Protein Isolation from Tomato Seed Meal, Extraction Optimization

Transcription

1 Proten Isolaton from Tomato Seed Meal, Extracton Optmzaton GEORGE N. LIADAKIS, CONSTANTINA TZIA, VASSILIKI OREOPOULOU, and CHRISTOS D. THOMOPOULOS ABSTRACT Water extracton of tomato seed meal protens was studed to fnd optmal condtons for proten extracton and solaton. A central composte desgn ncludng temperature, ph, tme and water/solds was used and second order models were employed. Optmum condtons were 5O C, ph.5, mn and water/solds = 3/l (v/w). Expermental values were extracton yeld (extracted proten to that n raw materal) 66.%, proten content of product 7.%, and total proten yeld (proten n solated product to that n raw materal) 43.6%. Estmated values were n good agreement wth expermental values. Optmum condtons were confrmed by a larger scale experment. Key Words tomato, proten extracton, seed meal, optmzaton INTRODUCTION ENVIRONMENTAL POLLUTION caused by food processng wastes could be reduced by approprate recovery of edble nutrents (Brch et al., 976; Green and Kramer, 979; Knorr, 983). Tomato processng wastes, prmarly skns and seeds, comprse to 3% of raw frut weght (Ben-Gera and Kramer, 969; Gesman, 98). Tomato seeds represent 5-55% of the pomace. Tomato pomace s manly dsposed of as anmal feed or fertlzer (Tsatsarons and Boskou, 975; Canella et al., 979; Cantarell et al., 989). A small fracton of the seeds s used by the ol ndustry (Canella et al., 979; Gesman, 98). About X lo6 metrc tons of tomatoes are processed nto products annually n Greece (NSSG, 99), generatng F=, tons of tomato seeds. The potental of tomato seeds as a food source has been reported (Ammerman et al., 963; Droulskos, 976; Kramer and Kwee, 977a,b; Abdel-Rahman, 98; Al-Wandaw et al., 985; Lasztty et al., 986; Rahma et al., 986). The approxmate composton of tomato seeds (dry bass) s fat l-%, proten 5- % and ash 3-7%. The hgh unsaturated fatty acd content of tomato seed ol (C,,,, %, C,,, 55-6%, C,,, %) and the nutrtve value of the proten compare favorably wth soybeans (Rymal, 973; Rymal et al., 974; Brodowsk and Gesman, 98; Lazos and Kalathenos, 988). The hgh lysne content (8- g/6 g N) of tomato seed proten (Rymal et al., 974; Cantarell et al., 989) makes t sutable for supplementng protens n cereal products (Brodowsk and Gesman, 98; Carlson et al., 98; Yaseen et al., 99). In addton the functonalty of tomato seed protens may have many uses n food systems (Kramer and Kwee, 977a; Moharram et al., 984; Doxastaks et al., 988a,b; Doxastaks et al., 988; Kosseoglu et al., 989). Tomato seeds lack antnutrtonal factors or toxc substances often found n other non-conventonal proten sources (Rahma et al., 986). Thus, the recovery and utlzaton of tomato seed proten for human consumpton has been studed (Kwee, 97; Canella et al., 979; Doxastaks et al., 988b; Cantarell et al., 989; Kosseoglu et al., 989). Proten has been solated from tomato seeds usng a 3-step process extracton, precptaton and dryng of proten precp- The authors are afflated wth the Laboratory of Food Chemstry & Technology, Dept. of Chemcal Engneerng, Natonal Techncal Unversty of Athens, 5 Heroon Polytechnou St., Zografou, 578 Athens, Greece. tate (Kramer and Kwee, 977b; Fazo et al., 983). Canella and Castrota (98) examned the effects of several ndvdual factors on proten extracton from tomato seed meal. Latlef and Knorr (983a,b) studed the proten precptaton step usng commercal tomato seeds. Our objectve was to determne the optmal condtons for proten extracton from defatted tomato