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E. T. Contis et al. (Editors) Food Flavors: Formation, Analysis and Packaging Influences © 1998 Elsevier Science B.V. All rights reserved

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Changes in Citrus hystrix oil during autooxidation F. Pudil, H. Wijaya*, V. Janda, J. Volfova, H. Valentova and J. Pokomy

Department of Food Chemistry and Analysis, Prague Institute of Chemical Technology, Technicka 5, CZ-166 28 Prague 6, Czech Republic

^Faculty of Agricultural Technology, Bogor Agricultural University, P.O. Box 220, Bogor 16002, Indonesia

Abstract The essential oil from Citrus hystrix is an interesting new raw material for the food and cosmetic industry. The composition of oil was analyzed by capillary gas chromatography. Changes due to autooxidation were studied at 40*^C and 60°C using a headspace autosampler. Volatiles were identified by GC-MS after solid-phase microextraction (SPME) sampling using a Carbowax coated fiber. Changes in the sensory profile of autooxidized oil were determined imder conditions specified by ISO standards, using unstructured graphical scales. Influences of a dihydropyridine antioxidant, Diludine, and of rosemary extracts on the course of oxidation of Citrus hystrix essential oil were determined; correlations between sensory and chromatographic data were calculated.

1. INTRODUCTION Citrus hystrix is a citrus plant which is widely used in various foods and beverages in Indonesia ("jeruk purut") and other Asian countries as a source of natural flavor. The leaves of Citrus hystrix^ called "som makrut" in Thailand and "suwangi limau" or "purut limau" in Malaysia, are used to give unique oriental flavour to soups, curries, and many other cookies and cakes. The flavor is similar to that of citrus flavours, but it is harsher, reminding the panelists of citrus leaves. This flavor source is ahnost unknown in the European countries. Investigations on Citrus hystrix oil composition have been akeady reported by several authors [1-7]. It was reported that the oil contained citronellal, citronellol and nerol. A recent study by Sato et al. [2] has confirmed citronellal as a principal component of the oil. Furthermore, the authors foimd that citronellal in the oil is present in the L-form. Discussion

708 on the oil constituents was also published in a review paper by Lawrence [3]. Processing methods to produce Citrus hystrix leaves flavor were compared and evaluated by Wijaya [4]. The earlier study on the Citrus hystrix oil composition was published by Pudil et al. [7].

2. MATERIALS AND CHEMICALS Citrus hystrix leaves were obtained from a local market in Kramat Jati, Jakarta. Indonesia.The oil was prepared from Citrus hystrix leaves by steam distillation at atmospheric pressure for 2 hours using a pilot plant modified oil separator trap. No terpene removal was made. Two samples were analyzed: Oil A, prepared in 1995 and oil B, prepared in 1996. The mixture C was a synthetic mixture containing only citronellal, citronellol and limonene in the ratio contained in Oil A. Rosemary extract was prepared by extracting dry rosemary leaves with acetone; the 1,4dihydropyridine derivatives, Diludine (2,6-dimethyl-3,5-diethoxycarbonyl-l,4-dihydropyridine, and OSI 7284 (2,6-dimethyl-3,5-dibutoxycarbonyl-l,4-dihydropyridine) were prepared in the Institute of Organic Synthesis (Director Prof. Dr. G. Duburs) in Riga, Latvia.

