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IJSTE - International Journal of Science Technology & Engineering | Volume 3 | Issue 06 | December 2016 ISSN (online): 2349-784X Determination of Characteristics and Vitamin C Content of Pepino Fruit (Solanum Muricatum)

using UHPLC MS/MS

Erny Rossanti Maruapey Sudarminto Setyo Yuwono Research Student Lecturer Department of Agricultural Product Technology Department of Agricultural Product Technology Faculty of Agricultural Technology, Brawijaya University, Faculty of Agricultural Technology, Brawijaya University, Malang, Indonesia Malang, Indonesia

Bambang Dwi Argo Lecturer Department of Agricultural Product Technology Faculty of Agricultural Technology, Brawijaya University, Malang, Indonesia

Abstract

Pepino fruit grows at the altitude of 2700 m asl which is a juicy fruit with various shapes, sizes and colours depending on cultivation process. Common physical appearances of pepino are yellow or golden beige background covered by purple lines that make this fruit visually more attractive than other exotic fruits available in markets. In Indonesia, there are two types of pepino circulating. Purple pepinos have purple rind, white spots, and dark purple stripes. White pepinos have greenish white or ivory rind, purples stripes and while they ripe turn yellowish. Ripe pepinos would be visible from their peculiar rinds and aromas. Purple pepino is orange, while white pepino is is pale yellow. Purple pepinos are easier to get and moslty cultivated, grow well in highlands. In East Java, they are cultivated mainly in Dieng while in East Java they have spread in three villages in Batu, Malang: Tulungrejo, Sumberrejo, and Bumiaji. Results of analysis showed these following values: water content 94.20%, ash content 0.532%, protein content 0.30%, fat content 0.06%, carbohydrate content 5.02%, Fe content 45.2 mg/100ml, K 4145 mg/100ml, soluble solid content 4.63%. Vitamin C content range was analyzed using iodide titration method showed a range of 14-55 mg/100ml while using UHPLC MS/MS gave duplo values in a row as foolow: 44.259 mg/100ml (AA=2,103 mg/100ml, DHAA=42.156 mg/100ml) and 44.324 mg/100ml AA=2,152 mg/100ml, DHAA=42,172 mg/100ml). Keywords: Vitamin C, Ascorbic Acid, Dehydroascorbic Acid, UHPLC MS/MS, Pepino (Solanum Muricatum) ______

I. INTRODUCTION

Pepino (Solanum muricatum), is a scrub originated from the highland of Andes in South America and was a very important plant during the pre-Colombus era [1]. During this era, pepino was not as excellent as other American Solanaceaes like paprika (Capsicum spp), potato (Solanum tuberosum), tobacco (Nicotiana Tabacum) and tomato (Solanum Lycopersicon). However, it remains an important local plant. Therefore, pepino production was specified to local markets in Andean countries like Columbia, Ecuador, Peru, Bolivia and Chile [1,2]. Pepino was exploited again for commercial during 1970-1980s. After the success of Kiwi fruit, New Zealand began to introduce exotic fruits [2]. with several cultivars. Such initiative then was followed by other countries like Spain, France, Italy, Netherland, US, Israel, Korea or Australia [1]. In some recent years, programs to expand pepino have been developed by other countries including Indonesia. Pepino grows at altitudes above 2700 m asl [3]. Also know as an aromatic herbal plant, pepino is also juciy fruit with various sizes, shapes and colours depending on cultivation process. When it is ripe, pepino can be consumed as a dessert such as salad and juice or ice cream. Pepino eventually became a commercial plant in New Zealan, Australia, Israel, Europe, and North America since it is an aromatic, sweet, juicy and nutritious fruit [4]. Pepino is climacteric plant because it is determined by respiration rate [5]. Ethylene production increases at ripening process and postharvest treatment using Ethepon [6]. However, pepino cultivars of El Camino [7] and Toma [8], showed low ethylene production non-climacteric ripening behavior. Harvesting period executed during perfect ripening would produce high quality pepino fruits [8,7]. Furthermore, storage conditions have big impacts on postharvest physiology and pepino shelf life [9]. According to Maroto et al [10], there is no significant change in total soluble solids content during ripening process. According to Lizana and Levano [5], pepino fruits can be stored at 10⁰C, if stored at 5⁰C or below, they would undergo damage faster and those harvested at early ripening process would be damaged faster than ripe fruit. According to El-Zeftawi et al. [6], pepino’s shelf life post harvest is one to two weeks at room temperature and shelf life would be extended if it is stored under 10⁰C with