seed meal, examnng smultaneously effects of temperature, ph, tme and water-tosolds rato. The effect of extracton condtons on proten yeld and on proten content of solated product was also determned. MATERIALS & METHODS Materals Tomato pomace was obtaned from a tomato processng plant (KO- PAIS S.A. Alartos, Greece). It was sundred (5-3O C, 3-4 days) and ground n a blender (Warng Commercal Blendor, Dynamcs Co., New Hartford, CO). The major part of the skns was removed usng a mm seve. The skns remanng on the seve were separated from seeds wth a fan blowng an upward arstream. The seed fracton was ground (Ultra- Centrfugal Mll, Type ZMl, F.K. Retsch GmbH & Co, Haan, Germany) to pass a mm seve. Tomato seed meal was prepared by defattng ground seeds wth n-hexane n a Soxhlet apparatus and grndng (Ultra- Centrfugal Mll, Type ZMl, F.K. Retsch GmbH & Co, Haan, Germany) to pass a.5 mm seve. Proten solaton from tomato seed Tomato seed meal ( g) was extracted wth deonzed water (lol- 3 rato) n a strred glass vessel. The ph of the suspenson (7.5-l S) was kept constant durng the extracton by adjustng wth OSN NaOH. Temperature (3-5 C) was regulated wthn -t. C by a water bath. The slurry was centrfuged at 6Xg for mn, the supernatant was collected and the ph was adjusted to the soelectrc pont (3.9) usng OSN HCl. The proten precptate was separated by centrfugaton at 6Xg for 5 mn and freeze dred. The sold resdue after proten extracton was dred at 6 C under vacuum and was used for proten determnaton. Isoelectrc pont (PI) The pi of tomato seed protens was determned as the ph value of maxmal precptaton. g of tomato seed meal was extracted as descrbed, under the condtons water-to-solds rato Ol (v/w), ph, 4o C, 3 mn. The p was found by ttratng alquots of the collected extract to specfc ph values and determnng the proten content of the supematant after centrfugaton. The proten content was determned accordng to the method of Lowry et al. (95). Analytcal methods Mosture, crude fat, ash, total detary fber and crude proten (Nx6.5) were determned accordng to standard methods (AOAC, 99). Mnerals were determned by atomc absorpton/emsson spectroscopy (Perkn-Elmer Model 38, Perkn Elmer Co., Norwalk, CT). Phosphorus was determned photometrcally by the ascorbc acd method (Hach Company, 989), after dgeston wth concentrated sulfurc acd and hydrogen peroxde (5%) n a Dgesdahl apparatus (Hach Company, Loveland, CO). Total sugars were measured accordng to the phenol-sulfurc acd method (Dubos et al., 956) usng glucose as standard. Volume SO, No. 3, 995-JOURNAL OF FOOD SCIENCE477

2 TOMATO SEED MEAL PROTEIN ISOLATION... Table l-varables and levels for central composte desgn Coded varables levelsa Varable Svmbol - - Temperature ( C) Xl x Tme (mn) x Water-to-solds rato (v/w) x4 Ol 5~ Ol 5l 3Ol a Passage from coded varable (X) level to natural varable (X) level s gven by the followng equatons x=5x + 4; x=x + 9.5; x3=x3 + 4; x4=(5x4 + O)l. Table -Proxmate composton and mneral content of tomato seed meal (dry bass) Tomato seed Tomato seed meal Mosture (%I Proten (%) Crude fat (%I 8..3 Ash (%) Total sugars (%).9 3. Total detary fber f%) 54. K fmg/loo g) 46 Na fmg/loo g) 7 Ca (mg/loo g) 94 Mg fmg/loo g) 49 P (mg/loo g) 93 Fe fmg/loo g) Mn (mg/loo g) 6 Cu fmg/loo g) Zn (mg/loo g) ~...,...,...,...I Fg. l-precptaton of extracted protens from tomato seed meal as related to ph. Expermental desgn and statstcal analyss The process varables (factors) and the responses (dependent varables) were defned from prelmnaty studes and publshed data (Latlef