Analysis of Flavonoids and Hydroxycinnamates in Citrus Processing Byproducts by High Performance Liquid Chromatography- Electrospray Ionization-Mass Spectrometry

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Proc. Fla. State Hort. Soc. 115:292-297. 2002. ANALYSIS OF FLAVONOIDS AND HYDROXYCINNAMATES IN CITRUS PROCESSING BYPRODUCTS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY- ELECTROSPRAY IONIZATION-MASS SPECTROMETRY

JOHN A. MANTHEY The study of the flavonoids in citrus depends on efficient USDA, ARS, SAA separations via HPLC and other chromatographic tech- Citrus and Subtropical Products Laboratory niques. The numerous flavonoids and the low concentrations 600 Avenue S, NW in which many of them occur make their individual detection Winter Haven, FL 33881 and analysis in citrus byproducts difficult by conventional chromatographic techniques. To overcome these difficulties, Additional index words. hesperidin, HPLC, orange peel, poly- HPLC-mass spectrometry (HPLC-MS) can be used (for re- methoxylated ﬂavones, molasses, LC/MS views see Barnes et al., 1999, 2002; Berhow 2002; Careri et al., 1998; Wolfender et al., 2001). This method permits highly se- Abstract. Electrospray ionization-high performance liquid chro- lective peak detection and accurate quantitation, even in cas- matography-mass spectrometry (ESI-HPLC-MS) greatly facili- es where chromatographic separations are incomplete. tates analyses of the complex phenolic constituents of citrus HPLC-MS has been used in a small number of previous anal- peel and associated citrus processing byproducts. The numer- yses of the main flavonoids in citrus (Careri et al., 1999; Dugo ous flavonoid glycosides and polymethoxylated flavones (PM- et al., 2000; He et al., 1997; Robards et al., 1997). In contrast, Fs) in these materials can be readily detected by positive ion this technique has not been applied to the analysis of the cit- ESI mode. Single ion monitoring (SIM) mode allows for selec- rus hydroxycinnamates, and very little study has also been tive detection and enhanced quantitation of the PMFs and oth- made of the minor flavonoid constituents. Yet, the high sensi- er flavonoid glycosides in spite of incomplete chromatographic peak separations. Flavonoid-O-glycosides tivity of HPLC-MS, in particular the operation of Selective Ion fragment with source cone voltages in ways that confirm struc- Monitoring (SIM), should allow the detection of many citrus tural assignments. These fragmentation patterns allow for the compounds that occur at very low concentrations. SIM im- detection of other trace-occurring chemical species with spe- proves the detection of citrus flavonoids by roughly 100 fold cific flavonoid components. Many of the hydroxycinnamates compared to typical scanning MS modes. Similar application are best detected by the ESI negative ion mode. However, the of SIM-MS was shown to greatly facilitate the detection of the fragmentation patterns obtained with positive ESI allow struc- liminoid glucosides in citrus processing byproducts (Schoch tural information to be obtained for individual components of et al., 2001). this complex group of compounds. This article describes the application of HPLC-MS to the analysis of the several