Determination of Characteristics and Vitamin C Content of Pepino Fruit (Solanum Muricatum) using UHPLC MS/MS (IJSTE/ Volume 3 / Issue 06 / 045) relative humidity of 90-95%. Ahymada and Cantwell [8], stated that pepino storage under 5⁰C results in severe damage on the surface and internal change of colours. These are the similar effects undergo by tropic and subtropic fruits stored under 12⁰C, with equal symptoms marked by abnormal ripening, uneven surface, change in colors, and increase in water loss [11]. Most common physical appearances of pepino are are yellow or golden beige background covered by purple lines that make this fruit visually more attractive than other exotic fruits available in markets [12]. In Indonesia, there are two types of pepino circulating. Purple pepinos have purple rind, white spots, and dark purple stripes. White pepinos have greenish white or ivory rind, purples stripes and while they ripe turn yellowish. Ripe pepinos would be visible from their peculiar rinds and aromas. Purple pepino is orange, while white pepino is is pale yellow. Purple pepinos are easier to get and moslty cultivated, grow well in highlands. In Cenral Java, they are cultivated mainly in Dieng while in East Java, according to local agency for industrial and trade, they have spread in three villages in Batu, Malang: Tulungrejo, Sumberrejo, and Bumiaji with total area up to 5 ha.

II. MATERIAL AND METHOD

Pepinos used in this study were fresh fruits with no dents, wounds and bruises with equal sizes. They were obtained from traditional market in Malang, East Java. Vitamin C analysis was performed using iodine titration method [13,14], and UHPLC MS/MS with column conditions: 20⁰C, mobile phase 0.2% formic acid with water rate 0.4ml/min, suing negative ions at 4000V capilary voltage, nebulizer pressure 60psi, dryer gas flow 11L/min and dryer temperature 350⁰C. Into samples was added 1% EDTA with 1:1 ratio, 1 ml of sample was centrifuged at 4500rpm for 5 minutes, filtered using 0,2µm filter and injected at 10⁰C sample temperature.

III. RESULTS AND DISCUSSION

Fe and K are essential soil nutrients required by by plants. Fe is one of micronutrients while K is one of macronutrients. The function of K element is to increase photosynthesis process, shaping stronger trunk, strengthening rooting, and increasing resistance to pest and disease attack. Fe is a micronutrient that plays an important role to the end result of of plant such as sweet taste of a fruit. Differences in Fe and K contents in research reports and literatures are caused by the differences of Fe and K soil availabilities in areas where pepinos grow. Area with higher soil Fe and K content is presumably due to excessive Fe and K fertilizers treatment by pepino farmer.

Fig. 1: Diversity of Size, Shape and Color of Pepino Fruit

Total soluble solids contents in this study ranged around 4.63% and are different from a research conducted by Arriola et al [15], with 9.5%. Hulme [16] said that fruits contain large amounts of carbohydrate as simple sugars like sucrose, glucose and fructose which become the source of total soluble solids. Therefore, it can be said that an increase in total solids contents will be in line with an increase in total soluble solids. Total soluble solids of material include reducing sugar, non-reducing sugar, protein, organic acids and pectins. The higher sucrose content in a ripe fruit, the higher is its total solid [17]. Total soluble solids content in pepino juice was obtained in a range of 5.3 ± 0,16⁰Brix which low sweetness level. This was due to the high water content of pepino fruits which reach 94.2% while other studies showed 90 – 92% [18,19,20]. Table – 1 Characterization of Pepino (Solanum muricatum) Characterization Result Literature Water content (%) 94.20 90 – 92 [18,19,20] Ash content (%) 0.42 - Protein (%) 0.30 0.1 – 0.93 [4,21] Fat (%) 0.06 - Carbohydrate (%) 5.02 7 [20] Fe (mg/mL) 0.45 0.002 – 0.0031 [4]

Determination of Characteristics and Vitamin C Content of Pepino Fruit (Solanum Muricatum) using UHPLC MS/MS (IJSTE/ Volume 3 / Issue 06 / 045)

K (mg/mL) 41.45 1.15 – 1.23 [4] Soluble solid (%) 4.63 9.5 [15] pH 4.5 - Vitamin C (mg/mL) 0.14 – 0.55 12.98 – 68 [4,19,20,15] Color : L* value 31.90 - a* value 2.20 - b* value 3.90 -

(a) (b) Fig. 2: Vitamin C Chromatogram (Ascorbic Acid and Dehydro-Ascorbic Acid) using UHPLC MS/MS Methods. A: Vitamin C content in pepino extract; B: Standard of vitamin C.

