Nutritive Components of Some Fresh and Refrigerated Fruits Ms. C. Sownthariya Research Scholar and Dr. P. Shanthi Assistant Professor, Department of Botany Holy Cross College (Autonomous) Tiruchirappalli-620002 Abstract An experiment was conducted to study the impact of refrigeration on some common fruits (Malus sylvestris, L., Psidium gujava, L., Citrus aurantium, L., Punica granatum, L., Achras sapota, L.,) under 10°C after 20 days. The present investigation was aimed to estimate the nutritive components (total sugar, reducing sugar and total amino acid content) and secondary plant product (total phenol) of some fresh and refrigerated fruits. Among the selected fresh fruits, P. gujava showed high level of reducing sugar (4.4 mg/g), total sugar (3.6 mg/g), total amino acid content (1.7 mg/g) and total phenol content (0.8 mg/g). Refrigeration, enhanced the total sugar, reducing sugar and total amino acid content, also decreased the total phenol content invariably in all selected fruits. After the storage period, C. aurantium registered the highest amount of reducing sugar (7.1 mg/g), total sugar (5.3 mg/g), total amino acid (2.2 mg/g) and total phenol content (0.1 mg/g). Exposure of fruits at low temperature (10ºC) affects the nutrient and physical nature of the fruit. Key Words : Fresh fruits, refrigerated fruits, total sugar, reducing sugar, total amino acid, total phenol. Introduction Fruits are the richest source of vitamins, minerals, dietery fibre and other nutrients that maintain the body with good health. It contains fibre in the form of cellulose, hemicelluloses, pectin and protopectin (Anuradha Subramanian, 1998). A fruit when exposed to extreme temperatures (either high or low), some reactions may be accelerated or retarded to excess and the result will be that, the general ripening pattern will be permanently disturbed by the under – production of substances which may be toxic (Hulme, 1970). Refrigeration or cold storage has relatively adverse effects on taste, texture, nutritive value and permits exchange of flavours between fruits and other foods, which affects the other attributes of fruits. Another common change in fruit during refrigerated storage involves loss of firmness and crispness. It preserves perishable fruits for days or weeks and must be stabilized against enzymatic changes during frozen storage. Too low temperature Academia and Society Volume 2 Number 2 can cause damage called “chill injury” to fruits when those are not physically damaged by freezing. In case of bananas and tomatoes, at storage temperatures of below 13°C slow down the activities of natural ripening enzymes and results in poor colours (Norman and Joseph, 1996).Temperate fruits such as apples may tolerate from 0-4˚C and tropical bananas behave abnormally at temperatures below 12˚C. In apples, low temperature injury usually appears as a browning in the cortical region, with streaks of darker brown in the vascular tissues (Hulme, 1970). Fruits spend a significant length of time from the day of harvest till they reach the consumer and undergo preservation by chemicals, hormones and low temperature exposure. The main goal of preservation is to prevent the growth of undesirable microbial flora and to avoid perish ability of fruits. But exposure of fruits to low temperatures for longer duration has a major effect in the deterioration of their nutrients. So, an experiment was designed to study the impact of refrigeration of some common fresh fruits (Malus sylvestris, L., Psidium gujava, L., Citrus aurantium, L., Punica granatum, L., Achras sapota, L.,) under 10°C for 20 days on certain biochemical components (total sugar, reducing sugar and total amino acid content) and secondary plant product (total phenol). Materials and Methods Fruits selected for the study were M. sylvestris (apple) of Rosaceae, P. gujava (guava) of the family Myrtaceae, C. aurantium (orange) of the family Rutaceae, P. granatum (pomegranate) of Punicaceae, A. sapota (sapota) of Sapotaceae. Fresh fruits mentioned above were purchased from the local Gandhi market, Tiruchirappalli. They were analyzed for certain biochemical components (total sugar, reducing sugar and total amino acid content) and secondary plant product (total phenol) at two different conditions. One set of each fruit was wrapped in polythene bags and stored in the refrigerator for 20 days at 10°C. After 20 days, the refrigerated fruits were examined for both the activities. Sample extraction Ethanolic extract : Hundred milligrams of the fruit sample was homogenized with 5ml of 80% ethanol and centrifuged at 2000rpm for 10 minutes. The pellet was extracted again with the same solvent and centrifuged again. The supernatant