classes of phenolic compounds in cit- There is a great deal of interest in increasing the value of rus, including the hydroxycinnamates, flavanone and flavone the U.S. citrus crop by developing value-added materials from glycosides, and the PMFs. the byproducts of citrus processing. One likely candidate of materials is the flavonoids which have exhibited a number of beneficial biological activities in animal trials and in numer- Materials and Methods ous cell culture studies. These beneficial activities include an- tioxidant, antiinflammatory, anticancer, and cardioprotective Sample Preparation. Whole dried peel was recovered from actions via a number of different mechanisms (for a review 20 Valencia and Hamlin fruit. The peel was lightly chopped ° see Manthey et al., 2001). Many of these beneficial activities in a blender and dried at 65 C for 2 d. The dried peel was reflect the protective effects of hesperidin, and presumably of ground to a fine powder with a coffee grinder. Extracts were the other flavanone and flavone glycosides in citrus, on the prepared by combining 300 mg peel with 10 mL dimethylsul- microvascular endothelium. Additionally, a recent report of foxide and shaken overnight. Concentrated molasses (45 ° the binding of citrus PMFs to human adenosine receptors in Brix or higher) was diluted 1:4 with water and allowed to set- ° vitro (Jacobson et al., 2002) suggests potential receptor-linked tle overnight at 4 C. The molasses was centrifuged at 10,000 activities for these compounds. The ramifications of adenos- gn for 60 min to remove the bulk of undissolved solids. The ine receptor binding are complex, and it is unknown at this partially clarified molasses was filtered through Grade 161 time what roles such putative PMF binding may play in animal glass fiber filters (Scientific Specialities Group, Mt. Holly physiology. Yet, the occurrence of the numerous beneficial Springs, Pa.). Clarified molasses was ultrafiltered through a biological actions of the flavonoid glycosides, as well as the Romicon model HF4 hollow fiber cartridge ultrafiltration sys- PMFs, thus far documented, supports the claim that there tem (Romicon, Inc., Woburn, Mass.) with a 10,000 Da molec- may be potential for the citrus flavonoids as high value nutra- ular weight cutoff. ceuticals and specialty ingredients. Chromatographic Methods. Measurements of citrus flavonoids were made with high performance liquid chromatography using solvent systems and columns selected for the best Mention of a trademark or proprietary product is for identification only resolution of specific groups of compounds. The PMFs were and does not imply a guarantee or warranty of the product by the U.S. De- analyzed with an Alltech Alltima C8 5µm analytical column partment of Agriculture. The U.S. Department of Agriculture prohibits dis- × crimination in all its programs and activities on the basis of race, color, (25 cm 4.6 mm i.d.). Elution conditions included a 2 solvent national origin, gender, religion, age, disability, political beliefs, sexual ori- gradient composed initially of 10 mm phosphoric acid/aceto- entation, and marital or family status. e-mail: [email protected] nitrile (90/10, v/v) and increased in a linear gradient to 60/

