Interntionl Journl of PhrmTech Reserch CODEN (USA): IJPRIF, ISSN: 0974-4304 Vol.8, No.4, pp 551-557, 2015 Anlysis of Totl Protein nd Non Protein Nitrogen in Wter nd Met (Cocos Nucifer L.) by using Kjeldhl Method Siti Morin Sing*, Lind Mrgt nd Jnsen Sillhi Fculty of Phrmcy, University of Sumter Utr Medn Indonesi, 20155 Abstrct : In Indonesi, coconut plm is one of the biggest contributors for the economy of the people nd ntion. As food, coconut wter nd coconut met contin some nutrients such s crbohydrte, ft, nd lso protein. During mturtion, chnges in protein content of coconut wter nd coconut met my hppen. The im of this study ws to determine the concentrtion of totl protein nd non protein nitrogen (NPN) in coconut wter nd coconut met, nd their chnges in young nd mture coconuts. The smples used were young nd mture coconuts which were obtined from villger s grden locted t Des Sintis, Kecmtn Percut Sei Tun, Kbupten Deli Serdng, Medn. Totl protein nd NPN determintion were done for coconut wter nd coconut met by using Kjeldhl method which is simple method for totl nitrogen determintion in protein nd other nitrogenous compounds. The result shows tht the totl protein contents in dry smples re: young coconut wter 2.19 g/100g; mture coconut wter 1.13 g/100g; young coconut met 4.14 g/100g; mture coconut met 2.87 g/100g. Totl protein contents in fresh smples re: young coconut wter 0.102 g/100g; mture coconut wter 0.028 g/100g; young coconut met 1.529 g/100g; mture coconut met 2.725 g/100g. NPN contents in dry smples re: young coconut wter 0.18 g/100g; mture coconut wter 0.13 g/100g; young coconut met 0.28 g/100g; mture coconut met 0.12 g/100g. NPN contents in fresh smples re: young coconut wter 0.008 g/100g; mture coconut wter 0.004 g/100g; young coconut met 0.103 g/100g; mture coconut met 0.117 g/100g. True protein contents in dry smples re: young coconut wter 1.22 g/100g; mture coconut wter 0.44 g/100g; young coconut met 2.66 g/100g, mture coconut met 2.22 g/100g. True protein contents in fresh smples re: young coconut wter 0.056 g/100g; mture coconut wter 0.011 g/100g; young coconut met 0.981 g/100g; mture coconut met 2.109 g/100g. The results show tht totl protein nd NPN contents in young coconut wter nd met re higher thn in mture coconut wter nd met. Keywords: coconut wter, coconut met, protein, non protein nitrogen. Introduction In Indonesi, coconut plm is one of the biggest contributors for the economy of the people nd ntion. All prts of coconut fruit, from its outer shell to its met, hve some benefits. As food, coconut wter nd coconut met contin high nutrients. The nutrients in coconut fruit hve the sme roles nd functions s in the nutrients in other food substnces. There re six nutrients tht must be contined in foods, such s crbohydrtes, proteins, fts, vitmins, minerl slts nd wter 1,2. wter is the liquid contined in coconut fruit. The min components re wter, potssium, smll mount of crbohydrtes, fts, proteins, nd minerl slts. Nitrogen cn be obtined from protein
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 552 contined in coconut wter. Although it is low, protein in coconut wter composed from 17 kinds of mino cids. Moreover, the protein content in coconut wter is directly bsorbble by the body. met hs some nutrients including proteins, fts, crbohydrtes, clcium, phosphorous, iron, scorbic cid, nd wter 3,4,5,6. Proteins re one of mcronutrient group. Proteins hve importnt role in the formtion of biomolecules. However, if the orgnism is lck of energy, proteins cn be used s energy source. The proteins re built up from simpler substnces clled mino cids, so free mino cids nd other relted simple nitrogenous compounds lwys occur in ssocition with proteins 7,8. The primry nutritionl importnce of protein is due to the fct tht they re the mjor source of essentil mino cids. The essentil mino cids