3. EXPERIMENTAL PROCEDURES 3.1. Oxidation of the Citrus hystrix oil A 0.1 mL portion of pure Citrus hystrix oil was placed m a 10 mL vial. An internal standard of 5 mg of n-decane was also added. The vial was sealed and conditioned at 40°C in a thermostat. A 0.1 mL sample of vapor phase was injected via a gas-tight Hamilton syringe into the gas chromatograph (using a headspace autosampler). Alternatively, the SPME sampling technique was used. To study the antioxidant efficiency, 1 mg of the antioxidant (1,4-dihydropyridines or rosemary extract) was added. 3.2. Gas liquid chromatography GLC analyses of oxidized Citrus hystrix oil were carried out in a the GC 8000 series gas chromatograph (Fisons Instruments) equipped with a flame ionization detector and a 60 m x 0.32 mm Supelcowax 10 capillary column fihn thickness (Supelco, USA). The column temperature was programmed from 50°C (constant for 2 min), at a rate 2°C/min to 220°C (and then at 220°C for 30 min). The injector temperature was 220°C and the detector temperature was 250°C. The carrier gas (helium) pressure was kept at 100 kPa. The input split ratio was 1 : 25. To determine retention indices, a mixture of n-alkanes was injected with the sample of Citrus hystrix oil.

709 3.3. Gas liquid chromatography - mass spectrometry (GLC-MS) GLC-MS analyses of oxidized Citrus hystrix oil were carried out on a MSD 800 mass spectrometer with a GC 8000 series gas chromatograph (Fisons Instruments). The energy of ionizing electron was 70 eV. The capillary column and the temperature program were as above. The compounds were identified on the basis of Aeir mass spectra (NIST library in the MassLab software package; Fisons) and retention data of pure standards.

I

3.4. Solid phase microextraction (SPME) A 0.065mm Carbowax - Divinylbenzene fiber for a manual holder (Supelco, USA) was used for extracting volatiles; the fiber was inserted into the vial (Figure 1). The extraction time was 10 min and the temperature was 40*^0. The time of desorption was 2 min at 220°C. At the same temperature the fiber was cleaned for 30 min.

Figure 1. The detail of SPME sampling 3.5. Sensory analysis The sensory analysis was performed according to international standard [8] in a test room provided with six standardized test booths [9]. The panel of 12 assessors consisted of selected and trained persons [10] with experience in the sensory profiling (2-4 sessions a week, 4 samples each) of at least 6 months. The sensory profile consisted of 24 -36 descriptors, and the intensities of partial flavor notes were rated by unstructured graphical scales [11]. Two samples were served at a time, consisting of the tested product supported on a piece of cotton, placed in a 500-mL ground-stoppered bottle. The absolute odour intensity was evaluated by sniffing, and rated using linear graphical scales.

3.6. Sociological study Each person evaluated the Citrus hystrix oil by sensory profiling and was familiar with the flavour, was served on a special sheet containing questions concerning the optimimi application of oil. Some application were preprinted (the same as shown in Table 2), and the respondents could add any other comments Aey judged as useful. The results were expressed in % of total responses.

710 4. RESULTS AND DISCUSSION

Citrus hystrix oil was slightly oxidized and shows peaks of oxidation products in addition to natural components (Figure 2). The chemical composition was similar to that published earlier [7] especially the major peaks (Table 1); only the content of citronellal was lower than reported in the literature. During autoxidation under simulated storage conditions, the content of citronellal decreased rapidly, and followed first order reaction kmetics (Figure 3A). For the GLC/MS analysis of oxidized oil, the device shown in Figure 1 was applied. No great difference was observed between the degradation of non-stabilized and stabilized samples. The formation of oxidation and other reaction products was affected by antioxidants. The formation of acetone was efficiently inhibited by tiie rosemary extract (Figure 3B), but both 1,4-dihydropyridines were ineffective. The same was observed in the case of linalool oxides formation (in this case, Diludine was found slightly better than OSI 7284), especially after long reaction times. In case of the formation of p-mentha-l,4(8)-diene and p-menth-8-en-3-ol, the 1,4dihydropyridine derivatives showed some moderate antioxidant activity as well. The last two compounds are typical intermediate derivatives, which are formed relatively rapidly, and decompose during the oxidation. Sensory profiles of two samples of Citrus hystrix oils are shown in Figure 4. The essential features are the same, but nevertheless, certain differences existed as their chemical composition was different. In both oils, citrus odour notes prevailed in the profile. The sensory profile of three main terpenic constituents are shown in Figure 5. They had a similar character as the essential oil, and the calculated contributions of citronellal, linalool and citronellol to the profile of oil A have been calculated; the results are compared with the three components under Mixture C. There were differences between the model mixture and the natural oil, showing that minor components markedly affected the sensory profile. Nevertheless, a significant correlation existed between the model mixture C and die natural material. The correlation coefficient was very high in case of oil A (r = 0.9451), but was lower in case of oil B (r = 0.7154); it should be considered, however, that the model mixture shnulated the composition of oil A. The correlation between the sensory profiles of the two oils and the profile of citronellal were very high (r = 0.9617 and 0.7905, respectively). Odor acceptabilities were determined as well, using unstructured graphical scales (0 % = very agreeable; 100 % = bad); they were rated 29 and 47 % in case of oil A and B, respectively, which is considered a favorable rating, especially in oil A.