292 Proc. Fla. State Hort. Soc. 115: 2002.

40, v/v) over 15 min. A final composition of 10 mm phosphoric acid/acetonitrile (55/45, v/v) was achieved by a subse- quent linear gradient over 35 min using a flow rate of 0.75 mL·min-1. Flavonoid glycosides were analyzed using the same column, but with a modified gradient comprised initially of 10 mL phosphoric acid/acetonitrile (90/10, v/v) and increased in a linear gradient to (75/25, v/v) over 15 min. A final composition of (67/33, v/v) was achieved with a linear gradient over 35 min with a flow rate of 0.75 mL·min-1. Instru- mentation included a Perkin-Elmer 250 binary LC pump with a Hewlett-Packard system 1050 autosampler, photodiode array (PDA) detector and ChemStation. The hydroxycinnamates were analyzed with a Whatman C18 5 µm analytical column (25 cm × 4.6 mm i.d.). Elution conditions included a 3 solvent gradient composed initially of 2% formic acid/water/methanol (5/85/10, v/v/v) and increased in a linear gradient to (5/73/22, v/v/v) over 15 min. A final composition of (5/0/95, v/v/v) was achieved with a linear gradient over 35 Fig. 1. HPLC of Polymethoxylated Flavones in Valencia 10 °Brix Ultrafil- -1 min using a flow rate of 0.75 ml·min . tered Molasses. Insert for 330 nm shows: S (sinensetin), N (nobiletin), H High Performance Liquid Chromatography-Electrospray Ioniza- (Heptamethoxyflavone), TM (tetramethylscutellarein), T (tangeretin). Sep- tion-Mass Spectral Analysis. HPLC-ESI-MS analyses were carried arations run with an Alltech Alltima C8 µm (25cm × 4.6mm) column. out with a Waters ZQ single quadrupole mass spectrometer equipped with a Waters 2695 HPLC pump and a Waters 996 PDA detector (Waters Corp., Milford, Mass.). PDA detection severely limited solubility of hesperidin results in its precipita- was monitored between 400-230 nm. Data handling was done tion during byproduct processing; hence, significant losses with MassLynx software (Micromass, Division of Waters occur in the concentrations of hesperidin in processed Corp., Beverly Mass.). HPLC conditions were as described byproducts such as the ultrafiltered molasses. Yet, as a re- above for the analysis of the hydroxycinnamates. Post column search tool, the use of molasses for the analysis of flavonoids split to the PDA detector and mass spectrometer was 10:1. MS in orange peel presents advantages due to the high concen- parameters were as follows: ionization mode, ES+; scan range trations of other soluble flavonoids, many of which are minor components in the peel. Concentrations of the main fla- 150-900 amu; scan rate, 1 scan/s; cone voltage, 20 eV. Peak ° identities were obtained by matching expected molecular vonoids in Valencia 10 Brix ultrafiltered molasses are listed weights, UV spectra, and elution properties with standards in Table 1. obtained commercially or from a library of standards at the HPLC-MS of the Flavonoid Glycosides in Molasses. In spite of Winter Haven Laboratory. the power of the HPLC separations presented above, there are still severe limitations in the accurate quantitation of many of the citrus flavonoids and hydroxycinnamates, partic- Results and Discussion ularly the minor constituents. Although citrus peel contains a number of major flavonoids, there are also large numbers of HPLC of the Phenolic Compounds in Molasses. The diversity in other minor-occurring flavone and flavanone glycosides and structures of the citrus flavonoids requires the use of multiple hydroxycinnamates. Additionally, in grapefruit and lemon sets of HPLC conditions to analyze these compounds in citrus processing waste streams. A number of normal and reversed phase HPLC column conditions have been reported for the separation and analysis of the citrus PMFs (Bianchini et al., 1987; Careri et al., 1999; Ooghe et al., 1994; Sendra et al., 1988). In our studies these compounds are resolved satisfac- torily with a C8 column (Fig. 1) and a gradient composed of 10mm phosphoric acid and acetonitrile (see Materials and Methods section). The flavonoid glycosides in this chromatogram show considerable overlap, and are better resolved by using a gradient with lower acetonitrile concentrations (Fig. 2). Even with this modified gradient, the earlier-eluting hydroxycinnamates remain poorly resolved. Satisfactory resolution of the hydroxycinnamates can be achieved with a C18 column and a gradient of 2% formic acid/water/methanol (Fig. 3). These HPLC methods were used to measure the concentrations of the main flavonoids in dried peel of Hamlin and Valencia oranges (Table 1). The main flavonoids in orange peel constitute flavanone disaccharides and trisaccharides, a Figure 2. HPLC of Flavonoid Glycosides in Valencia 10 °Brix Ultrafiltered flavone-C-glycoside (6,8-di-C-glucosylapigenin), and the late- Molasses. Chromatogram measured at 285 nm with an Alltech Alltima C8 5 eluting PMFs. Hesperidin, occurring at nearly 2.5% of the µm (25cm × 4.6mm) column and modified 10mM phosphoric acid/acetoni- peel dry weight, is clearly the main constituent. However, the trile gradient.

