Continuous extraction of prebiotics from jackfruit seeds

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1 Songklanakarn J. Sc. Technol. 32 (6), , Nov. - Dec Orgnal Artcle Contnuous extracton of prebotcs from jackfrut seeds Veeraphong Bhornsmthkun, Pakamas Chetpattananondh*, Ram Yamsaengsung, and Kulchanat Prasertst Department of Chemcal Engneerng, Faculty of Engneerng, Prnce of Songkla Unversty, Hat Ya, Songkhla, Thaland. Receved 30 June 2009; Accepted 20 September 2009 Abstract Prebotcs are functonal foods wth health-promotng propertes that are currently used n many developed countres, such as the Unted States, Japan, and the EU. The synthess method s stll the man commercal producton method. There are only a few drect extractons of natural olgosacchardes from plants n Thaland due to the lack of extracton devces. Ths research ams to desgn and construct the contnuous extractor and study the optmum condtons of prebotcs extracton from jackfrut seed. Jackfrut seeds were extracted wth 50% ethanol as a solvent. The response surface methodology was appled for expermental desgn to study the effects of temperatures (40-60 C), extracton tmes (15-45 mn), and L/S ratos (6:1-10:1 v/w) n laboratory scale contnuous extracton. The extracton effcency was based on the extracton yeld and the amount of nonreducng sugar, whch s expected to be prebotcs. The optmum condton was the extracton tme of 15 mn at 60 C and L/S rato 10:1 (v/w), whch gave the maxmum non-reducng sugar content of mg/g extract from RSM modelng. Ths optmum condton was appled for plot scale contnuous extracton. The plot scale contnuous extracton unt composes of three 70-L stanless steel extracton tanks equpped wth an ndrect steam chest for process heatng. The heatng tank s an 88-L stanless steel vessel. Each extracton pot s connected to a soluton pot. After extracton the soluton was pumped to a large evaporaton tank (60 L) and a small evaporaton tank (7 L), respectvely. Wth three-stage extracton the average extracton yeld was 20.25% and the average non-reducng sugar content was 400 mg/g extract. Keywords: contnuous extracton, jackfrut seed, olgosaccharde, prebotcs, response surface methodology 1. Introducton A prebotcs s defned as nondgestble food ngredent(s) that benefcally affects host health by selectvely stmulatng the growth and/or actvty of one or a lmted number of bactera n the colon (Gbson and Roberfrod, 1995). The stmulated bactera should be of a benefcal nature, namely bfdobactera and lactobacll (Gbson et al., 1999). At least three crtera of prebotcs are requred: (1) the substrate must not be hydrolyzed or absorbed n the stomach or small ntestne, (2) t must be selectve for benefcal commensally bactera n the colon such as the bfdobactera, * Correspondng author. Emal address: pakamas.p@psu.ac.th (3) fermentaton of the substrate should nduce benefcal lumnal/systemc effects wthn the host (Mannng and Gbson, 2004). Prebotcs are typcally carbohydrates, such as olgosacchardes (Van Loo et al., 1999). Prebotcs are used n many developed countres, such as the U.S.A., Japan, and the EU. The market for prebotcs n food s growng rapdly. A 2007 report on the world prebotcs market states that there are over 400 prebotcs food products and more than 20 companes producng olgosacchardes and fbers used as prebotcs ( template/fs/the-world-prebotc-ingredent-market.pdf). The European prebotcs market s currently worth 87 mllon, and wll reach mllon by 2010 ( Prebotcs can also be purchased n supplement form wth some prebotcs commandng as much as 700 per kg of supplement capsules. The strct market for added prebotcs

