Formation of Volatile Sulfur Compounds in Broccoli Stored under

Transcription

1 J. Japan. Soc. Hort. Sci. 65 (4) : Formation of Volatile Sulfur Compounds in Broccoli Stored under Anaerobic Condition Kazuhiro Dan1, Setsuko Todoriki2, Masayasu Nagata1 and Ichiji Yamashita 1 National Research Institute of Vegetables, Ornamental Plants and Tea, Ministry of Agriculture, Forestry and Fisheries, Ano, Are, Mie National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Kannondai, Tsukuba, Ibaraki 305 Summary The development of an off-flavor in anaerobically kept broccoli was studied by sealing the heads in a 100-ƒÊm-thick polyethylene film and storing them at 20 Ž for 4 days. O2 and CO2 concentrations in the package were less than 0.5% and more than 20%, respectively, within 8 hours after storage; undesirable odors developed rapidly under these anaerobic conditions. Ethanol, acetaldehyde, methanethiol, and dimethyl disulfide were identified as the volatile compounds in the headspace of the anaerobic package. Volatile sulfur compounds, such as methanethiol and dimethyl disulfide, are the main contributors to an off-odor. When the activity of C-S lyase, a key enzyme for the formation of volatile sulfur compounds, was measured, no significant changes were observed in C-S lyase activity between an anaerobic packaged and nonpackaged broccoli during storage. The electrolyte leakage from an anaerobically packaged broccoli increased to a greater extent than that of nonpackaged broccoli during storage. In addition, the level of free fatty acid in the microsomal membrane was higher in an anaerobically packaged broccoli than in the nonpackaged broccoli. These results suggest that, under anaerobic condition, the volatile sulfur compounds are formed by the deterioration of cellular membrane lipids and loss of intracellular compartmentation, allowing the enzyme-substrate reaction to proceed. Introduction Atmosphere modification, by lowering O2 and/or increasing CO2 concentrations, maintains the quality of many fresh fruits and vegetables and extends their storage life (Isenberg, 1979; Kader, 1986; Kader et al., 1989; Smock, 1979; Zagory and Kader, 1988). Packaging with polymeric films of specific gas permeabilities creates a beneficial modified atmosphere (MA) which extends the storage life of various fruits and vegetables (Kader et al., 1989; Zagory and Kader, 1988). The composition of the atmosphere in the package depends on the respiration rate of the commodity and the gas permeation rate of the film. However, exposure of fresh commodities to levels below their O2 tolerance limit or exposure to levels above their CO2 tolerance limit or both, increase anaerobic respiration and the development Received; March Accepted; July 17, of an off-odor (Kader, 1986). When the respiration rate increases because of an increased holding temperature, O2 concentration decreases and CO2 concentration increases in the MA package to levels that result in the development of off-odors unless the gas permeabilities of the film is sufficient (Kader, 1986). Broccoli is a highly perishable vegetable. Packaging broccoli by using polymeric films has retarded the deterioration and enhanced the retention of nutrients and color (Barth et al., 1993a; Barth et al., 1993b). Atmospheric concentrations of 2.5% O2 and 6% CO2 maintained the visual quality of broccoli beyond 4 weeks of storage without risking the development of unpleasant odors and disorders (Makhlouf et al., 1989). An unpleasant odor occurred in broccoli stored under less than 0.5% O2 or more than 10% CO2 (Kasmire et al., 1974). It is a factor in causing marketing losses in broccoli, but the mechanism of odor formation under anaerobic condition is not yet 867

