OPTIMIZATION OF PREBIOTICS AND PHENOLIC COMPOUNDS EXTRACTION FROM JACKFRUIT SEEDS USING RSM

The 5 th PSU-UNS Internatonal Conference on Engneerng and 78 Technology (ICET-2011), Phuet, May 2-3, 2011 Prnce of Songla Unversty, Faculty of Engneerng Hat Ya, Songhla, Thaland 90112 OPTIMIZATION OF PREBIOTICS AND PHENOLIC COMPOUNDS EXTRACTION FROM JACKFRUIT SEEDS USING RSM Supansa Pasan 1,*, Ram Yamsangsung 1, Paamas Chetpattananondh 1 a Prnce of Songla Unversty, Faculty of Engneerng, Thaland *e-mal: supansa_no@hotmal.com Abstract: In ths study, response surface methodology (RSM) was used to optmze the extracton condton of prebotcs and phenolc compounds from jacfrut seeds usng 50% ethanol. Box-Behnen desgn wth four varables and three levels: extracton tmes (30-90 mn), extracton temperatures (30-90 C), sold to lqud ratos (1:10-1:20 w/v) and tme of dryng at 60 C (0, 12 and 24 hrs) was nvestgated. The RSM was applcable to predct the hghest yeld of non-reducng sugar contents, but not for phenolc compounds. The optmum condtons were reached by extracton wth fresh seed n a sold to lqud rato 1:20 w/v at 90 C for 30 mn. Under these condtons, the non-reducng sugar was 26.45 mg glucose/g dred seed. Key Words: Prebotcs/ Phenolc compounds/ Extracton/ Response surface methodology 1. INTRODUCTION The jacfrut (Artocarpus heterophyllus Lam.) s a speces of tree n famly Moraceae. It s natve to Inda and grown wldly many parts of Southern and Southeast Asa, such as Bangladesh, Burma, Sr Lana, Malasa, Indonesa, Phllppnes and Thaland [1] ncludng Brazl and other countres that there are humd tropcal and near-tropcal clmates. In Thaland, we can fnd jacfrut around the year and the best tme to fnd t s around the end of rany season n October or November. The green frut s cooed as a vegetable. Addtonally, the rpe fruts are normally eaten fresh or used n ce cream and also processng nto canned and snacs products [2] and the resdual seeds from those processes are mostly dscarded, except sometmes they are boled or roasted for consumpton. Prevous research found that the jacfrut seeds contaned prebotcs [3] and phenolc compounds [4]. Prebotcs are non-dgestble food ngredents that stmulate the growth and actvty of bactera n the dgestve system that benefcally affect the host by mprovng ts ntestnal mcrobal balance. Prebotcs are carbohydrate. The composton of food classfed as prebotcs nclude olgosacchardes and polysacchardes, such as fructo-olgosaccharde (FOS), galacto-olgosaccharde (GOS), nuln and xylo-olgosaccharde, whch are non-reducng sugar. The structure of nuln and FOS are shown n Fg. 1. (A) (B) Fg.1. Structure of nuln (A) and FOS (B) Phenolc compounds or polyphenols are physologcal functon, ncludng antoxdant, antmutagenc and anttumour actvtes [5]. Phenolc compounds have nhbton of carcnogeness and mutageness. Generally, structure of phenolc compounds comprse an aromatc rng, bearng one or more hydroxyl substtuents. Two structures of phenolc compounds are shown n Fg. 2. (A) (B) Fg.2 Structure of gallc acd (A) and caffec acd (B)

79 Both prebotcs and phenolc compounds are found n vegetables and fruts. They can be obtaned by several methods, e.g. supercrtcal flud extracton, mcrowave-asssted extracton and solvent extracton. Solvent extracton s wdely used as t s effcently and economcally operated. Nuallaong et al. [3] studed extracton of prebotcs from jacfrut seed. The nvestgated parameters were extracton temperatures (30, 50 and 60 C), extracton tmes (30, 60, and 120 mn), sold to lqud ratos (1:8, 1:10 and 1:15, w/v) and types of solvent (dstlled water, ethanol 50% and ethanol 95%). The optmum condton was extracton tme 120 mn, extracton temperature 30 C, sold to lqud rato 1:15 w/v and use of 50% ethanol as a solvent. Soong [4] reported that phenolc compounds were extracted from jacfrut seeds by usng 50% ethanol. Therefore, ths wor ams to optmze the extracton condton of prebotcs and phenolc compounds from jacfrut seeds usng 50% ethanol. The studed parameters nclude tme, temperature, sold to lqud rato and tme of dryng at 60 C by usng a three-level, four-varable Box-Behnen Desgn (BBD). 