2 636 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , 2010 ngredents n functonal foods are as defned, n the EU, U.S.A. and Asa, totals some 25,000 tones, whch s forecast to rse n volume by more than 6% per year. However, the European market remans relatvely small at 880m n terms of fnshed product sales values. Market potental though s consdered hgh. ( reports/report2. pdf) Jackfrut (Artocarpus heterophyllus, lamk) s wdely cultvated n Thaland. In 2003, Thaland has jackfrut produced 828,611 tons, wth jackfrut seed as a by-product of about 120,000 tons (Department of Agrcultural Extenson, 2003). Jackfrut seed was selected as an agrcultural materal n ths study. Among the dfferent exstng technques, sngle pot extracton n batch mode s the most wdely practced one n the herbal ndustry. The dsadvantages of sngle pot extracton nclude hgh solvent consumpton, long extracton tme, and low extracton effcency. Mcrowave or ultrasonc extracton provdes better yeld and faster speed but suffers from hgh-energy cost. Another attractve alternatve s multstage countercurrent extracton, whch combnes crculatory dynamc extracton and contnuous countercurrent extracton technologes (Shen, 2001). Studes on mult-stage countercurrent extracton have been reported as early as 1980 and snce then the processng technque and equpment have been under contnuous development (Shen and Da, 1997). Wang et al. (2004) nvestgated many technologes for extracton of glycyrrhzc acd from lcorce, ncludng batch sngle pot extracton, batch double pot extracton, mcrowaveasssted extracton, ultrasonc extracton, Soxhlet extracton, room temperature extracton, and mult-stage countercurrent extracton. It was found that the mult-stage countercurrent extracton process offers the hghest glycyrrhzc acd extracton yeld. The stage numbers of contnuous extracton have a slght affect on extracton yeld or extract concentraton, as shown n the theoretcal extracton yeld reach of 93.3% and 98.4% wth three-stage and fve-stage, respectvely (Wang et al., 2004). An extracton unt developed n Thaland s not avalable and mported unts are extremely expensve. For example, the cost of Dgmaz extractor RWBL model (10 L) of Olds College School n Alberta Canada, whch comprses of a 10-L extracton tank, solvent tank, condenser, dstllate vessel and EMSR measurement and control unt (Fgure 1) s currently 7,500,000 baht. So, ths research ams to develop a mult-stage countercurrent extracton process that s sutable for Tha agrcultural products and can be appled to use n the ndustres. In addton, the extracton condton of prebotcs from jackfrut seeds was optmzed usng RSM technque. 2. Materals and Methods 2.1 Sample preparaton Prebotcs extracton from 32 Tha crops usng 50% (v/v) ethanol as a solvent was pre-studed and the plants gven hgh amount of ndgestble polysaccharde were selected. Jackfrut seed gave hgh amount of ndgestble Fgure 1. Schematc of the Dgmaz-extractor RWBL model 10 L, A s Plant casng, B s Electrcal-control cabnet, C s Vacuum pump, 1 s Extractor, 2 s Solvent vessel / Extract tank, 3 s Dstllate vessel, 4 s Coolng unt, and 5 s Heat exchanger (Terry, 2006).