Proc. Fla. State Hort. Soc. 115: 2002. 293

Fig. 3. HPLC of Phenolic Compounds in Valencia 10 °Brix Ultrafiltered Molasses. Resolution of early-eluting hydroxycinnamates achieved with a Whatman Partisil 5 column (25cm × 4.6mm). Fig. 4. Mass Spectra of Hesperidin at Increasing Positive Electrospray Ion- ization Cone Voltages. Waters ZQ mass spectrometer operated with scan mode: 1 s intervals, 150-900 amu. the profiles of phenolic compounds in the peel also include numerous courmarins and psoralens (D’Amore and Calapaj, positive ESI voltage of 20 electron volts (eV), the mass spec- 1965; Fisher and Nordby, 1965; Stanley and Vannier, 1957; trum shows mainly the molecular ion plus proton [M+H]+ ion Stanley et al., 1965; Tatum and Berry, 1979). In many cases, at 611 atomic mass units (amu). At 40eV there are the lower the low concentrations of these flavonoids and related phe- intensity signals at 633 amu representing [M+Na]+, and addi- nolic compounds, and the multiple peak overlaps in the chro- tionally at 465 and 303 amu for hesperetin-7-glucoside and matograms for many of these compounds make their hesperetin aglycone respectively. detection and quantitation difficult by conventional HPLC The 2 flavanone trisaccharides in orange peel further il- methodologies. However, with the high sensitivity and selec- lustrate the usefulness of CID to confirm the chemical struc- tivity of HPLC-MS, many of these limitations are overcome. tures of the flavonoid glycosides in citrus. The mass spectrum In addition to the excellent sensitivity and selectivity of of narirutin-4’-glucoside (Fig. 5) at 20eV shows the molecular HPLC-MS, these measurements can provide structural infor- ion [M+H]+ at 743. The loss of the glucose at the 4’-position mation through fragmentation by in-source collision-in- to yield narirutin is detected at 581. Increased cone voltage duced-dissociation (CID). The use of HPLC-MS to elucidate key components of chemical structures of flavonoid glycosides has been previously reported (Cuyckens et al., 2001; Grayer et al., 2000; Waridel et al., 2001; Wolfender et al., 2001). The extent of CID is largely influenced by the strength of the voltage applied in the ionization source. Fortunately, many citrus flavonoids are fragmented by this method in ways that provide a great deal of information about their structures, hence assisting in confirming compound identifica- tions. The example of hesperidin (Fig. 4) shows that at a

Table 1. Flavonoids in dried orange peel and 10 °Brix ultraﬁltered molasses. Concentrations in peel (ppm dry weight) are averages of measurements of 3 replicate extractions. Concentrations in molasses (ug·mL-1) are averages of 3 replicate measurements by photodiode array UV detection.

Valencia Hamlin Valencia Compound Peel Peel Molasses 6,8-di-C-glucosylapigenin 1015 725 107 Narirutin-4-glucoside 1459 2566 227 Hesperetin trisaccharide 1131 2055 183 Narirutin 3340 3967 480 Hesperidin 23777 25756 777 Isosakuranetin 2705 2961 88 Sinensetin 409 195 38 Nobiletin 505 350 37 Heptamethoxyﬂavone 204 118 22 Tetramethylscutellarein 166 97 12 Fig. 5. Mass Spectra of Narirutin-4’-glucoside at Increasing Positive Elec- trospray Ionization Cone Voltages. Waters ZQ mass spectrometer operated Tangeretin 75 49 5 with scan mode: 1 s intervals, 150-900 amu.