2 868 K. Dan, S. Todoriki, M. Nagata and I. Yamashita clear. The objective of our present study is to propose the mechanism by which an undesirable odor forms in broccoli when stored under anaerobic conditions. Materials and Methods Plant materials and packaging Broccoli (Brassica oleracea L. var. italica cv. Haitsu) was grown in the field under normal cultural practices. Broccoli heads were harvested, selected for uniformity, and stored under anaerobic conditions. A head (350 to 400 g) was placed in a 100-ƒÊm-thick low density polyethylene bag (30 cm x 30 cm), sealed with heat sealer, and held at 20 Ž for 4 days. Nonpackaged heads (control) were also stored at the same temperature for 4 days. Relative humidity was maintained near 100% by humidifiers in a storage room. To determine volatile compounds from broccoli flower buds, 20 g of flower buds was sealed in 300-ml glass bottle and held at 20 Ž for 48 hr. Head space analysis 02 and CO2 concentrations in the package and in the sealed glass bottle were analyzed with a gas chromatograph (GC-14B, Shimadzu Co. Ltd.) with a 1.8 m WG-100 column (GL Science Co.) and a thermal conductivity detector (TCD). The column temperature was 80 Ž and the temperatures of the injector and detector were 100 Ž and 120 Ž, respectively. Volatile compounds of the headspace in the package and sealed glass bottle were identified with a gas chromatograph (GC-17A, Shimadzu Co. Ltd.) equipped with a flame ionization detector (FID) or with a flame photometric detector (FPD). The gas samples were injected into a 60 m x 0.53 mm (i. d.) DB-WAX column (J&W Scientific) with a helium carrier flow of 5 ml/min. The column temperature was 50 Ž for 10 min, increased to 150 Ž at 3 Ž/min, and held at 150 Ž for 10 min. Injector and detector temperatures were 220 Volatile compounds in the headspace were identified by their GC retention time and GC/MS spectra. GCMS-QP1100EX system (Shimadzu) was equipped with a 60 m ~ 0.25 mm (i. d.) DB-WAX column (J&W Scientific). The column was held at 50 Ž for 10 min, increased to 150 Ž at 3 Ž/min, and held at 150 Ž for 10 min. Mass spectra were obtained by electron ionization at 70 ev. The ion source temperature was 250 Ž. C-S lyase assay Flower buds (1 g) were homogenized in a chilled mortar and pestle in 3 ml of cold 0.1 M phosphate buffer (ph 7.0), containing 3% (w/v) polyvinylpolypyrrolidone (PVP). The homogenate was filtered through 4 layers of gauze and centrifuged at 10,000 ~ g for 20 min at 4 Ž. The supernatant was assayed for C-S lyase activity as described by Hamamoto and Mazelis (1986). Proteins were determined by the method of Bradford (1976), using BSA as standard. Electrolyte leakage Flower buds were cut lengthwise into two division with a razor. One gram of broccoli flower buds were shaken for 1 hr in a 100-ml flask containing 50 ml of deionized water at 25 Ž. Electrical conductivity of the sample medium was measured using Conductivity Meter (AOL-10, DKK Co.). Total electrolyte leakage was obtained by boiling samples for 5 min. Results were expressed as percentage of total electrolytes. Isolation of microsomal membrane A 20-g sample of flower buds was homogenized with a chilled mortar and pestle in 60 ml of grinding buffer, containing 0.25 M mannitol, 25 mm HEPES-Tris (ph 7.6), 5 mm EGTA, 5 mm EDTA, 5 mm DTT, 1% (w/v) PVP, and 0.001% BHT. The homogenate was filtered through four layers of gauze and centrifuged at 10,000 ~ g for 20 min. The supernatant was centrifuged at 100,000 x g for 60 min. The pellet was used as a source of microsomal membranes. The microsomal pellet was resuspended in 4 ml of deionized water, and the aliquot suspension was immediately used for lipid extraction. Extraction and analysis of lipids Lipids were extracted from microsomal membranes by the method of Bligh and Dyer (1959). To avoid deterioration of membrane lipids, 0.01% BHT was added to the extraction solvents. Transesterification of fatty acids was carried out by incubating the lipids in anhydrous methanol containing 5% HCl for 3 hr at 100 Ž. Fatty acid methyl