must be supplied in the diet either s free mino cids or s constituents of the dietry proteins. It is well known tht proteins differ in their nutritive vlues due to the vribility of mino cids composition, digestibility nd vilbility of the digested proteins. To be bsorbed, proteins must be broken down to individul mino cids or smll peptides. The finl results of the proteins digestion re minly in the form of mino cids, nd they re directly bsorbed in 15 minutes fter mels 9,10,11. Chnges in contents nd chemicl composition of coconut during mturtion hve been reported. The chemicl composition chnges in coconut wter from six to nine months coconut fruits hve been reported. The protein nlysis results show tht protein content decreses from 9.5 g/l to 6 g/l. The chemicl composition chnges in coconut met hve lso been reported. mets were tken from four vrieties of coconut fruit in mturtion rnks s follows 5, 7, 9, 11, 13 nd 14 months old. The nlysis protein result shows tht protein contents in four vrieties of coconut met increse t 7 months old but grdully decrese t 9 until 14 months old 12,13. The im of this study ws to determine totl protein nd non protein nitrogen (NPN) chnges in coconut wter nd met from young nd mture coconut fruit. The method used for nlysis of totl protein nd NPN is Kjeldhl method. Experimentl Apprtus Apprtus used in this study were nlyticl blnce, Kjeldhl flsk, gs stove, wter hose, liebig condenser, sttive, clmps, Whtmnn filter pper 42, hot plte, mgnetic stirrer, luminium cup, qurtz snd, sptul, desicctor, oven, burette nd lbortory glsswre. Mterils wter nd met used in this study were derived from young nd mture nuts of coconut plm plnted on villger s grden locted t Des Sintis, Kecmtn Percut Sei Tun, Kbupten Deli Serdng, Medn. All chemicl mterils used in this study were 98% concentrted sulfuric cid, cupric sulfte, potssium sulfte, distilled wter, 40% sodium hydroxide b/v, 0.02 N sulfuric cid, methyl red, methylene blue, phenolphthlein, 0.02 N sodium hydroxide nd 10% trichlorocetic cid b/v. Smples Preprtion Young coconut wter: 20 g qurtz snd nd 20 g young coconut wter were plced in weighed luminium cup, which then were dried in the oven t 70 C for 3 hr, cooled in the desicctor nd weighed. The luminium cup nd smple were dried gin in the oven for 30 min, cooled in desicctor nd weighed. This tretment ws repeted until constnt weight (the difference of consecutive weighing is less thn 0.2 mg) ws chieved. 5 g of qurtz snd nd dry young coconut wter mixture which ws equl to 0.25 g dry young coconut wter ws then used for totl protein nd NPN determintion in young coconut wter 14. Mture coconut wter: 20 g qurtz snd nd 20 g mture coconut wter were plced in weighed luminium cup, which then were dried in the oven t 70 C for 3 hr, cooled in the desicctor nd weighed. The luminium cup nd smple were dried gin in the oven for 30 min, cooled in desicctor nd weighed. This tretment ws repeted until constnt weight (the difference of consecutive weighing is less thn 0.2 mg) ws
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 553 chieved. 10 g of qurtz snd nd dry mture coconut wter mixture which ws equl to 0.25 g dry mture coconut wter ws then used for totl protein nd NPN determintion in mture coconut wter 14. Young coconut met: 15 g young coconut met were mshed nd plced in weighed luminium cup, which then were dried in the oven t 70 C for the first 1 hr, then t 105 C for the next 2 hr, cooled in the desicctor nd weighed. The luminium cup nd smple were dried gin in the oven for 30 min, cooled in desicctor nd weighed. This tretment ws repeted until constnt weight (consecutive weighing difference is less thn 0.2 mg) ws chieved. 