3 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , polysaccharde and moreover the extract could selectvely stmulate the growth and/or actvty of 3 knds of probotcs, Lactobacllus acdopllus, Lactobacllus plantarum and Bfdobacterum bfdum (Paboon, 2007). The jackfrut seeds used n the experments are Tongprasert speces, whch were obtaned from Tesco Lotus, Hat Ya. The jackfrut seeds were prepared by washng, dryng, slcng, mllng and sevng nto a sze of 2.00 mm. The prepared samples were frozen at -20 C and stored at ths temperature. 2.2 Contnuous extracton procedure Extracton procedure n ths work was modfed from the contnuous extracton process by Wang et al. (2004). The non-reducng sugar concentraton gradents of the sample slurry and the extract n dfferent extracton pots are llustrated n Fgure 2. The sample slurry and the extract are represented by the symbols crcle ( ) and square ( ), respectvely, whle the number of star symbols * nsde the crcles or the squares represents the relatve concentraton of nonreducng sugar content n them. All the extracts transfer along the drecton ndcated by the arrows, whle the sample slurry or extract dscharges are expressed by ( ). The contnuous extracton process could be dvded nto two stages: the condtonng stage followed by the extracton stage. The condtonng stage conssts of Preprocess 1 and Pre-process 2. In the sample condtonng stage, jackfrut seeds were mxed wth the approprate amount of 50% (v/v) ethanol n each extracton pot. The sample slurry nsde the extractor was contnuously extracted for a predetermned tme perod. After the completon of the above condtonng operaton, the extracton stage started accordng to the sequence of steps llustrated by the flow dagram n Fgure 2. Processes 1 3 n the dagram llustrate the mass transfer of non-reducng sugar content between the solvent and the sample slurry n each step. Each step conssts of four basc operatons. For example, n process 1 the operatons nclude (1) sample extracton for a pre-set tme; (2) dscharge of sample slurry from unt A and collecton the extract from unt C; (3) transfer of solvents n the drectons of A C, B A; (4) addton of jackfrut seed sample and fresh solvent to unts A and B, respectvely. Processes 1 3 llustrate the operatonal sequence of the frst cycle of the process. Ths s followed by the second cycle n whch the sequence of operatons from processes 1 to 3 was repeated. 2.3 Analytcal analyss The yeld of prebotcs was calculated as a percentage of the weght of extracton per weght of seeds (dry bass) wth Extracton yeld (%) = (extracted weght/dry raw materal weght) 100%. The total sugar content was determned by the reacton of sugars wth phenol n the presence of sulfurc acd usng glucose as a standard (Dubos et al., 1956). The reducng sugar content was determned by modfed dntrosalcylc acd method usng glucose as a standard (Mller, 1959; Robertson et al., 2001), and non-reducng sugar content was calculated as followng Non reducng sugar Total sugar Re ducng sugar. 2.4 Expermental desgn for contnuous extracton at laboratory scale Response surface methodology (RSM) was employed to optmze multple varables to predct the best performance condtons wth a mnmum number of experments (Lngyun Extracton Unt Before Extracton Preprocess 1 After Extracton Solvent Exchangng Preprocess 2 Process 1 Process 2 Process 3 Process 4 Unt A Unt B Unt C **** **** **** ** ** ** ** *** *** * * ** *** * **** ** * * ** ** *** *** **** ** ** *** * *** *** * * ** ** *** * **** ** ** ** ** *** *** * * ** *** * **** ** * * ** ** *** *** Fgure 2. Flow dagram of the three-stage contnuous extracton process, symbols crcle ( ) s raw materal, symbols square ( ) s extracted solvent, number of star symbols * nsde the crcles or the squares s the relatve concentraton of non-reducng sugar content. ** **

4 638 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , 2010 et al., 2006). The optmzaton process frst entals dentfyng the most mportant factors n the extracton usng the fractonal factoral desgn, then focusng on the crtcal subset of factors. The steepest ascent desgn was used to determne the drecton towards predcted hgher responses (Lngyun et al, 2006). Ths research nvestgates the effect of three ndependent varables (temperature, resdent tme, and lqud to sold rato) usng a Box-Behnken desgn (BBD) to optmze the crtcal factors and maxmze the non-reducng sugar content. The quadratc model for predctng the optmal pont was expressed accordng to 3 2 A0 AX A X 3 Y A X X (Eq. 1) j j 1 where Y s the response varable, A 0 s the center pont of the system, A, A, A j are the regresson coeffcents of varables for lnear, quadratc, and nteracton terms, respectvely, and X and X j are ndependent varables ( j). Each experment was performed n duplcate and the average of non-reducng sugar content was taken as the response, Y. The range of ndependent varables and ther levels are presented n Table 1. The ndependent varables and ther ranges were chosen based on prelmnary expermental results. The varables were coded accordng to the followng equaton X x x 0, (Eq. 