and Knorr, 983a; Rustom et al., 99). The process varables (xr) were temperature (x,), ph (x,), and tme of extracton (x3 and water-to-solds rato (x4). Each varable was coded at fve levels -, -,,, (Table ). Selected responses whch evaluate the extracton process ncluded proten extracton yeld (EY) defned as the rato of total extracted proten to total proten n the raw materal, expressed as percentage. Also ncluded were proten content of the product (PR) and total proten yeld (TY) defned as the rato of total proten n the solated product to total proten n the raw materal, expressed as percentage. A central composte desgn (CCD) was arranged to allow for fttng of a second-order model (Cohran and Cox, 957; Adler et al., 975). The CCD combned the vertces of a hypercube whose coordnates were gven by the factoral desgn (runs -6) wth the star ponts (runs 7-4). The star ponts were added to the factoral desgn to provde for estmaton of curvature of the model (Joklegar and May, 987). Seven replcates at the center pont of the desgn (runs 5-3) were used to allow for estmaton of the pure error sum of squares. All experments were carred out n a randomzed order to mnmze any effects of extraneous factors on the observed responses. The regresson coeffcents Table 3-Central composte desgn arrangement and responses (l-3) and expermental runs at the optmum condtons (3-39) Varable levelsa Responses Run Xt X X3 X4 EY PR TY a Coded varables and the ANOVA tables were computed usng the Data analyss-regresson opton of EXCEL 5. (Mcrosoft Corporaton) program. Optmzaton Optmum extracton condtons were estmated by the steepest ascent method (Adler et al., 975) usng a computer program wrtten n BASIC. The program usng the ftted model for each response searched the expermental space for optmum responses that were generated by feasble combnatons of all factors smultaneously. Several expermental runs were conducted at the predcted optmum condtons; these runs n combnaton wth the earler ones were used for estmaton of new coeffcents for ftted models. Contour plots Varables wth sgnfcant lnear terms were chosen for axes of contour plots for each response. Contour plots were generated by assgnng constant (zero) values to two of the four varables and solvng the ftted equatons as a quadratc equaton n the remanng two varables. RESULTS & DISCUSSION Dred tomato pomace conssted of about 53% seeds and 47% skns (weght bass). The seeds were separated from skns, not only because they contaned most of the protens but also because the essental amno acd content and the bologcal value of seed protens are hgher than those of skn protens (Lasztty et al., 986). The seeds had a proten content of 5.5% (dry bass) consdered adequate for proten recovery. Seeds were defatted, snce tomato seed ol s recognzed as an edble ol (Canella et al., 979). The proxmate composton of tomato seeds and tomato 476-JOURNAL OF FOOD SCIENCE-Volume 6, No. 3, 995

3 l Table 4--Regresson coeffcents for the ftted second-order models Coeffcents bo bl bz b3 b4 bll bzz b33 b44 blz h3 h b3 ba b34 EY x*** **** Intal models PR TY EY x -.64* -..84***.35****.7****.356.8** **.69***.46**** x** ** ** -.7** * *.53** New models PR **.755** *** **** ** TY **.89****.7**.93**** -.47*x ** -.7 PCO. l * P<O.O5 l ** P<O.Ol l *** PO. Table 5-F values, coeffcent of determnaton and coeffcent of varaton for the ft of expermental data to models Intal models New models EY PR TY EY PR TY F values 3.9*** 4.4*** 3.97*** 7.5**** **** R CV(%) l ** P<O.Ol l *** P<O.OOl Table 6bserved and predcted responses at