0.25 g dry young coconut met ws then used for totl protein nd NPN determintion in young coconut met 14. Mture coconut met: 5 g mture coconut met were mshed nd plced in weighed luminium cup, which then were dried in the oven t 70 C for the first 1 hr, then t 105 C for the next 2 hr, cooled in the desicctor nd weighed. The luminium cup nd smple were dried gin in the oven for 30 min, cooled in desicctor nd weighed. This tretment ws repeted until constnt weight (consecutive weighing difference is less thn 0.2 mg) ws chieved. 0.25 g dry mture coconut met ws then used for totl protein nd NPN determintion in mture coconut met 14. Regents Preprtion Regents used in this study were 40% sodium hydroxide b/v, 0.02 N sulfuric cid, 0.02 N sodium hydroxide, 10% trichlorocetic cid (TCA) b/v, ctlyst mixture of cupric sulfte nd potssium sulfte, mixed indictor methyl red-methylene blue. 40% sodium hydroxide b/v ws prepred by dissolving 40 g of sodium hydroxide pellets in 100 ml crbon dioxide-free distilled wter. 0.02 N sulfuric cid ws prepred by diluting 1.4 ml sulfuric cid 98% with distilled wter in 1000 ml volumetric flsk. 0.02 N sodium hydroxide ws prepred by dissolving 0.8 g of sodium hydroxide with crbon dioxide-free distilled wter in 1000 ml volumetric flsk. 10% TCA b/v ws prepred by dissolving 100 g of TCA in distilled wter d 1000 ml. Ctlysts for digestion ws prepred by mixing cupric sulfte nd potssium sulfte (1:1). Mixed indictor methyl red-methylene blue ws prepred by dissolving 100 mg of methyl red + 30 mg of methylene blue in 60 ml lcohol 95%, then diluted d 100 ml boiled distilled wter 14,15. Stndrdiztion of Sodium Hydroxide Solution 0.02 N 0.1 g of oxlic cid (C 2 H 2 O 4.2H 2 O) molr mss=126 ws weighed nd plced into 250 ml erlenmeyer flsk, which then 25 ml distilled wter ws dded. After it ws dissolved, 2 drops of phenolphthlein indictor ws dded. Sodium hydroxide which would be stndrdized ws then used for titrtion. The titrtion ws done until the solution chnged to pink 14. Determintion of N-Totl nd Totl Protein Content Dry smple ws weighed nd plced into Kjeldhl flsk, then 2 g of ctlyst mixture nd 3 ml of concentrted H 2 SO 4 were dded. Digestion ws done for bout 3 hr until the color of the digest is cler green. After it ws cooled, 10 ml of distilled wter ws dded nd the solution ws trnsferred into erlenmeyer flsk. 15 ml of 40% NOH were dded to the solution until its color chnged to blck nd then it ws distilled. 25 ml of 0.02 N H 2 SO 4 nd 3 drops of mixed indictor were dded into the receiving flsk. The distillte were titrted with 0.02 N NOH until the distillte chnged from purple to green. Crry out blnk determintion in the sme wy without the smple 14. N-totl content ws clculte using the following expression: where, N NOH = Normlity of NOH fter stndrdiztion Totl protein content ws clculted using the following expression:
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 554 where, conversion fctor for coconut = 5.3. N-totl nd totl protein contents in fresh smples were clculted by converting N-totl nd totl protein contents in dry smples to N-totl nd totl protein contents in fresh smples. Seprtion of Protein from Non Protein Nitrogen Seprtion of protein from NPN ws done by precipitting protein in the smples using 10% TCA. Dry smple ws weighed nd plced into 200 ml glss beker. 50 ml of distilled wter ws dded nd llowed to stnd for 30 min. 10 ml of 10% TCA ws dded, llowed to stnd for 30 min, nd then filtered. The precipitte which contined true protein ws wshed twice with TCA solution 17. Determintion of N-Protein nd True Protein Content True protein content ws determined fter seprtion process from NPN. The nitrogen content in protein precipitte obtined ws determined by using Kjeldhl method, s ws done for the determintion of totl protein. Protein precipitte ws plced into Kjeldhl flsk. 2 g of ctlyst mixture nd 3 ml of concentrted H 2 SO 4 were dded. The next procedure is s sme s the procedure for totl protein determintion 17. N-protein content ws clculted by using the following