2. MATERAILS AND METHODS 2.1 Materals and chemcals Tongprasert-jacfrut seeds were obtaned from Tesco Lotus Department Store n Hat Ya, Songhla. Fresh seeds were cleaned wth water and grndng before szng by seve shaer. Ethanol, sodum hydroxde and concentrate sulfurc acd were purchased from lab-scan analytcal scence (laboratory grade, Thaland). Sodum potassum tatrate and sodum carbonate were purchased from Ajax Fnechem Pty Ltd. (NSW, Australa ). D-glucose anhydrous and gallc acd were from Sgma-Aldrch (Stenhem, Germany). Sodum sulfte and Foln- Cocateu reagent were purchased from Merc (Darmstadt, Germany). 3,5-dntrosalcylc acd was from Flua Cheme (Buchs, Swtzerland). Phenol was from Fsher Scencetfc (Loughborough, UK). 2.2 Extracton of prebotcs and phenolc compounds Fresh seeds (60% mosture content) were ground by blender and szed to 1.0 2.0 mm. The seeds were extracted wth 50% ethanol at studed condton usng 250 ml-glass bottle shaen at 200 rpm n ol bath (Memmert: SV 1422). The extracted soluton was fltered by vacuum flter (SIBATA: Crculatng Asprator WJ-20) and solvent evaporated by rotary vacuum evaporator (Buch: Vacuum pump V-700). The extract was then freeze dred by Vacuum Freeze Dryer (Flex-Dry µp). 2.3 Determnaton of non-reducng sugar 2.3.1 Determnaton of total sugar The total sugar concentraton was determned usng Modfed phenol sulfurc method [6]. The assay was calbrated wth D-glucose anhydrous standards from 0 ppm to 600 ppm. Sample or glucose standard soluton (25 µl) was added to 5 %w/v phenol soluton (25µL) and concentrate sulfurc acd (125 µl). Then soluton was shaen for 30 second at 500 rpm.the samples and standard were boled for 30 mn and then coolng on ce for 10 mn before readng the absorbance at 492 nm by mcroplate reader (Bote: Power Wave XS). The concentraton of sugar n the samples were calculated by comparson wth the standard curve of glucose and expressed as glucose equvalents. 2.3.2 Determnaton of reducng sugar Reducng sugar concentraton was determned usng the Modfed dntrosalcylc acd (DNS) method [7]. The assay was calbrated wth D-glucose anhydrous standards from 0 ppm to 600 ppm. Sample or glucose standard soluton (100 µl) was added to DNS soluton (100 µl). Then soluton was shaen for 30 second at 500 rpm. The samples and standard were boled for 30 mn and then coolng on ce for 10 mn before readng the absorbance at 575 nm by mcroplate reader. The concentraton of reducng sugar n the samples were calculated by comparson wth the standard curve of glucose and expressed as glucose equvalents. 2.3.3 Determnaton of non-reducng sugar Non-reducng sugar contents was determned usng ths equaton NRS = TS RS (1) Where NRS s non-reducng sugar (mg glucose/g dred seed), TS s total sugar (mg glucose/g dred seed) and RS s reducng sugar (mg glucose/g dred seed). 2.4 Determnaton of total phenolcs Total phenolc content was determned accordng to the Foln-cocateu method [2]. The assay was calbrated wth gallc acd standards from 0 ppm to 1200 ppm. Sample or gallc acd standard soluton (2 µl) was added to water (158 µl) and Foln-Cocateu reagent (10 µl). 10 µl of saturated Na 2 CO 3 (35%) (Redel-de Haen) was added to the mxture. Then soluton was shaen for 30 second at 500 rpm. The reacton was ept n the dar for 2 hours before readng ts absorbance at 765 nm. The concentraton of total phenolcs n the samples were calculated by comparson wth the standard curve of gallc acd. 2.5 Expermental desgn A Box-Behnen desgn (BBD) was employed wth four ndependent varables: extracton tme, extracton temperature, rato of jacfrut seeds to ethanol and tme of dryng at 60 C by the sngle factor method. The range and center pont values of three ndependent varables presented n Table 1 were based on the results of prelmnary experments. Nonreducng contents (mg glucose/g dred seed) and total phenolc contents (mg GAE/g dred seed) were selected as the responses for combnaton of the ndependent varables gven n Table 2.