were pooled. Total Sugar (Anthrone method) (Dubios, et al., 1956) To 1ml of protein free carbohydrate solution, 4ml of the anthrone reagent (0.2% in conc. H2SO4) was ISSN : 2393-9419 eISSN : 2393-8919 13 added and rapidly mixed. The reaction mixture was heated for 5 minutes in a boiling water bath at 100°C with a marble on the top of the test tube to prevent loss of water by evaporation. Suitable reagent blank was prepared. The colour intensity was measured at 620nm in a spectronic-20. The amount of total soluble sugar was calculated by referring to a standard curve of D-glucose and the values were expressed as mg/gram fresh tissue. Reducing Sugar (Nelson - Somogyi method) (Mahadevan and Sridhar, 1982) To 1 ml of the extract, 1 ml of distilled water and 1ml of alkaline copper tartrate reagent was added and boiled until the development of orange colour. Along with the sample, blank was boiled (1 ml distilled water and 1 ml alkaline copper tartrate reagent). After boiling, to the cooled tubes, 1 ml of arsenomolybdate reagent was added and made upto 10 ml using distilled water. The colour developed was read at 620 nm in a colorimeter. The amount of reducing sugar was calculated using the glucose standard graph and the values were expressed as mg glucose/gram fresh tissue. Results and Discussion Morphological Variation The fruits selected for the study showed a wide range of morphological difference after refrigeration (Plate-1). Due to refrigeration, there was complete discolouration, shrinkage of outer peel, poor firmness and pitting was noted invariably in all the selected fruits. Also, there was a characteristic watery breakdown in M.sylvestris, A.sapotaandC. aurantium.Futhermore, Norman and Joseph (1996). Jeanelle Boyer and Rui H Lin (2004) reported internal browning, soggy breakdown, rind disorders, depressions and browning in fruit husk, loss of weight and shrinkage, poor firmness and peel colour, pitting, watery breakdown, internal browning, internal discolouration, decayas characteristic injuries on different fruits such as apple, pomegrantate, grapes, orange, squash, sapota, guava, watermelon, lemon and mango during refrigeration. Plate-1 Fresh and Refrigerated Fruits (After 20 Days at 10°C) Total Free Amino Acid (Moore and Stein, 1948) To 1ml of alcoholic extract, 0.5ml of acetate buffer was added, followed by 1ml of 1% Ninhydrin. The reaction mixture was heated for 15 minutes in a boiling water bath at 100°C for colour development. It was then cooled and the volume was diluted to 10ml with distilled water. For blank, 0.5ml distilled water was taken and all the reagents were added and carried out as above. The colour intensity was measured at 570nm. The amount of free amino acids was calculated by referring to a standard curve of L-leucine and the values were expressed as mg/gram fresh tissue. Total Phenol (Mahadevan& Sridhar, 1982) 1ml of folin-ciocalteau reagent was added to 1ml of alcoholic extract of the sample. 2ml of 20% sodium carbonate was added and heated for one minute. After cooling, the solution was made upto 10ml with distilled water. A blank was prepared by adding all the reagents except the sample. The absorbancy was read at 650 nm in spectrophotometer. A standard graph was prepared from different concentrations of catechol. The amount of total phenol in the sample was expressed as mg catechol / gm fresh tissue. Academia and Society Volume 2 Number 2 ISSN : 2393-9419 eISSN : 2393-8919 14 Biochemical Compounds In the present study, biochemical compounds such as total sugar, reducing sugar, total amino acid and total phenol recorded a wide range of nutrient content. The variation in the nutritive compounds of the selected fruits were presented in Table – 1. Table – 1 Biochemical components of selected fresh & refrigerated fruits glucose. In apple, during low temperature storage, fructose was reduced to D-glucitol. Also, Dahot and Hanif (1996), studied the properties of invertases in Achras sapota, Linn. The fruit showed 6.5% invertase activity at 20°C and also the enzyme hydrolyses sucrose, raffinose and stachyose but not inulin, cellobiose and maltose. This provides a scientific evidence of increase in the total sugar content during refrigerated condition, could be attributed to the enhanced activity of invertase like enzyme in the fruits under investigation. Our results are also in line with the report of Biale (1960), he stated that, in Citrus fruit, as a result of metabolism of cell wall polysaccharides, the sugar content may show an initial increase on cold storage and prolonged storage result in decrease in the concentration. Reducing Sugar Total Sugar The total sugar content estimated in the selected fruits, exhibited