expression: where, N NOH = Normlity of NOH fter stndrdiztion True protein content ws clculted using the following expression: where, conversion fctor for coconut = 5.3. N-protein nd true protein contents in fresh smples were clculted by converting N-protein nd true protein contents in dry smples to N-protein nd true protein contents in fresh smples. Determintion of Non Protein Nitrogen Content NPN content ws clculted by subtrcting N-protein from N-totl. NPN content in smples ws clculted using the following expression: NPN content expressed s percent of totl nitrogen ws clculted by using the following expression: Dt Anlysis using Sttistics Nitrogen nd protein contents in ech smple were nlyzed using t test stndrd devition method. Stndrd devition ws clculted using the following expression: SD = Dt ws rejected if t vlue t tble t the confidence intervl of 99% (α = 0,01). t vlue ws clculted by using the following expression: t vlue = where, SD = Stndrd devition X = Protein content
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 555 = Men of the protein content n = number of determintions The ctul protein content ws clculted using the following expression: μ = ± t tble x where, µ = Actul protein content = Men of the protein content SD = Stndrd devition n = number of determintions 18,19. Results nd Discussion Smple Identifiction Result The smple identifiction result shows tht smples used for nlysis re coconut fruit (Cocos nucifer L.) from fmili Areccee. Totl Protein, True Protein nd Non Protein Nitrogen Contents in Smples The results of N-totl, N-protein, totl protein, true protein nd NPN determintion in young coconut wter, mture coconut wter, young coconut met nd mture coconut met re shown in Tble 1. Tble 1. N-Totl, N-Protein, Totl Protein, True Protein nd Non Protein Nitrogen Contents in Smples Smple Young Wter Mture Wter Young Met Mture Met Kdr (g/100g) N-Totl N-Protein Totl Protein True Protein NPN 0,41±0,0666 0,23±0,0285 2,19±0,3439 1,22±0,1563 0,18 0,019±0,0034 b 0,011±0,0018 b 0,102±0,0156 b 0,056±0,0076 b 0,008 b 43,90 * 0,21±0,0285 0,08±0,0232 1,13±0,1362 0,44±0,1130 0,13 0,006±0,0013 b 0,002±0,0023 b 0,028±0,0033 b 0,011±0,0018 b 0,004 b 61,90 * 0,78±0,0360 0,50±0,0750 4,14±0,1974 2,66±0,3986 0,28 0,288±0,0127 b 0,185±0,0276 b 1,529±0,0732 b 0,981±0,3286 b 0,103 b 35,90 * 0,54±0,0390 0,42±0,0441 2,87±0,2076 2,22±0,2183 0,12 0,514±0,0370 b 0,397±0,0418 b 2,725±0,1912 b 2,109±0,4636 b 0,117 b Ech vlue represents n verge of six determintions (n=6) : content in dry smple b : content in fresh smple * : content expressed s percent of totl nitrogen in smple 22,22 * Totl protein nd true protein content in young nd mture coconut wter re significntly different, s well s in young nd mture coconut met. Totl protein content mesured on dry weight in young coconut wter (2.19 g/100g) is higher thn in mture coconut wter (1.13 g/100g), likewise, totl protein content in young coconut met (4.14 g/100g) is higher thn in mture coconut met (2.87 g/100g). True protein obtined fter seprtion from NPN decreses. True protein in young coconut wter (1.22 g/100g) is higher thn in mture coconut wter (0.44 g/100g), s well s true protein in young coconut met (2.66 g/100g) is higher thn in mture coconut met (2.22 g/100g). Totl protein, true protein, nd NPN content in young coconut met is lower thn in mture coconut met if mesured on fresh smples, this is due to the higher wter content in young coconut met thn in mture