80 The varables were coded accordng to the followng quaton: X x x = 0. (2) x Where X was a coded value of the varable; x was the actual value of varable; x 0 was the actual value of the x on the center pont; and x was the step change value. Data from the BBD were determned by multple regresson to ft the followng quadratc polynomal model. 2 0 1 = 1 = 1 > j Y = β + B X + B X + B X X. (3) Where Y s defned as the response functon, β 0 s the constant. B, B and B j are the lnear coeffcent, the quadratc coeffcent and the cross-product coeffcent, respectvely. X and X j are levels of the ndependent varables whle equals to the number of the tested factors (=3). The ftted polynomal equaton s expressed as 3-D surface and contour plots n order to vsualze the relatonshp between the expermental levels of each factor and response and to nfer the optmal condtons. The analyss of varance, regresson coeffcents of ndvdual lnear, quadratc and nteracton terms were determned. The regresson coeffcents were used to mae statstcal calculatons to generate surface and contour plots from the regresson models. The P-values of less than 0.05 were consdered to be statstcally sgnfcant. Table 1. The ranges and correspondng levels of ndependent varables used for expermental desgn. Factors Symbol Level coded uncoded -1 0 1 Extracton tme X t x t 30 60 90 (mn) Extracton X T x T 30 60 90 Temperature ( C) Rato of X R x R 10 15 20 Jacfrut seeds to 50% ethanol (v/w) Tme of dryng X D x D 0 12 24 at 60 C (hrs) 3. RESULTS AND DISCUSSION 3.1 Statstcal analyss and the model fttng The value of non reducng sugar (Y n ) and total phenolc contents (Y T ) at dfferent expermental combnaton for coded varables s gven n Table 2. The non reducng sugar and total phenolc contents ranged from 3.82 to 25.25 mg glucose/g dred seed and 1.19 to 2.94 mg GAE/g dred seed, respectvely. The maxmum value was found by extracton of fresh seed at extracton tme 75 mn, extracton temperature 90 C and rato of 50% ethanol to jacfrut seeds 15:1 (v/w). The applcaton of RSM offers an emprcal j j relatonshp between the response varable (non reducng sugar contents and total phenolc contents) and test varables under consderaton. The RSM optmzaton s more advantageous than the tradton sngle parameter optmzaton n that ts saves tme and raw materals. By applyng multple regresson analyss on the expermental data, the response varables are related by followng second-order polynomal equaton (4). 2 Yn = 19.50 2.117 X D + 0.0701 X D (4) + 0.0044 X X T R Table 2. The Box-Behnen desgn matrx and the results for extracton yeld of non-reducng sugar and total phenolc compounds. Run Coded Varable levels Response X t X T X R X D Y n Y T 1-1 0-1 0 10.41 1.10 2-1 -1 0 0 6.25 2.04 3 0 1 0-1 25.25 2.20 4 0 1-1 0 3.82 2.05 5 0-1 1 0 10.34 1.34 6 0-1 -1 0 12.50 1.77 7 0 0-1 1 8.24 1.09 8 0-1 0 1 5.57 1.45 9 1 0 1 0 7.07 2.03 10 0 0 1 1 12.94 2.57 11-1 0 0 1 20.02 1.88 12-1 1 0 0 8.48 1.97 13 1 0 0 1 14.73 1.98 14 0-1 0-1 17.07 1.19 15 0 0 0 0 7.15 1.68 16 1 0 0-1 23.26 2.40 17 0 0 1-1 22.78 1.51 18 1 0-1 0 5.15 2.21 19 0 0 0 0 7.65 2.94 20 1 1 0 0 11.58 2.02 21 0 0-1 -1 23.31 1.81 22 0 0 0 0 7.07 1.90 23 0 1 0 1 16.86 2.13 24 0 1 1 0 13.70 2.64 25-1 0 1 0 8.92 1.68 26 1-1 0 0 3.92 