High Fe content in samples allowed natural oxidation to occur in fruits. Gerald [22] stated that vitamin C as ascorbic acid is unstable toward Fe and a way to keep it stable is to be put in an enclosed and neutral pH condition. Factor that is expected to be the cause of composition difference among analyses with literature is different harvest age. Harvesting at perfect ripening time will generate high quality pepinos [8,23]. Moreover, storage conditions highly affect postharvest physiology and pepino shelf life [9]. Lizana and Levano [5] summarized that proper storage temperature is at 10⁰C. Storage at 5⁰C or below would accellerate fruit damage. Based on colours analysis, pepino jas brightness values of L* 31.9±2.9, a* 2.2±0.9 dan nilai b* 3.9±1.38. Positive a* value obtained indicates that samples have red color type and negative b* value obtained indicates blue color type. Vitamin C analysis using iodine titration with 6 replications gave a range of vitamin C value of 14-55 mg/100ml while UHPLC MS/MS gavein a row duplo values of 44,259 mg/100ml (AA=2,103 mg/100ml, DHAA=42,156 mg/100ml) dan 44,324 mg/100ml (AA=2,152 mg/100ml, DHAA=42,172 mg/100ml). Mean value of vitamin C in pepino fruits was 44.295 mg/100ml with ascorbic acid (AA) content of 2.128 mg/100ml or 4.80% and dehydroascorbic acid (DHAA) content of 90.39% or 95.19%. Results showed that DHAA content was higher than AA as shown in Figure 2 chromatogram. According to Rahmawati [24], askobase (ascorbic acid oxidase) is an enzyme that works at a range of pH 4.0 – 7.0 with optimal activity at 5.6 – 6.0 and catalyzes ascorbic acid oxidation either in natural or synthetic form but does not catalyze other compounds like cysteine, gluthatione, tyrosine, and phenol. In intact fruits, ascorbase enzyme is separated from vitamin and very controlled. However, when there is a change in cell’s structure due to mechanical process then ascorbase is activated and the available oxygen induces oxydative breakdown of vitamin C [25]. Tissue damage due to destruction is easily affected by air and therefore allows vitamin C as AA easily oxidized become DHAA as stated by Ashari [26], that commonly damage and loss of vitamin are due to tissue damage and air exposure. Moreover, other cause of oxidated AA to DHAA is a high Fe content in samples that allowed an increase ascorbase activity in vitamin C breakdown. Furthermore, high water content in pepino fruits that reached 94.2% and supported by very water soluble characteristic of vitamin C causes vitamin C is easly lose due to surface damage or while cutting the fruit. The effect of water activity to vitamin C stability has been demonstrated by some experts that the rate of vitamin C damage in food would increase

Determination of Characteristics and Vitamin C Content of Pepino Fruit (Solanum Muricatum) using UHPLC MS/MS (IJSTE/ Volume 3 / Issue 06 / 045) in line with the increasing water activity [25]. These conditions are the causes of lower value of AA than DHAA in samples. Due to one electron removal from ascorbic acid which then rapidly changes to DHAA [27,22]. This is supported by George [28] statement that AA is very easily oxidized reversibly to DHAA but both compounds have activities as vitamin C.

IV. CONCLUSION

Results of analysis showed water content of 94.2%, ash content 0.425, protein 0.3%, fat 0.06%, carbohydrate 5.02%, Fe 45.2 mg/100ml, K 4145 mg/100ml, soluble solid 4.63% and vitamin C analysis using iodine titration with 6 replications gave the range of vitamin C content was 14-55 mg/100ml while using UHPLC MS/MS gave in a row duplo values of 44,259 mg/100ml (AA=2,103 mg/100ml, DHAA=42,156 mg/100ml) and 44,324 mg/100ml (AA=2,152 mg/100ml, DHAA=42,172 mg/100ml).

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