3 J. Japan. Soc. Hort. Sci. 65(4) : esters were identified with a gas chromatograph (GC-17A, Shimadzu Co. Ltd.) with a capillary column (30 m x 0.25 mm i. d., DB-225, J&W Scientific) and an FID. The column temperature was 190 Ž and that of the injector and detector was 235 Ž. Pentadecanoic acid was used as an internal standard for quantification. Free fatty acids their concentrations are shown in Table 1. Ethanol and acetaldehyde could be detected in the headspace of the package within 24 hr and their concentrations increased to ppm and 48.2 ppm, respectively, by day-4 of storage. Small amounts of methanethiol and dimethyl disulfide were detected in the early period of storage; the concentration of (FFAs) were determined as their 9-anthryldiazomethane (Funakoshi Co., Tokyo, Japan) derivatives by high-performance liquid chromatography as described by Nimura and Kinoshita (1980). Results The O2 concentration in the packages of broccoli dropped to below 0.5% and the CO9 concentration increased to more than 20% within the first 8 hours. The O2 concentration remained at the same level until the end of the storage, whereas the CO2 concentration continued to increase, exceeding 30% after 4 days of storage (Fig. 1); an undesirable off-odor was emitted when the package was opened. The main volatile components in the headspace of the anaerobic package were ethanol, acetaldehyde, methanethiol, and dimethyl disulfide (Fig. 2); Fig. 1. Changes in oxygen and carbon dioxide concentration of atmosphere within the polyethylene bag (100 Values are means }SE for n=4. Fig. 2. GC-FID chromatogram of volatile compounds in the headspace from packaged broccoli head stored for 4 days at 20 Ž. 1. methanethiol: 2. acetaldehyde; 3. ethanol: 4. dimethvl disulfide.

4 870 K. Dan, S. Todoriki, M. Nagata and I. Yamashita methanethiol increased greatly, attaining a maximum of ppm by day-2 of storage and then decreased. The concentration of dimethyl disulfide also increased, reaching a maximum 3 days after packaging. In a subsequent study using only flower buds, the same volatile compounds were formed under anaerobic condition. The build up ethanol, acetaldehyde, methanethiol, and dimethyl disulfide by the sealed broccoli buds is shown in Table 2. Thus, we confirmed that flower buds can develop volatile compounds, including methanethiol and dimethyl disulfide. The activity of C-S lyase in the flower buds during storage is shown in Fig. 3. During the 4 days of storage, there was no significant difference in activity of C-S lyase between the anaerobically packaged and nonpackaged broccoli. The percentage of electrolyte leakage from flower buds of nonpackaged broccoli increased gradually during storage (Fig. 4), whereas that from flower buds of anaerobically packaged broccoli was faster. The fatty acid contents in total lipid of microsomal membrane were 3.59,ƒÊmol per g of tissue fresh weight on day-o and 2.11 ƒêmol on day-2 of anaerobically packaging, whereas it was 2.05 ƒê mol on day-2 of the nonpackaged broccoli. The initial (day-0) fatty acid content in phospholipid of the microsomal membrane was 2.49 ƒêmol/g of tissue fresh weight. This value declined to 1.10 ƒê mol/g of tissue fresh weight (anaerobic packaging) and 1.19 ƒêmol/g of tissue fresh weight (nonpackaging) after 2 days in storage. The FFA content in the microsomal membrane was 0.049,ƒÊmol/g of fresh tissue weight in anaerobic broccoli which Table 1. Concentrations of volatile compounds in the anaerobic package containing broccoli head held at 20 Ž Fig. 3. Changes in activities of C-S lyase in packaged and nonpackaged broccoli during storage at 20 Ž. One unit of enzyme activity produces 1 ƒêmol pyruvate/ min at 30 Ž. Values are means } SE for n= 4 Table 2. Concentrations of volatile compounds in the glass bottle containing broccoli buds held at 20 Ž.