2.06 27-1 0 0-1 22.21 1.93 When Y n s non-reducng sugar contents (mg glucose/g dred seed) and Y T s total phenolc contents (mg GAE/g dred seed). The result of the non-reducng sugar contents and the total phenolc contents were analyzed usng the SAS pacage for analyss of varance (ANOVA). The coeffcent of determnaton and adjusted determnaton coeffcent of the non-reducng sugar contents were 0.792 and 0.765, respectvely. Ths confrms that the model s well ftted wth the results. On the other hand, those values of the total phenolc contents were 0.339 and 0.441, respectvely. Therefore the model cannot be nterpreted the yeld of

81 phenolc compounds. The regresson coeffcents determned usng F-test and p-value are shown n Table 3. The p-value s used as tool to chec the sgnfcance of the coeffcent, whch n turn may ndcate the pattern of the nteracton between the varables. The smaller s the p-value, the more sgnfcant s the correspondng coeffcent. It can be seen that the lnear coeffcent (X D ), a quadratc term coeffcent (X D *X D ) and cross product coeffcents (X T *X R ) were sgnfcant, wth very small P-values (P<0.05). Table 3. Test of sgnfcance for regresson coeffcents Effect Estmate Stand t rato P value error X D -2.117 0.241-8.772 <0.0001 X D *X D 0.07010 0.00942 7.441 <0.0001 X T *X R 0.00442 0.00185 2.392 0.02530 4.2 The optmum extracton condton Fg. 3 and Fg. 4 demonstrate the 3-D response surface plot and the contour plot at varyng ratos of solvent to seed and extracton temperatures at fxed tme of dryng at 60 C (0 level). It can be seen that non-reducng sugar contents ncrease slghtly wth ncreasng rato of solvent to seed and sgnfcantly ncreased when the extracton temperature ncreased. From our results the studed extracton tme dd not show sgnfcant effect on the non-reducng sugar contents. The extracton of olgosacchardes from chcpea seeds revealed smlar results that extracton tme 30 and 60 mn were not sgnfcantly dfferent [8]. The results ndcated that extracton for 30 mn was enough. Fg. 4. Response surface (2D) analyss for extracton of non-reducng sugar contents from jacfrut seeds wth respect to rato of solvent to seed and temperature Fg. 3. Response surface(3d) analyss for extracton of non-reducng sugar contents from jacfrut seeds wth respect to rato of solvent to seed and temperature. Fg. 5. Response surface (3D analyss for extracton of non-reducng sugar contents from jacfrut seeds wth respect to tme of dryng at 60 C and temperature Fg. 5 and Fg. 6 show the 3-D response surface plot and the contour plot at varyng tmes of dryng at 60 C and extracton temperatures at fxed rato of solvent to seed (0 level). The non-reducng sugar contents was hghest wth extracton of fresh seed and reduced rapdly when dred seed at 60 C for 12 hr. On the other hand, non-reducng sugar contents returned hgher when dryng at 60 C for 24 hr. Ths s because extracton wth fresh seed gave hghest nonreducng sugar content, but lowest extract yeld. The maxmum extract yeld was obtaned wth dryng for 24 hr. However, wth dryng process the reducng sugar content ncreased as some polysacchardes or olgosachardes were broen down [9]. The mosture contents of dryng jacfrut seeds for 12 and 24 hr were 27.7% and 3.5%, respectvely. At hgh mosture content the gelatnzaton of starch was observed by