294 Proc. Fla. State Hort. Soc. 115: 2002.

(40eV) produces the naringenin aglycone [M+H]+ fragment at 273 amu. The molecule identified as hesperetin trisaccharide exhibits a [M+H]+ at 773 and a main fragment at 611 amu (hesperidin, [M+H]+) (Fig. 6). At increasing cone voltage, signals at 465 and 303 amu appear, indicating hesperetin-7-glucoside and hesperetin, respectively. The ion at 773 is consistent with a additional glucose, hence, the assignment of hesperetin trisaccharide. Previous analyses of hydrolysis products of this molecule confirm the presence of hesperetin, 2 glucose molecules, and a rhamnose substituent (data not presented). In the SIM mode, the mass spectrometer monitors only at specific atomic mass units instead of continuously scanning over wide mass ranges, as is typically the case in the scan mode. The high sensitivity of the mass spectrometer operated in the SIM mode can be used to detect many of the compounds in citrus that occur at very low concentrations. The flavone glycosides, rhoifolin and diosmin are two compounds that occur at the lower levels of HPLC UV detection and the scan mode for the mass spectrometer (Fig. 7). However, when the Waters ZQ mass spectrometer was used in the SIM mode, the detection of these minor constituents was vastly improved - as evidenced by the significant improvement in the signal-to- noise ratios in the total ion current chromatograms for both Fig. 7. HPLC-MS Detection of Diosmin and Rhoifolin in Valencia 10 °Brix of these compounds. Ultrafiltered Molasses. A: Signal for rhoifolin at 579 amu from data collected HPLC-MS of the PMFs in Molasses. An analysis by HPLC-MS during scanning mode: 1 s interval, 150-900 amu. B: Signal for diosmin at 609 of the PMFs in oils from a number of different varieties of cit- amu from data collected during scanning mode: 1 s interval, 150-900 amu. C: rus has been previously reported (Dugo et al., 2000). These Signal for rhoifolin at 579 amu from SIM measurements. D: Signal for di- measurements, made with positive mode atmospheric pres- osmin at 609 amu from SIM measurements. sure chemical ionization (APCI), evaluated fragmentation patterns of the PMFs over wide voltage ranges, and provided nobiletin measured at several positive ionization voltages. structural confirmations of the PMFs. Similarly, CID in posi- This fragmentation occurred primarily at voltages greater tive ESI mode for the PMFs produce losses of 30 amu, repre- than 20eV, and hence, optimal [M+H]+ signal detection for senting losses of methoxy groups. This is shown in Fig. 8 for the PMFs was achieved at 20eV. HPLC-ESI-MS at 20eV selectively detected and provided accurate quantitation of the partly resolved PMFs in Valencia ultrafiltered molasses (Fig. 9). With these chromatographic conditions, overlap occurred between sinensetin and querce- tagetin hexamethylether, and additional overlap occurred

Fig. 6. Mass Spectra of Hesperetin trisaccharide at Increasing Positive Fig. 8. Mass Spectra of Nobiletin at Increasing Positive Electrospray Ion- Electrospray Ionization Cone Voltages. Waters ZQ mass spectrometer oper- ization Cone Voltages. Waters ZQ mass spectrometer operated with scan ated with scan mode: 1 s intervals, 150-900 amu. mode: 1 s intervals, 150-900 amu.

Proc. Fla. State Hort. Soc. 115: 2002. 295

quantified (Fig. 9). Close agreement occurred between the quantifications made by the HPLC-MS and analyses made in- dependently with the reversed phase C8 column described for Fig. 1. Hydroxycinnamates. HPLC-MS measurements provided ex- tremely sensitive detection and structural analysis of the hydroxycinnamates in orange peel molasses. These compounds thus far reported in citrus occur mainly as esters with galactar- ic and glucaric acids (Risch and Herrmann, 1988). A nearly complete mixture of these compounds in ultrafiltered molasses was recovered by anion exchange and reversed phase chromatography (Manthey and Grohmann, 2001). Results of HPLC-ESI-MS indicated that analyses of this mixture were best achieved with negative ionization. Increased strength of the negative ionization dramatically increased the signal in- tensities for the molecular ions of most of these compounds. Figure 10 illustrates the sharp increases in the signal-to-noise ratios in the total ion-curent chromatograms of the hydroxycinnamates, as the ionization energies were decreased from - 10 to -60eV. Positive ESI yielded weak intensity [M+Na]+ signals, negligible signals for [M+H]+, but typically produced fragment ions for the hydroxycinnamic acid components (da- Fig. 9. HPLC-MS of Polymethoxylated Flavones in Valencia 10 °Brix Ultra- filtered Molasses. Separations (measured at 330 nm (bottom)) made with a ta not shown). Hence, in spite of the superior sensitivity of Whatman Partisil 5 column (25cm × 4.6mm) and gradients with 2% formic negative ESI for the detection of the hydroxycinnamates, pos- acid/water/methanol. Signals for specific amu values plotted from data col- itive ESI is useful in confirming key chemical features of these lected during scanning mode 1 s interval, 150-900 amu, +20eV, positive ESI. compounds.