the optmum condtons EY PR TY Pont Predcted Observeda Predcted Observeda Predcted Observeda -,,, ,, a Mean values of four replcates seed meal were compared (Table ). Proten content of tomato seeds was hgher but ash and crude fat content were slghtly lower than reported values (Brodowsk and Gesman, 98; Latlef and Knorr, 983a; Moharram et al., 984). Ths may be due to dfferences n tomato cultvars and processes. Tomato seed meal was rch n proten (3.3% d.b.) and compared favorably wth other olseed meals as a potental nonconventonal source of proten (Yazcoglu et al., 98 ; Ladaks et al., 993). Accordng to Cantarell et al., (989), tomato seed meal s more sutable for proten solates producton because of hgh fber content. Proten precptaton was done at the p whch was 3.9 (Fg. ). Other values of tomato seed protens p have been reported to be between 3.8 and 4.6 (Kramer and Kwee, 977b; Canella and Castrota, 98; Latlef and Knorr, 983a,b; Fazo et al., 983). Proten extracton yeld (EY), proten content of the product (PR) and total proten yeld (TY) obtaned by dfferent combnatons of the extracton condtons were compared (Table 3). Wdely dspersed values of EY were obtaned wth dfferent combnatons of extracton condtons, varyng from 4.8 to 57.3%. TY also showed wdely dspersed values, from 4.5 to 4%. As proten extracton ncreased, more protens were n soluton, and more protens could be precptated, contrbutng to total yeld ncrease, thus explanng the same pattern of EY and TY values. However, PR of solated products dd not vary much, rangng from 67.7 to 76.%, relatvely hgh for such products (Kramer and Kwee, 977b; Tchorbanov et al., 986). Nevertheless, these products were characterzed as concentrates. Model fttng Regresson coeffcents for the ftted models were compared (Table 4). ph and water-to-solds rato were the most sgnfcant 8.5 I 9.5 Fg. -Contour plots for EY as related to ph and water-to-solds rato; the other two varables were fxed at zero coded levels. factors for all models. Especally, for the PR model, ph showed hghly sgnfcant (P <.) lnear and quadratc terms. Ths mples that ph and water-to-solds rato were the predomnant factors for modelng of proten solaton from tomato seed meal, as reported by Rustom et al., (99) for peanuts. Note that temperature was not a sgnfcant factor n any models; Canella and Castrota, (98) reported a smlar effect, however, others have reported that temperature nfluenced proten extracton from protenaceous sources (Drawert et al., 979; Rustom et al., 99). The adequacy of each model was tested by the lack of ft test and the coeffcent of determnaton R (Table 5). All three models were sgnfcant by the F-test at the % confdence level and none of the models exhbted lack of ft. Our three models for EY, PR and TY showed R values of 77.4, 79.5 and 77.6% respectvely, adequate for models of ths type. Reproducbltes of our models were very good (CV 4.5,.8 and 8.6% respectvely). Optmzaton Ftted models were ntroduced as objectve functons n an optmzaton program based on the steepest ascent method. Predcted optmum EY values were 7.3 and 67.5 at (-,,,) and (,,-,) respectvely (varables n coded levels). For PR optmum values of 8.4 and 75. were predcted at (,-.9,-,-) and (-,.7,,) respectvely. For TY the predcted optmum values were 5. and 5.5 at (,,-,) and (-.5,,,) respectvely. As seen from these values, both EY Volume 6, No. 3, 995-JOURNAL OF FOOD SCIENCE479