a tremendous increase during the refrigerated condition. The total sugar content present in the 5 selected fruits, ranged between 1.5to 5.5mg/g fresh tissue (Fig-1). The highest amount was recorded in C. aurantium during fresh (3.3 mg/g) and refrigerated conditions (5.3 mg/g). It was followed by P. Gujava which showed 3.6mg/g in fresh condition and 5mg/g under refrigeration. Also, fresh P. granatum and A. sapotarecorded 2.3 and 2.5 mg/g of total sugar content respectively. Whereas, the lowest amount (1.8 mg/g) was recorded in fresh M. sylvestris. Under refrigeration, A. sapota, M. sylvestris and P. granatum recorded 3.9, 3.6 and 3.2 mg/g respectively. The sugar concentration in fruits is often determined by the difference between reducing sugar content before and after inversion. According to the report of Hulme (1970), invertase, is an enzyme which hydrolyses sucrose into fructose and Academia and Society Volume 2 Number 2 Similar to the total sugar, the highest amount of reducing sugar among the selected fruits was estimated from the fresh fruit of P. gujava (4.4mg/g fresh tissue) followed by P. granatum (3.1 mg/g). In other fruits, the amount of reducing sugar was significantly low in C. aurantium (1.1 mg/g), A. sapota (1.6 mg/g) and M. sylvestris (1.8 mg/g) (Fig-2). Invariably, in all the selected fruits, the reducing sugar content showed a remarkable increase except in P. gujava which revealed only a slight change. In refrigerated condition, the maximum amount of reducing sugar (7.08mg/g) was recorded in C. aurantium, followed by P. granatum (5.7 mg/g), M. sylvestris (5.3 mg/g), P. gujava (4.9 mg/g) and A. sapota(4.6 mg/g).It is evident that, whenever a non-reducing sugar is hydrolysed, an equal amount of reducing sugaris replaced, during refrigeration. Our result finds an evidence of Biale(1960). He reported that, mango showed a large increase in ISSN : 2393-9419 eISSN : 2393-8919 15 sucrose concentration and a small proportion of reducing sugars, as starch is hydrolysed. Later during cold storage, sucrose tends to disappear to be replaced by an equal amount of reducing sugar. According to the report of Albert and John (1983), the invertase activity increased in the flavedo tissue of marsh grape fruit (Citrus paradisi, Macf.) stored at 5°C, which in turn paralleled the reducing sugar level i.e., sucrose level was inversely proportional to invertase level. Therefore, after refrigeration, the invertase activity might be one of the possible reasons for the remarkable increase in the reducing sugar content of the present study. Our result on the reducing sugar content of refrigerated fruit also finds scientific evidence of Hulme, 1970. In fruits like passion fruit, cherry (Hulme, 1970), tomato (Dalal et al., 1965) and grape fruit (Kliewer, 1965), reducing sugar often increase steadily throughout their growth and maturation was reported. Total Amino Acid When compared to the fresh fruits, the total amino acid content of selected fruits exhibited an increase in their amount after refrigeration (Fig-3). The increase was found maximum in C. aurantium and P. granatum (2.2mg/g fresh tissue) followed by P. gujava (2.1 mg/g), M. sylvestris (0.8 mg/g) and A. sapota (0.7 mg/g). Among the fresh fruits, the maximum was registered in P. gujava (1.7 mg/g) followed by P. granatum (1.2 mg/g), C. aurantium (0.8 mg/g), M. sylvestris (0.7 mg/g) and the lowest was estimated from the fruit of A. sapota (0.5 mg/g). Increase in the amino acid content of selected fruits in the present investigation might be due to the metabolic changes which could include the enzymatic following scientific evidence. Hulme (1970) opined that, the experimental study of enzyme systems in fruits is different because of the low pH and the presence of polyphenols. Undoubtedly, the free amino acids are to be regarded as metabolic equilibrium with the processes of protein synthesis and degradation (Jacek Patykowski et al., 2007). Total Phenol Phenol is a secondary plant product acts as an antioxidative biomolecule in living systems. In the present study, a reverse trend was noticed with regard to total phenol content of the fruits after refrigeration when compared to all other nutritive components. In all the fruits, the total phenol content in terms of catechol was comparatively higher in fresh fruits than after refrigeration. However, the quantity of phenol estimated from all the fruits showed that it was markedly lower than total sugar, reducing sugar and total free amino acid. The amount of total phenol present in fresh fruits ranged between 0.1 mg/g to 0.7 mg/g. The maximum of 0.7 mg/g of total phenol was recorded in fresh P. gujava, which is two-fold greater than the fresh