5 J. Japan. Soc. Hort. Sci. 65 (4) : Fig. 4. Changes in percentages of electrolyte leakage in packaged and nonpackaged broccoli during storage at 20 Ž. Values are means } SE for n=4. Fig. 5. Changes in the ratio of free fatty acid (FFA) to total fatty acid (TFA) of microsomal membranes from packaged and nonpackaged broccoli stored at 20 Ž. Values are means } SE for n =3. Table 3. Fatty acid composition of free fatty acid in microsomal membranes from packaged and nonpackaged broccoli stored at 20 Ž. was significantly higher than the 0.029,ƒÊmol/g of fresh tissue weight in the nonpackaged heads. The FFA to total fatty acid (TFA) ratio of microsomal membranes from nonpackaged broccoli increased with storage time (Fig. 5). The FFA to TFA ratio of microsomal membranes from anaerobic packaged broccoli was about 2 times higher than that of nonpackaged broccoli. The percentage of polyunsaturated free fatty acids (18:2, linoleic acid and 18:3, linolenic acid) in FFA of microsomal membrane from anaerobic packaged broccoli was significantly higher at day-2 than those on day-0 and nonpackaged broccoli (Table 3). Discussion Changes occurred in the atmosphere in the polyethylene package: the 02 concentration decreased to below 0.5% and CO2 concentration increased by more than 20% within the first 8 hours following sealing (Fig. 1). Broccoli heads held under this anaerobic condition developed strong off-odors within 2 days. Ethanol and acetaldehyde evolved within one day after storage (Table 1). This formation of ethanol and acetaldehyde may be the result of anaerobic respiration. Sulfur containing volatile compounds methanethiol and dimethyl disulfide which also formed under this anaerobic condition (Table 1) are included among the major contributors to the undesirable odor of broccoli stored under anaerobic condition (Di Pentima et al., 1995; Forney et al., 1991; Hansen et al., 1992). Two mechanisms for the formation of dimethyl disulfide are proposed (Chin and Lindsay,

6 872 K. Dan, S. Todoriki, M. Nagata and I. Yamashita Fig 6. Proposed mechanism for the formation of dimethyl disulfide. 1994)(Fig. 6): one does not require oxygen, the dimethyl disulfide forming from dehydration and chemical disproportionation of methyl sulfenic acid. Chin and Lindsay (1994) support this proposition, stating that dimethyl disulfide is produced in both air- and nitrogen-saturated disrupted cabbage tissues without significant difference. This indicates that oxidation of methanethiol is not the predominant mechanism for the formation of dimethyl disulfide. The second mechanism requires oxygen for the oxidation of methanethiol. Reaction of the sulfenic acid is the prominent mechanism for the formation of methanethiol and dimethyl disulfide following the action of C-S lyases. We observed no significant changes in C-S lyase activity between packaged and nonpackaged broccoli during storage (Fig. 3). These results suggest that the formation of sulfur-containing volatile compounds might not be because of an increase in the activity of C-S lyase. Several plant families are characterized by the presence of a significant amount of nonprotein sulfur amino acids, such as S -methyl-l-cysteine sulfoxide (Marks et al., 1992). Much of the odor and flavor characteristic of these plants after tissue rupture is due to the degradation of these amino acids by C-S lyases. Wellknown examples of these plants are garlic, onion, cabbage, cauliflower, and broccoli (Boelens et al., 1971; Chin and Lindsay, 1993; Chin and Lindsay, 1994; Marks et al., 1992). Much of the characteristic odor and flavor associated with these plants is due to the degradation of the cysteine derivatives by C-S lyase in the tissues but separated from the substrate until the tissue is physiologically and/or physically damaged. Lancaster and Collin (1981) reported that alliinase and alkyl cysteine sulphoxides are prevented from reacting in the intact onion protoplast because of compartmentation of the alliinase in the vacuoles and alkyl cysteine sulphoxides in the cytoplasm. Membrane deterioration is a fundamental feature of senescence in plant tissue. Deschene et al.