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 556 coconut met. From sttisticl test results, totl protein nd true protein contents in fresh young nd mture coconut wter re significntly different, s well s in young nd mture coconut met. The higher totl protein content in young coconut wter nd met thn in mture coconut wter nd met is in the greement with the study conducted by Viglir et l. (2006) tht protein content in coconut wter decreses from 9.2 g/l to 6 g/l s the coconut fruit mtures, nd lso in greement with Kurin nd Peter (2009) tht protein content in coconut met increses t 7 months old nd then decreses grdully until 12 months old. Futhermore, the study conducted by Ass et l. (2010) lso shows tht totl protein content in coconut met from 4 vrieties of coconut plm decrese during nuts mturtion. The decresing of protein content in coconut wter nd met is probbly cused by the incresing of other nutrients like fts nd crbohydrtes which increse more rpidly thn the proteins, thus protein content in mture coconut wter nd met is lower 12,13,20. In this study, NPN content obtined in young coconut wter (0.18 g/100g) is higher thn in mture coconut wter (0.13 g/100g), nd in young coconut met (0.28 g/100g) is higher thn in mture coconut met (0.12 g/100g). The decresing of NPN content is probbly cused by the chnges of free mino cids to form proteins during nut mturtion. In most foods, 95% of totl nitrogen exists s protein nd free mino cids, this cn be considered s wy to mesure mino cids content. This shows tht free mino cids content in young coconut wter nd met is higher thn in mture coconut wter nd met, thus young coconut wter nd met is better to be consumed s mino cids source becuse free mino cids is esier to be bsorbed by the gstrointestinl trct 21. Most proteins re digested into mino cids, the rest re digested into tripeptide nd dipeptide. Protein digestion or hydrolisis begins in the stomch by chloride cid nd pepsin. Becuse food sty in stomch for short time, proteins re only digested to polypeptide, proteose, nd pepton mixture. The digestion continues in smll intestines by mixture of protese enzyms like trypsine, chymotrypsine, crboxypeptidse, nd elstse. These enzyms brek the protein from polypeptide to smll peptides like tripeptide, dipeptide nd mino cids. Proteolytic enzyms in stomch nd smll intestines finlly digest most proteins in foods into free mino cids. The end results of the protein digestion which minly in form of mino cids re then bsorbed directly in 15 minutes fter mels 11. If expressed s percent of totl nitrogen, it turns out tht NPN content is considerble. The NPN content expressed s percent of totl nitrogen from the highes to the lowest re mture coconut wter (61.90 g/100g), young coconut wter (43.90 g/100g), young coconut met (35.90 g/100g) nd mture coconut met (22.22 g/100g). The significnce of nonprotein, orgnic, nitrogenous compounds in foods hs been pprecited only in recent yers. These compounds include mino cids, mines, mides, quternry nitrogen compounds, purines, pyrimidines nd N-nitrosmides. They contribute to nutritionl vlue, flvor, color nd other importnt food ttributes. They provide source of nutrients nd growth fctors tht re importnt in mlting, brewing nd pnry fermenttion 22. Conclusion Totl protein contents in dry smples re: young coconut wter 2.19 g/100g; mture coconut wter 1.13 g/100g; young coconut met 4.14 g/100g; mture coconut met 2.87 g/100g. Totl protein contents in fresh smples re: young coconut wter 0.102 g/100g; mture coconut wter 0.028 g/100g; young coconut met 1.529 g/100g; mture coconut met 2.725 g/100g. NPN contents in dry smples re: young coconut wter 0.18 g/100g; mture coconut wter 0.13 g/100g; young coconut wter 0.28 g/100g; mture coconut met 0.12 g/100g. NPN contents in fresh smples re: young coconut wter 0.008 g/100g; mture coconut wter 0.004 g/100g; young coconut met 0.103 g/100g; mture coconut met 0.117 g/100g. True protein contents in dry smples re: young coconut wter 1.22 g/100g; mture coconut wter 0.44 g/100g, young coconut met 2.66 g/100g, mture coconut met 2.22 g/100g. True protein contents in fresh smples re: young coconut wter 0.056 g/100g; mture coconut wter 0.011 g/100g; young coconut met 0.981 g/100g; mture coconut met 2.109 g/100g.