711 During autoxidation under simulated storage conditions, the content of citronellal decreased rapidly, and followed first order reaction kinetics (Figure 3A). For the GLC/MS analysis of oxidized oil, the device shown in Figure 1 was applied. No great difference was observed between the degradation of non-stabilized and stabilized samples. The formation of oxidation and other reaction products was affected by antioxidants. The formation of acetone was efficiently inhibited by die rosemary extract (Figure 3B), but both 1,4-dihydropyridines were ineffective. The same was observed in the case of linalool oxides formation (in this case, Diludine was found slightly better than OSI7284), especially after long reaction times. In case of the formation of p-mentha-l,4(8)-diene and p-menth-8-en-3-ol, the 1,4dihydropyridine derivatives showed some moderate antioxidant activity as well. The last two compounds are typical intermediate derivatives, which are formed relatively rapidly, and decompose during the oxidation. Sensory profiles of two samples of Citrus hystrix oils are shown in Figure 4. The essential features are the same, but nevertheless, certain differences existed as their chemical composition was different. In both oils, citrus odour notes prevailed in the profile. The sensory profile of three main terpenic constituents are shown in Figure 5. They had a similar character as the essential oil, and the calculated contributions of citronellal, linalool and citronellol to the profile of oil A have been calculated; the results are compared with the three components under Mixture C. There were differences between the model mixture and the natural oil, showing that minor components markedly affected the sensory profile. Nevertheless, a significant correlation existed between the model mixture C and the natural material. The correlation coefficient was very high in case of oil A (r = 0.9451), but was lower in case of oil B (r = 0.7154); it should be considered, however, that the model mixture simulated the composition of oil A. The correlation between the sensory profiles of the two oils and the profile of citronellal were very high (r = 0.9617 and 0.7905, respectively). Odor acceptabilities were determined as well, using unstructured graphical scales (0 % = very agreeable; 100 % = bad); they were rated 29 and 47 % in case of oil A and B, respectively, which is considered a favorable rating, especially in oil A.

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713 Table 1 The main identified components of slightly oxidized Citrus hystrix oil Figure 1), Peak No. RT(min) RI Identification Note acetone 825 6.63 1 7.61 2 methanol 891 ethanol 934 8.37 3 chloroform 9.79 4 air contamination 989 n-decane 10.01 5 internal standard 1000 10.83 6 1023 a-pinene 1028 11.00 7 a -phellandrene 1107 14.27 8 P-pmene 1123 14.94 9 P -phellandrene 1166 17.16 10 p -myrcene a -terpinene 1182 17.99 11 limonene 1198 19.05 12 c/5-ocimene 1240 21.25 13 1255 21.85 14 y-terpinene ^an5-ocimene 1257 22.29 15 p-mentha-1.4(8)-diene 1274 23.46 16 3-hydroxycyclopentanone 1342 27.69 17 4-methyl-(2-methyl1356 28.53 18 1 -propenyl)-tetrahydropyrane 2,2,6-trimethylcyclohexanone 1361 28.84 19 octahydro-3A-methyl-cw-2H1371 29.61 20 inden-2-one cw-linalool oxide 1446 34.51 21 /ra/w-linalool oxide 1475 36.39 22 citronellal 1485 37.15 23 ? 2-cubebene sesquiterpene, M==204 1528 39.79 24 linalool 1552 41.40 25 ? p-menth-8-en-3-ol 1568 42.42 26 7 p-menth-8-en-3-ol 1575 42.89 27 ? caryophyllene sesquiterpene, M=204 1590 43.86 28 terpinen-4-ol 1599 44.43 29 citronellyl formate 1617 46.33 30 citronellyl acetate 1636 48.61 31 geranyl acetate 1681 54.33 32 citronellol 1684 54.76 33 ? nerolidol sesquiterpene, M=222 1840 34 59.23