4 TOMATO SEED MEAL PROTEIN ISOLATION Temperature ( C) Temperature ( C) 5 Fg. 3-Contour plots for PR as related to (A) temperature-p-l, (B) temperature-water-to-solds rato, (Cl ph-water-to-solds rato; the other two varables were fxed at zero coded levels. 7.5 C and TY showed ther optmum values at the same factor regon. Because proten extracton s the predomnant step for proten solaton, we decded to examne further the optmum condtons for EY. Observng optmum EY condtons, note that maxmum EY values were obtaned at the hgh level of ph and water-tosolds rato, low temperature-hgh tme level as well as hgh temperature-low tme level. As s well known, proten solublty ncreases as ph ncreases above 7.5 (Kwee, 97; Kramer and Kwee, 977b; Canella and Castrota, 98; Latlef and Knorr, 983a; Fazo et al., 983). In addton ncreased water-to-solds rato facltates proten extracton. The combnatons of hgh temperature-short tme as well as low temperature-long tme gve better results of proten extracton, avodng proten denaturaton.,. In order to verfy the predcted values a seres of expermental runs at the optmum condtons was conducted, at the ponts (-,,,) and (,,-,) (Table 3). The observed value for EY compared favorably wth the predcted one only at (,,-,). Because of the dscrepances found between predcted and observed values, we decded to reevaluate the coeffcents of ftted equatons usng all 39 expermental runs (l-3 CCD runs optmum ponts runs) (Table 4). For all models no essental change was observed for sgnfcant factors and nteractons; coeffcents had slghtly modfed values, keepng the same sgn. The reftted equatons were also adequate. All adequacy tests were mproved for EY and TY models, especally R* beng 94. and 89.6% for EY and TY respectvely. Only PR model showed a slght decrease n R (Table 5). Predcted values from the reftted equatons (Table 6) and observed values were n very good agreement. The (,,-,) pont was selected as condtons gvng the maxmum EY and TY values. Contour plots Varables wth sgnfcant effect were chosen as axes for contour plots for each response; the other varables were fxed at the central (zero) level. (Fg. -). EY ncreased wth ncreasng water-to-solds rato and ph level (Fg. ). The hghest EY resulted at hgh ph and waterto-solds rato. PR showed maxmum values at temperature >4O C (Fg. 3A,B), ph between (Fg. 3A,C) and low water-to-solds rato (Fg. 3B,C). TY ncreased as ph (Fg. 4A,C) and water-to-solds rato (Fg. 4B,C) ncreased. A sgnfjcant nteracton between ph and water-to-solds rato was observed for TY (Fg. 4C). A larger scale experment was conducted at optmum condtons (,,-,). Tomato seed meal (3 g) was treated followng the same procedure. The experment gave results n close agreement wth those of the small scale, wth an EY of 64.%, and a TY of 4.%. The product proxmate composton and the. sold resdue remanng after proten extracton were analyzed (Table 7). The economc feasblty of proten solaton process also depends on utlzaton of by-products. The whey remanng after proten precptaton could be concentrated or 48O-JOURNAL OF FOOD SCIENCE-Volume 6, No. 3, 995

5 I I I I I Temperature ( C) Temperature ( C) $ 5 L 4 z= y ; k. 3 5 Fg. 4-Contour plots for TY as related to (A) temperature-ph, (B) temperature-water-to-solds rato, (C) ph-water-to-solds rato; the other two varables were fxed at zero coded levels Table 7-Proxmate composton and mneral content of tomato seed proten concentrate and sold resdue after proten extracton (dry bass) Tomato seed Sold proten cone resdue Crude proten (%I Ash (%) Total sugars (%).8.7 Total detary fber (%) K (mg/loo g) 93 4 Na (mg/loo g) Ca (mg/loog) 55 4 Mg (mg/loo g) 8 P (mg/loo g) Fe (mg/loo g) 5 3 Mn b-w/ g) 6 Cu b-w/ gl Zn (mg/loo g) 3 5 spray-dred to recover protens and the sold resdue of the extracton could be used as anmal feed. CONCLUSION OPTIMUM EXTRACTION of tomato seed protens wth water for proten solaton, could be acheved by extractng one part of tomato seed meal wth 3 parts of water (w/v rato) at ph.5 at 5O C for mn. These condtons resulted n extractng 66.