 63 a* Butanol 119 a 102 a 21 b** 32 b** 2-methylbutanol 163 a** 163 a** 45 b** 62 b** Hexanol 173 a** 159 a** 49 b** 43 b** Total volatile abundance 50 a** 75 a* 16 b** 24 b** *p < 0.05, **p < 0.01: mean comparison with the amount of volatiles in fruit before transferring to 20 C. y Mean values (n = 30) in same row that are not followed by the same letter are significantly different (p < 0.05). treatments over the chilled storage period were 35% and 40% of that detected from RA fruit, respectively (Table 2). However, total volatile abundance in 1-MCP-pretreated fruit was lower than those stored in CA after transferring to 20 C (Fig. 1), being 16% compared to 75% remaining after transfer to 20 C, respectively (Table 1). Inhibition of total volatile abundance by the 1-MCP + CA combination treatment was the most severe compared to either treatment alone (Fig. 1, Tables 1 and 2). CA and modified atmosphere (MA) is reported to decrease volatile production in most fruits, including apple (Guadagni et al., 1971; Mattheis et al., 1998; Patterson et al., 1974; Saftner et al., 2003), because metabolic activity is reduced. Production of lipid-derived volatiles is inhibited by low O 2, while volatiles that arise from amino acid catabolism are negatively impacted by high CO 2 (Brackmann et al., 1993; Fellman et al., 2000). The mechanism of how 1-MCP inhibits volatile production is not yet clear, but in many cultivars of apple, 1-MCP inhibits production of volatiles (Fan and Mattheis, 1999, 2001; Lurie et al., 2002; Rupasinghe et al., 2000) perhaps through inhibition of ethylene production and related events. Aroma production during fruit ripening is considered to be an ethylene-mediated response (Abeles et al., 1992), as evidenced by reduced aroma in 1-MCP-treated plums, and by the fact that propylene application restored aroma (Abdi et al., 1998). Table 2. Relative amount (%) of major esters, alcohols and total volatile abundance of Gala apples from three treatments compared with that of the RA-stored control (as 100%) averaged over 1-6 months storage at 1 C.,y Component CA 1-MCP 1-MCP+CA Butyl acetate 29 a** 32 a** 12 b** 2-methylbutyl acetate 109 ab 129 a 89 b Hexyl acetate 33 a** 32 a** 13 b** Butanol 24 ab** 34 a** 16 b** 2-methylbutanol 77 a 102 a 89 a Hexanol 75 a 91 ab 123 b Total volatile abundance 35 a** 40 a** 21 a** *p < 0.05, **p < 0.01: mean comparison with the amount of volatiles in fruit from RA storage. y Mean values (n = 30) in same row that are not followed by the same letter are significantly different (p < 0.05). Fig. 2. Butyl acetate volatile abundance (FID response peak height) of Gala apples pretreated or not with 1-MCP and stored in CA or RA at 1 C (Solid line), sub-samples were subsequently transferred to 20 C for an additional In apple fruit, esters are the most significant contributors to aroma (Mattheis et al., 1998; Plotto et al., 2000; Ueda et al., 1993). Of these, three esters (butyl acetate, hexel acetate and 2-methylbutyl acetate) have been identified as characteristic aroma compounds of Gala apples (Mattheis et al., 1998; Plotto et al., 2000; Young et al., 1996). Normally butyl acetate is the most abundant ester in RA stored Gala apple. This volatile increased during first two months storage, then kept relatively consistent until a sharp drop was observed at the end of six months storage at 1 C (Fig. 2); at same time, the flesh firmness declined to 48 N (Table 3), which is considered below commercial acceptability (Bai et al., 2002a; Ueda et al., 1993). Similar to the total volatile abundance, transfer of fruit to 20 C caused butyl acetate to decline sharply to 27% of the amount present when the fruit were in cold storage (Fig. 2, Table 1). Butyl acetate levels in apples from CA, 1-MCP and 1- MCP + CA were 29%, 32% and 12% of the amount detected in RA fruit averaged over the 6 months cold storage, respectively (Table 2). Transferring the fruit to 20 C for a week caused a further decline (Fig. 2, Table 1) in butyl acetate levels. The effect of CA and/or 1-MCP treatments and subsequent storage temperature increase (from 1 to 20 C) on hexyl acetate was similar to butyl acetate (Tables 1 and 2). The inhibition of 1-MCP treatment on butyl acetate and hexyl acetate levels was stronger than that of CA after transferring the fruit from 1 C to 20 C storage. The strongest inhibition was Table 3. Firmness and titratable acidity content of Gala apples after 6 months storage at 1 C. Treatment Firmness (N) TA (%) Initial (before storage) 75 a 0.33 a RA 48 c 0.17 c CA 58 b 0.26 b 1-MCP 67 a 0.28 ab 1-MCP+CA 72 a 0.31 a Mean values in same column that are not followed by the same letter are significantly different (P < 0.05). 402 Proc. Fla. State Hort. Soc. 116: 2003.