7 J. Japan. Soc. Hort. Sci. 65(4) : (1991) reported that membrane deterioration, such as phospholipid breakdown, was strongly inhibited when broccoli was stored under controlled atmosphere (5% CO2, 3% O2) conditions. Zhuang et al. (1995) reported that suitable MA storage reduced the rate of decrease of polyunsaturated fatty acid and the rate of increase of lipid peroxidation in broccoli. Our results suggest that anaerobic treatment accelerated membrane deterioration compared to natural senescence of broccoli under aerobic condition. The FFA to TFA ratio of both nonpackaged and packaged broccoli increased 2 days after storage, but the ratio of anaerobic packaged broccoli was about 2 times higher than that of nonpackaged broccoli (Fig. 5). Significant membrane degradation is reflected by a large increase in electrolyte leakage and a rise in the FFA to TFA ratio (Fig. 4, 5). FFA accumulation in membrane bilayer has been correlated with the appearance of gel phase domains (Barber and Thompson, 1983; McKersie et al., 1989; Yao et al., 1991). The occurrence of gel phase lipid domains in the membrane causes packing imperfections that result in the loss of selective membrane permeability, particularly to ions and small molecules. Presumably, the loss of membrane integrity contributes to the loss of intracellular compartmentation, decreased membrane enzyme activity, and release of hydrolytic enzymes (Barber and Thompson, 1980). Intracellular compartmentation of H+ and other ions has important roles in the maintenance and regulation of homeostasis of the cell (Guern et al., 1991). The gradient of electrochemical potential of H + across the membrane is utilized as an energy source. The gradient of H + is maintained by a transporting system across the membrane, such as the ATP-dependent H + -pump. Macri et al. (1991) reported that FFAs, such as 18:2 and 18:3, drastically inhibited ATP-dependent H + -pump in microsomes of pea stems. In our study, the percentages of 18:2 and 18:3 in FFA of microsomal membrane from MA packaged broccoli were significantly higher than those from the initial and nonpackaged broccoli. The accumulations of 18:2 and 18:3 in the membrane might have disturbed normal compartmentation of ions and small, organic molecules by suppressing the activity of a membrane-bound enzyme, such as ATP-dependent H+-pump. In conclusion, the most obvious characteristic difference between anaerobic packaged and nonpackaged broccoli was membrane leakage. This phenomenon indicates that, under anaerobic condition, sulfur containing volatile compounds, such as methanethiol and dimethyl disulfide are formed by the deterioration of cellular membrane lipids and the loss of intracellular compartmentation, thus, allowing enzymes and their substrates to react. Literature Cited Barber, R. F. and J. E. Thompson Senescencedependent increase in the permeability of liposomes prepared from bean cotyledon membranes. J. Exp. Bot. 31 : Barber, R. F. and J. E. Thompson Neutral lipids rigidify unsaturated acyl chains in senescing membranes. J. Exp. Bot. 34 : Barth, M. M., E. L. Kerbel, A. K. Perry and S. J. Schmidt. 1993a. Modified atmosphere packaging affects ascorbic acid, enzyme activity and market quality of broccoli. J. Food Sci. 58 : Barth, M. M., E. L. Kerbel, S. Broussard and S. J. Schmidt. 1993b. Modified atmosphere packaging protects market quality in broccoli spears under ambient temperature storage. J. Food Sci. 58 : Bligh, E. G. and W. J. Dyer A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 : Boelens, M., P. J. Valois, H. J. Wobben and A. Gen Volatile flavor compounds from onion. J. Agric. Food Chem. 19 : Bradford M. M A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72 : Chin, H.-W. and R. C. Lindsay Volatile sulfur compounds formed in disrupted tissues of different cabbage cultivars. J. Food Sci. 58 : Chin, H.-W. and R. C. Lindsay Mechanisms of formation of volatile sulfur compounds following the action of cysteine sulfoxide lyases. J. Agric. Food Chem. 42 : Deschene, A., G. Paliyath, E. C. Lougheed, E. B. Dumbroff and J. E. Thompson Membrane deterioration during postharvest senescence of broccoli florets: modulation by temperature and controlled atmosphere storage. Postharvest Biol. Technol. 1 : Di Pentima, J. H., J. J. Rios. A. Clemante and J. M. Olias Biogenesis of off-odor in broccoli storage under low-oxygen. J. Agric. Food Chem. 43 : Forney, C. F., J. P. Mattheis and R. K. Austin Volatile compounds produced by broccoli under

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9 J. Japan. Soc. Hort. Sci. 65(4) :