82 heatng at 65 C [10]. Gelatnzaton mght obstruct the extracton process. Fg. 6. Response surface (2D) analyss for extracton of non-reducng sugar contents from jacfrut seeds wth respect to tme of dryng at 60 C and temperature 4. CONCLUSION The extracton of non-reducng sugar from jacfrut seeds was able to optmze by a statstcal response surface methodology. However the RSM model was not applcable for extracton of phenolc compounds. From the equaton of response varables, the tme of dryng has the most sgnfcant effect on the non-reducng sugar contents. The extracton temperature and sold to lqud rato also have postve effect, whle the studed extracton tme does not show sgnfcantly effect. The optmum condtons for extracton of non-reducng sugar were extracton wth fresh seed n a sold to lqud rato 1:20 w/v at 90 C for 30 mn, whch gave 26.45 mg glucose/g dred seed. 5. ACKNOWLEDGMENT Ths research has been fnancally supported by Prnce of Songla Unversty and the Natonal Research Councl of Thaland (NRCT). The Graduate School at Prnce of Songla Unversty has provded partal fundng. The Department of Chemcal Engneerng and the Faculty of Engneerng, Prnce of Songla Unversty are gratefully acnowledged for other supports. 6. REFERENCE [1] B. T. Ong, S.A. H Nazmah, A. Osman, S. Y. Que, Y. Y. Voon, D. Mat Hashm, P. M. Chew and Y. W. Kong. Chemcal and Flavour Changes n Jacfrut (Artocarpus heterophyllus Lam.) Cultvar J3 durng Rpenng, J. Postharvest Bol. Technol., 2006, Vol. 40, pp. 279-286. [2] C. K. Pua, N. S. A. Hamd, C. P. Tan, H. Mrhossen, R. B. A. Rahman and G. Rusul, Optmzaton of Drum Dryng Processng Parameters for Producton of Jacfrut (Artocarpus heterophyllus) Powder usng Response Surface Methodology, J. Food Sc. Technol., 2010, Vol. 43, pp. 343-349. [3] S. Nualla-ong, P. Chetpattananondh and R. Yamsaengsung, Extracton of Prebotcs from Jacfrut Seeds, Khon aen J. Eng., 2009, Vol. 36, No. 3, pp. 213-220. [4] Y. Y. Soong, and J. Phlp Barlow, Antoxdant Actvty and Phenolc Content of Selected Frut Seeds, J. Agrc. Food. Chem., 2004, Vol. 88, pp. 411 417. [5] E. Mddleton, C., Kandaswam, and T. C. Theohardes, The effects of plant flavonods on mammalan cells: mplcatons for nflammaton, heart dsease and cancer, J. Pharma. Revew, 2000, Vol. 52, pp. 673-751. [6] M. Dubos, K. A. Glles, J. K. Hamlton, P. A. Rebers, and F. Smth, Colormetrc Method for Determnaton of Sugars and Related Substances, J. Anal. Chem., 1956, Vol. 28, pp. 350-356. [7] G.L.Mller, Use of Dntrosalcylc Acd Reagent for Determnaton of Reducng Sugar, J. Anal. Chem., 1959, Vol. 31, pp. 426-428. [8] X. Xang, L. Yang, S. Hua, W. L, Y. Sun, J. Zhang and X. Zeng, Determnaton of olgosaccharde contents n 19 cultvars of chcpea (Ccer aretnum L) seeds by hgh performance lqud chromatography, J. food chem., 2008, Vol. 111, pp. 215-219. [9] B. A. Jason, E. Mathew, E. Judd and S. H. Brent, Hydrolyss of olgosacchardes from dstllers gans usng Organc-Inorganc hybrd mesoporous slca catalysts J. Bo. Technol., 2008, Vol. 99, pp. 5226-5231. [10] Q. Lu, G. Charlet, S. Yelle and J. Arul, Phase transton n potato starch gelatnzaton at hgh mosture level, J. food res. Inter., 2002, Vol. 35, pp. 397-407.