% of the protens contaned n tomato seed meal, at a total proten yeld of 43.6%. Isolated product had a proten content of 7%. REFERENCES Abdel-Rahman. A.Y. 98. The chemcal consttuents of tomato seed;. Food Chem. 9 3&38. Adler,, Y.P., Markova, E.V., and Granovsky; Y.V The Des& of Ezpenments to Fnd tmal Con&ons. Mu Publshers, Moscov, Russa. Al-Wandaw, H., Ab B ul-rahman, M., and Al-Shakhly, K Tomato processng wastes as essental raw materals source. J. Agrc. Food Chem Ammerman, C.B., Arrngton, L.R., Loggns, P.E., McCall, J.T., and Davs, G.K Nutrtve value of dred tomato pulp for rumnants. J. Agrc. Food Chem AOAC. 99. Ofcal Methods of Anal ss, 5th ed. Assocaton of Offcal Analytcal Chemsts, Washngt.on,.D 6 Ben-Gera. I. and Kramer. A The utlzaton of food ndustres wastes. Adv. Fobd Res Brch, G.G., Parker, K.J., and Worogan, J.T Food from Waste. Appled Scence Publshers Ltd., London, England. Brodowsk, D., and Gesman, J.R. 98. Proten content and amno acd composton of proten of seeds from tomatoes at varous stages of rpeness. J. Food Sc , 35. Canella, M., Cardnal, F., Castrotta, G., and Nappuc, R Caratter chmc de sem d Domodoro d dfferent vareta. Rv. Ital. Sost. Grasse Canella, M. and Castrota, G. 98. Proten composton and solublty of tomato seed meal. Lebensm. Wss. u.-technol Cantarell, P.R., Parma, E.R., and Caruso, J.G.B Composton and amno acd profles of tomato seeds from cannng wastes. Acta Almentara Carlson, B.L., Knorr, D., and Watkns, T.R. 98. Influence of tomato seed ;t,,.,, on the quahty of wheat flour breads. J. Food Sc , Cohran, W.G. and Cox, G.M Expermental Desgns. John Wley & Sons Inc., New York. Doxastaks, G., Kosseoglou, V., and Boskou, D. 988a. Emulsfjrng propertes of tomato seed protens obtaned from processng waste. Scences des Alments Doxastaks, G., Kosseoglou, V., and Boskou, D. 988b. Functonal propertes of tomato seed proten obtaned from processng waste. In Fronters of Flauor, Proceedngs of the 5th Internatonal Flavor Conference, Port Karras, Chalkdk, Greece, l-3 July 987, G. Charalambous (Ed.), p Elsever Scence Publshers B.V., Amsterdam, Netherlands. Volume 6, No. 3, 995-JOlJRNAL OF FOOD SCIENCE48

6 TOMATO SEED MEAL PROTEIN ISOLATION... Doxastaks, G., Kosseoglou, V., Boskou, D., and Stalds, G Adsorpton behavour of tomato seed protens at the corn ol-water nterface. Lebensm. Wss. u.-technol Drawert, F., El-Refa, A.A., and Radola, B.J Extracton of cottonseed protens wth urea. Chem. Mkrobol. Technol. Lebensm Droulskos, N.J Nutrtonal evaluaton of the proten of dred tomato pomace n the rat. Br. J. Nutr Dubos,, M., Glles, K.A., Hamlton, J.K., Rebers, P.A., and Smth, F Colonmetrc method for determnaton of sugars and related substances. Anal. Chem Eggers, L.K Some bochemcal and electron mcroscopc studes of the proten present n seeds recovered from tomato cannery waste. Ph.D. dssertaton. The Oho State Unv., Columbus, OH. Fazo, G., Arcoleo, G., and Prrone, L Sul contenuto proteco de seme d pomodoro. Rv. Sot. Ital. Sc. Alment Gesman, J.R. 98. Proten from tomato seeds. Oho Report on Research and Development. 66(6) Green, J.H. and Kramer, A Food Processng Waste Management. AVI Publshng Co. 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