Siti Morin Sing et l /Int.J. PhrmTech Res. 2015,8(4),pp 551-557. 557 The results obtined in this study show tht the totl protein nd true protein contents in young nd mture coconut wter is significntly different, s well s in young nd mture coconut met. Totl protein, NPN nd true protein contents in young coconut wter re higher thn in mture coconut wter determined on either fresh or dry smples. Totl protein, NPN nd true protein contents in young coconut met re higher thn in mture coconut met determined on dry smples, while on fresh smples, the contents in young coconut met re lower thn in mture coconut met. References 1. Wrisno. Budi Dy Kelp. Yogykrt: Knisus. 2007; 9-12. 2. Rukmn, H.R. Anek Olhn Kelp. Yogykrt: Knisus. 2007; 26. 3. Sutrminingsih, L. Pelung Ush Nt De Coco. Yogykrt: Knisus. 2008; 23. 4. Pmbyun, R. Teknologi Pengolhn Nt De Coco. Yogykrt: Knisus. 2006; 19. 5. Tietze, H., Arthur E., Jut S. : Rediscovered s Medicinl Food. New South Wles: Hrld W. Tietze Publishing Pty/Ltd. 2006; 27. 6. Khomsn, A. Rhsi Seht dengn Mknn Berkhsit. Jkrt: Komps. 2009; 63. 7. Sudrmdji, S., Bmbng H., Suhrdi. Anlis Bhn Mknn dn Pertnin. Yogykrt: Liberty. 1989; 119, 141 142, 144. 8. Duckworth, R.B. Fruits nd Vegetbles. Oxford: Pergmon Press Ltd. 1966; 14. 9. Jeon, I. dn Willim G.I. Anlyzing Food for Nutrition Lbelling nd Hzrdous Contminnts. New York: Mrcel Dekker, Inc. 1994; 141, 160, 162. 10. Eschlemn, M.M. Introductory Nutrition nd Diet Therpy. Phildelphi: J.B. Lippincott Compny. 1984; 104. 11. Almtsier, S. Prinsip Dsr Ilmu Gizi. Jkrt: PT Grmedi Pustk Utm. 2004; 90-93. 12. Viglir, R., Ver L. S., dn Ulysses F. N. Biochemicl Profile of Wter from Plms Plnted in An Inlnd Region. Journl de Peditri. 2006; Vol 82: 310. 13. Ass, R.R., Louis K. K. J., Alexi P., Jen N., dn Ernest K. Physicochemicl Chrcteristics of Kernel During Fruit Mturtion of Four Cultivrs (Cocos nucifer L.). Africn Journl of Biotechnology. 2010; Vol. 9(14): 2140. 14. Sudrmdji, S., Bmbng H., Suhrdi. Prosedur Anlis Bhn Mknn dn Pertnin. Yogykrt: Liberty. 1984; 51-53, 123-124. 15. Ditjen POM. Frmkope Indonesi. Edisi ketig. Jkrt: Deprtemen Kesehtn RI. 1979; 744, 748. 16. Jones, D.B. Fctors for Converting Percentges of Nitrogen in Foods nd Feeds into Percentges of Proteins. Wshington, D.C.: United Sttes Deprtment of Agriculture. 1941; 14. 17. Ktoch, R. Anlyticl Techniques in Biochemistry nd Moleculr Biology. New York: Springer. 2011; 332-333. 18. Hrmit. Petunjuk Pelksnn Vlidsi Metode dn Cr Perhitungnny. Mjlh Ilmu Kefrmsin. 2004; Vol I (3): 122. 19. Hrvey, D. Modern Anlyticl Chemistry. Toronto: McGrw-Hills Compnies, Inc. 2000; 85. 20. Kurin, A. dn Peter K.V. Commercil Crops Technology. New Dehli: New Indi Publishing Agency. 2007; 196-197. 21. McGilvery, R.W. dn Gerld W.G. Biokimi Sutu Pendektn Fungsionl. Edisi ketig. Surby: Airlngg University Press. 1996; 910. 22. Pomernz, Y. dn Clifton E. M. Food Anlysis: Theory nd Prctice. USA: Aspen Publisher, Inc. 2000; 749. *****