between nobiletin, heptamethoxyflavone, and tetramethyls- Literature Cited cutellarein. In spite of these overlaps, the specific mass-cur- Barnes, S., C.-C. Wang, M. Kirk, M. Smith-Johnson, L. Coward, N. C. Barnes, rent chromatograms of each of these compounds were G. Vance, and B. Boersma. 2002. HPLC-mass spectrometrometry of isofla- obtained from the total ion current chromatogram data, and vonoids in soy and the American groundnut, Apios Americana. p. 77-88. In therefore, these compounds were individually resolved and B. S. Buslig and J. A. Manthey (eds.). Flavonoids in Cell Function. Kluwer Academic/Plenum Publishers. NY. Barnes, S., C.-C. Wang, M. Smith-Johnson, and M. Kirk. 1999. Liquid chromatography-mass spectrometry of isoflavones. J. Med Food 2:111-117. Berhow, M. A. 2002. Modern analytical techniques for flavonoid determination. p. 61-76. In B. S. Buslig and J. A. Manthey (eds.). Flavonoids in Cell Function. Kluwer Academic/Plenum Publishers. NY. Bianchini, J. P., E. M. Gaydou, A. M. Siouffi, G. Mazerolles, D. Mathieu, and T. L. Phan. 1987. Optimization of the separation of polymethoxylated flavones in reversed phase chromatography. Chromatographia 23:15-20. Careri, M., L. Elviri, and A. Mangia. 1999. Validation of a liquid chromatography ion spray mass spectrometry method for the analysis of flavanones, flavones, and flavonols. Rapid Commun. Mass Spec. 13:2399-2405. Careri, M., A. Mangia, and M. Musci. 1998. Overview of the applications of liquid chromatography-mass spectrometry interfacing systems in food analysis: naturally occurring substances in food. J. Chromatography 794:263-297. Cuyckens, F., R. Rosenberg, E. De Hoffmann, and M. Claeys. 2001. Structure characterization of flavonoid O-diglycosides by positive and negative nano-electrospray ionization ion trap mass spectrometry. J. Mass Spec- trometry 36:1203-1210. D’Amore, G. and R. Calapaj. 1965. Fluorescent substances in the essences of lemon, bergamot, tangerine, bitter orange, and sweet orange. Rass. Chim. 17:264-269. Dugo, P., L. Mondello, L. Dugo, R. Stanceanelli, and G. Dugo. 2000. LC-MS for the identification of oxygen heteroxyclic compounds in citrus essen- tial oils. J. Pharm. Biomed. Anal. 24:147-154. Fisher, J. F. and H. E. Nordby. 1965. Isolation and spectral characterization of coumarins in Florida grapefruit peel oil. J. Food Sci. 30:869-873. Grayer, R. J., G. C. Kite, M. Abou-Zaid, and L. J. Archer. 2000. The application of atmospheric pressure chemical ionization liquid chromatography- mass spectrometry in the chemotaxonomic study of flavonoids: character- Fig. 10. HPLC-MS of Hydroxycinnamates Isolated from Valencia 10 °Brix ization of flavonoids from Ocinmum gratissium var. gratissium. Phytochem. Ultrafiltered Molasses. Separations made with a Whatman Partisil 5 column Analysis 11:257-267. (25cm × 4.6mm) and gradients with 2% formic acid/water/methanol. Sig- He, X.-G., L.-Z. Lian, and M. W. Bernart. 1997. High-performance liquid nals represent total ion counts for data collected during scanning mode. MS chromatography-electrospray mass spectrometry in phytochemical analy- conditions included negative electrospray ionization at -10 to -60 eV, 1 s in- sis of sour orange (Citrus aurantium L). J. Chromatography 791:127-134. terval, 150-900 amu. Bottom chromatogram monitored at 330 nm with pho- Jacobson, K. A., S. Moro, J. A. Manthey, P. L. West, and X.-D. Ji. 2002. Inter- todiode array. actions of flavones and other phytochemicals with adenosine receptors. p.