4 Fig methylbutyl acetate volatile abundance (FID response peak height) of Gala apples pretreated or not with 1-MCP and stored in CA or RA at 1 C (Solid line), sub-samples were subsequently transferred to 20 C for an additional Fig methylbutanol volatile abundance (FID response peak height) of Gala apples pretreated or not with 1-MCP and stored in CA or RA at 1 C (Solid line), sub-samples were subsequently transferred to 20 C for an additional the 1-MCP + CA treatment combination at 1 C, with a relatively small loss after transferring the fruit to 20 C probably because the volatile levels were already minimum, and there was nothing left to lose (Fig. 2, Tables 1 and 2). CA and/or 1-MCP influence on 2-methylbutyl acetate showed a different pattern compared to butyl acetate and hexyl acetate. Sometimes 2-methylbutyl acetate was higher in fruit treated with CA and/or 1-MCP, and at other times, it was lower during storage at 1 C (Fig. 3). There was no difference between the average 2-methylbutyl acetate levels in RA fruit and the treated fruit at 1 C averaged over the cold storage period (Table 2), although transferring the fruit to 20 C resulted in a greater loss of 2-methylbutyl acetate in 1-MCP-treated fruit, with or without CA, than those in RA and CA alone (Fig. 3, Table 1). These results indicate that there was a difference in which 1-MCP and CA affected straight- and branch-chain esters. 2 kpa O kpa CO 2, the CA condition in this research, offered a low enough O 2 level to reduce lipid-derived esters (straight chain); but the CO 2 level was not high enough to reduce amino acid-derived (branch-chain) ester production (Brackmann et al., 1993). On the other hand, 1-MCP influenced ester production in a similar way to low O 2 at 1 C. Alcohols also play an important part in fruit aroma, as volatile compounds in their own right, and as substrates of ester volatiles. Figure 4 shows the changes of butanol concentra- Fig. 4. Butanol volatile abundance (FID response peak height) of Gala apples pretreated or not with 1-MCP and stored in CA or RA at 1 C (Solid line), sub-samples were subsequently transferred to 20 C for an additional Fig. 6. Plots of canonical discriminant analysis from GC volatile data. Gala apples were pretreated or not with 1-MCP and stored at CA or RA at 1 C for 6 months, and subsequently transferred to 20 C for 1 week. R = regular air: C = CA; M = 1-MCP; X = 1-MCP + CA. Proc. Fla. State Hort. Soc. 116:

5 Fig. 7. Plots of canonical discriminant analysis from electronic nose data. Gala apples were pretreated or not with 1-MCP and stored at CA or RA at 1 C for 6 months, and subsequently transferred to 20 C for 1 week. R = regular air: C = CA; M = 1-MCP; X = 1-MCP + CA. tion in fruit from the different treatments, stored at 1 C and subsequently transferred to 20 C. The inhibitory effect of CA and/or 1-MCP on butanol levels in the fruit at 1 C was similar to butyl acetate (Figs. 2 and 4, Table 2). Transferring fruit to 20 C for a week caused a further decrease in butanol levels in fruit treated with 1-MCP and 1-MCP+CA, as was the case for butyl acetate, but caused increases in the butanol amounts in RA and CA fruit (Fig. 4, Table 1). A similar patterns was observed for 2-methylbutanol and hexanol (Fig. 5, Table 1). These results indicate that production of alcohol volatiles in fruit treated without 1-MCP is increased by increasing the storage temperature. Alcohols have lower Henry s law coefficients than esters (Bai et al. 2002b), and enhanced alcohol production tends to lead to accumulation rather than evaporation. In contrast, esters tend to evaporate out of the fruit because of the much higher Henry s law coefficients for these compounds. Canonical discriminant analysis of headspace GC and electronic nose data separated the four treatments of RA, CA, 1-MCP and 1-MCP+CA (Figs. 6 and 7), indicating that the aroma profile was different in apples from each treatment. The differences in aroma profile increased with increasing storage time, and especially after transfer to increased storage temperature (20 C) for 1 week. Sensory analysis might provide further information, but 1-MCP had not yet been approved at the time of this experiment. Literature Cited Abdi, N., W. B. McGlasson, P. Holford, M. Williams, and Y. Mirahi Response of climacteric and suppressed-climacteric plums to treatment with propylene and 1-methylcylcopropene. 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