C. aurantium (0.1 mg/g) followed by, fresh P. granatum and M. sylvestris (0.3 mg/g), A. sapota (0.2 mg/g) was observed. The same after refrigeration showed a range of about 0.1mg/g to 0.2mg/g of phenol which was much lower than in the fresh fruits (Fig-4). The decline in the total phenol content in the present study might be due to the enhanced polyphenolic oxidase activity. According to Hulme (1970), the phenol content was reported to be more in fresh fruit than in other parts of the plant. The amount of phenolics in selected fruits vary widely, based on this, phenol confer resistance against pathogens (Cruickshank and Perrin, 1964, Kamran et. al., 2009; Kumar, et.al., 2010). action on various stored products of the fruits. The observed change in the study is in agreement with the Academia and Society Volume 2 Number 2 ISSN : 2393-9419 eISSN : 2393-8919 16 Thus, the fresh fruit containing phenol could be advantageous and desirable for health of human beings rather than the consumption of the refrigerated fruits. 8. 9. Conclusion From this study, it is concluded that the fresh fruits are an excellent source of nutrients with several health benefits which contained rich amount of nutritive components in terms of total sugars, reducing sugars, total amino acids and total phenol. The morphological degradation, discolouration, water loss in the fruits are important contributors of textural deteriotation due to refrigeration. Thus, refrigeration has adverse effects on taste, texture, nutritive value and involves loss of firmness and crispness. The higher concentration of phenol content in fresh fruits confers resistance against microbial pathogens. Also the increase in the enzyme activity during refrigeration reduced the complex polymers into simpler forms mediated by polyphenol oxidase and phenol peroxidase. This biochemical mechanism interferes with the shelf- life and in turn the palatability of the fruits. Hence, exposure of fresh fruits to low temperatures and storing for longer duration has significant effect on the deterioration of nutritive components. Therefore, prolonged storage of fruits under refrigeration affects its nutrient quality and human health when consumed. 10. 11. 12. 13. 14. 15. References 1. 2. 3. 4. 5. 6. 7. Albert, C., Purvis and John D. Rice.1983. Low temperature induction of invertase activity in grape fruit flavedo tissue. J. Phytochemistry. 22(4): 831-834. Anuradha Subramanian.1998. Concise Food Science. Fruits and vegetables. Soundariya Publications. pp. 52-53. Biale, J.B. 1960. The post harvest biochemistry of tropical and subtropical fruits. Adv.Fd.Res.10: 293-254. Cruickshank, I.A.M. and Perrin, D.R.1964. Biochemistry of Phenolic compounds. Academic Press, New York and London. pp.511-544. Dahot, M.U. and Hanif Noomrio, M. 1996. Purification and some properties of invertases from Achras sapota fruit. J. Islamic Academy of Science. 9(2):31-36. Dalal, K.B., Salunkhe, D.K., Boe, A.A. and Olsen, L.E.1965. Certain physiological and biochemical changes in the developing tomato fruit. J. Fd. Sci. 30: 504-508. Dubios, M., Gilles, K.A., Hamilton, J.K., Roberts, P. A. and Smith, F. 1956. A colorimetric method for the determination of sugars. Nature.168-167. Academia and Society Volume 2 Number 2 16. 17. Hulme. A.C. 1970. The Biochemistry of fruits and their products. Academic Press Inc, New York. Jacek Patykowski, Alina Majczak, Katarzyna Bergier, Maria Sklodowska. 2007. Ascorbate content and peroxidase activities in apple fruits during storage. J. Fruit and Ornamental Plant Research.15: 21-33. Jeanelle Boyer and Rui H Liu. 2004. Apple phytochemicals and their health benefits. J.Nutrition.3: 5. Kamran Ghasemi, Yosef Ghasemi and Mohammad Ali Ebrahimzadeh. 2009. Antioxidant activity, phenol and flavonoid contents of 13 citrus species peels and tissues. Pak. J. Pharm. Sci. 22(3) : 277-281. Kliewer, W.M. 1965, Changes in concentration of glucose, fructose and total soluble solids in flowers and berries of Vitis vinifera. Am. J. Enol Vitic. 16:101-110. Kumar, D., Kumar ,S., Singh, J., Narender, Rashmi, Vashistha, B.D., Singh, N.2010. Free radical scavenging and analagesic activities of Cucumissativus, L. fruit extract. J. Young Pharmacists. 2:365-368. Mahadevan, A. and Sridhar, R. 1982. Method in physiological plant pathology. Sivakami Publications, Chennai. Moore, S. and Stein, W.H. 1948. Photometric ninhydrin methods for use in the chromatography of amino acids. J. Biol. Chem. 176: 367 -388. Norman N. Potter, Joseph H. Hotchkiss. 1996. Food science., 5�ℎ edn., CBS Publishers and Distributors, New Delhi. Srilakshmi. B. 2008. Food Science. , 4th edn, New Age international (P) limited publishers. ISSN : 2393-9419 eISSN : 2393-8919 17