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Figure 3. Changes of selected components during autooxidation of Citrus hystrix oil under conditions described above, A - citronellal, B - acetone, C, D - cis, /ra«5-linalooloxides, E p-mentha-l,4(8)-diene, F - p-menth-8-en-3-oL

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Areas of possible applications of this oil are shown in Table 2. In Indonesia, Citrus hystrix oil is generally considered as suitable for the application in foods, but the best application field according to the answers given by our sensory panel was assigned to cosmetic products in case of oil A, but some food products could be successfully flavoured as well. The reverse rank was foimd in the case of oil B. The application would thus depend on the actual composition of the material.

Table 2 Possible applications oi Citrus hystrix oils (% of total responses). Application Perfumes Cosmetic creams Toothpastes Fruit beverages Liqueurs Candy

Oil A 48 52 47 39 36 37

OilB 29 33 31 41 35 40

5. CONCLUSION 1. Essential oil from Citrus hystrix is shnilar to other citrus oils, consisting mainly of terpenes and related compounds. 2. The odor character is influenced by main terpenic components, but minor compounds also affect its sensory profile. 3. The area of application depends on the actual chemical composition and thus the odour profile of the sample. 4. During oxidation, the odour character is affected marginally, and typical oxidation products of monoterpenes represent main oxidation products. 5. Antioxidants, such as rosemary extract or 1,4-dihydropyridines, not only inhibit oxidation of Citrus hystrix oil, but also influence the composition of oxidation products.

6. REFERENCES 1 E. Gildmeister and F. HofBnann, Die Atherischen Ole, Band V, Akademie Verlag, Berlin (1959). 2 A. Sato, K Asano and T. Sato, J. Essent. Oil Res. 2 (1990) 179. 3 B. M. Lawrence, Perfumer <& Flavorist 18 (1993) 43. 4 C, H. Wijaya, Oriental Natural Flavor: Liquid and Spray-Dried Flavor of "Jeruk Purut" {Citrus Hystrix DC) Leaves. In: Food Flavors: Generation, Analysis and Process Influence. Ed.: G. Charalambous, p. 235, Elsevier (1995).

718 5 B. M. Lawrence, J. W. Hogg, S. J. Terhune and V. Podimuang, Phytochemistry 10 (1971) 1404. 6 C. Moreuil and R. Huet, Fruits 28 (1973) 703. 7 F. Pudil, H. Wijaya and V. Janda, Chemical Composition of Citrus Hystrix Oil. In: XVIII th International Symposium on Capillary Chromatography. Eds.: P. Sandra and G. Devos, Vol. II, 20.5. - 24.5.1996, Riva del Garda, p. 1027 (1996). 8 ISO 6658: Sensory analysis - Methodology - General Guiddance, ISO, Geneva, 1985. 9 ISO 8589: Sensory analysis - General guidance for the design of test rooms. ISO, Geneva, 1988. 10 ISO 8586: Sensory analysis - General guidance for the selection, training and monitoring of assessors. ISO, Geneva, 1989. 11 ISO 6564: Sensory analysis - Flavour profile. ISO, Geneva, 1985. 12 ISO 4121: Sensory analysis - Grading of food products by methods using scale categories. ISO, Geneva, 1988.