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Proc. Fla. State Hort. Soc. 115:297-300. 2002. POSTPROCESSING DIP MAINTAINS QUALITY AND EXTENDS THE SHELF LIFE OF FRESH-CUT APPLE

JINHE BAI AND ELIZABETH A. BALDWIN1 ketable quality within a day, because of severe browning accom- ° USDA, ARS panied by a sharp decrease of hue angle (h ab), and lightness (L*), Citrus & Subtropical Products Lab., and an increase in a* and b* values. Slices dipped in the aqueous 600 Avenue S N.W. solution plus additives maintained cut surface color, inhibited ethylene production, maintained firmness, and maintained the Winter Haven, FL 33881 major aroma of apple. However, these slices exhibited green mold after 8 days of storage. Addition of soybean oil emulsion re- Additional index words. browning, volatile, ﬁrmness, sanitizer, duced water loss, whereas chitosan and CMC did not, although coating, minimally processed water loss was not a problem for polyethylene packaged products. The wedges dipped in coatings lost several major volatiles Abstract. An aqueous solution with hypochlorite as a sanitizer, so- of apple compared to those dipped in the aqueous solutions. dium erythorbate (isoascorbate), n-acetylcysteine and 4-hexylre- These results suggest that a dip with a sanitizer, firming agent, sorcinol as reducing and anti-browning agents, and Ca and reducing/anti-browning agents is beneficial of fresh-cut ap- propionate as a firming agent was developed for postprocessing ple quality. Addition of film-formers led to loss of important aro- dip of fresh-cut ‘Gala’ apple. The additional effect of edible coat- ma compounds while chitosan did not reduce decay as has been ing materials to the aqueous solution of additives was also inves- reported for whole fruits. tigated. The edible coating film-forming agents were soybean oil emulsion, chitosan and carboxymethyl cellulose (CMC), which were expected to form a protective layer on the cut surface of the Fresh-cut fruit products have not developed commercially apple wedges, decreasing water loss and other deteriorating fac- compared to fresh-cut vegetables, which have already had a tors due to cutting. Apple slices were dipped in aqueous solu- multi-billion dollar sales annually in the United States (Lami- tions of sanitizer, with or without anti-browning and firming kanra, 2002). Fresh-cut fruits usually have a much larger agents (additives), and with or without film-formers. Treated slic- wound-surface area, softer texture, and higher susceptibility es were then allowed to drain for 1 h at 5.5 °C before placement in to enzymatic and nonenzymatic browning than do vegetables. perforated polyethylene bags (20 × 18 cm, thickness 30 m, with Various approaches have been used to minimize the quality ° ten 1.5 mm holes) and storage at 5.5 °C for up to 14 days. Slices deterioration in fresh-cuts. Hypochlorite (50-200 µL·L-1), dipped in water (control, containing hypochlorite only) lost mar- used as a sanitizer, significantly retards microbial population on the surface of cut products during processing and subse- Mention of a trademark or proprietary product is for identification only quent storage (Ayhan et al., 1998; Watada et al., 1996). Calci- and does not imply a guarantee or warranty of the product by the U.S. De- um salt dip, including CaCl2, Ca propionate and Ca chelate, partment of Agriculture. The U.S. Department of Agriculture prohibits dis- maintains or improves tissue firmness and crispness (Luna- crimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual ori- Guzman et al., 1999; Saftner et al., 2002), while Ca propi- entation, and marital or family status. onate also functions as a preservative to retard mold, for 1Corresponding author. which purpose it is used in the bread industry. Browning con-

Proc. Fla. State Hort. Soc. 115: 2002. 297