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. 2 3 j 2.5 Contnuous extracton of prebotcs from jackfrut seeds at laboratory scale The laboratory scale contnuous extracton system comprsed of three 250-mL stanless-steel vessels. 20 g of prepared jackfrut seeds were well mxed wth 50% (v/v) ethanol n each vessel by shaken at 200 rpm n ol bath. At the frst stage the prepared jackfrut seeds were extracted wth fresh solvent and ths extracted solvent was used for the next stage. The extracton process was shown n Fgure 2. After extracton, the solvent was evaporated by a rotary vacuum evaporator. The extract was analyzed for total sugars and reducng sugars. The RSM wth Box-Behnken Desgn was used to decde the optmum condton. 2.6 Contnuous extracton at plot scale The schematc of a plot scale contnuous extracton nstrument s llustrated n Fgure 3. Photograph of the contnuous extractor s represented n Fgure 4 and photograph of the evaporator s llustrated n Fgure 5. The nstrument conssts of three extracton unts labeled as A C. All unts had the same confguraton and dmensons, consstng of an extracton tank, a soluton tank, and a pump. The extracton unt was run n a close loop confguraton by lettng the solvent from the soluton tank flow down to the extracton tank, mxng wth the plant materal loaded n the seved tank va nozzle. After extracton the soluton was pumped to a large evaporaton tank and a small evaporaton tank, respectvely. The extracton was carred out wth a solvent to raw materal rato of 10:1 (v/w) at 60 C for 15 mn. Table 1. The Box-behnken expermental desgn and results of laboratory scale contnuous extracton. Run Temperature Resdent tme L/S rato non-reducng sugar content ( C) (mn) (v/w) (mg/g extract)

5 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , Fgure 3. 3-D schematc of contnuous extracton nstrument. Fgure 4. Photograph of contnuous extractor. 3. Results and Dscusson Fgure 5. Photograph of evaporator. 3.1 Contnuous extractons at laboratory scale The expermental results of laboratory scale contnuous extracton are n Table 1. From the expermental results the extracton wth L/S rato of 8 at 60 C for 15 mn gave maxmum amount of non-reducng sugar content. By applyng multple regresson analyss on the expermental data, the response varable and the test varable are related by the followng second-order polynomal equaton: Y = X 37.36X 1.163X X1X 2 0.7X 2 X 3 (Eq. 4) Where the non-reducng sugar content (Y) can be expressed as a functon of extracton temperature (X 1 ), resdent tme (X 2 ) and solvent to raw materal rato (X 3 ). Ths polynomal model equaton was found to be adequate for predcton wthn the range of expermental varables as the determnaton coeffcent, R 2 s The closer the value of R 2 to the unty, the better the emprcal model fts the actual data (Lee et al, 2006). The P-value s used as a tool to check the sgnfcance of each coeffcent, whch n turn may ndcate the pattern of the nteractons between the varables. The smaller s the value of P, the more sgnfcant s the correspondng coeffcent. It was found that the lnear coeffcents (X 1, X 2 ), a quadratc term coeffcent (X 2 1 ) and cross product coeffcent (X 1 X 2 ) were sgnfcant, wth very small P values (P < 0.05). The other term coeffcents were not sgnfcant (P > 0.05). The ftted polynomal equaton s expressed as surface and contour plots n order to vsualze the relatonshp between the response and expermental levels of each factor and to deduce the optmum condtons (Trven et al, 2001). In the plots two contnuous varables were developed for the non-reducng sugar content, whle another varable was held constant at ts respectve zero level (centre value of the testng ranges) (Wu et al, 2007). Fgure 6 and Fgure 7 show the effect of resdent tme and extracton temperature on nonreducng sugar content. The non-reducng sugar content reduced wth ncreasng of resdent tme, but ncreased wth ncreasng of extracton temperature. The maxmum nonreducng sugar content was reached at extracton temperature of 60 C and resdent tme of 15 mn. The effect of extracton temperature and solvent to raw materal rato on nonreducng sugar content s presented n Fgure 8 and Fgure 9. Hgher extracton temperature leaded to greater amount of non-reducng sugar content whle the solvent to raw materal rato dd not show sgnfcantly effect. The optmum extracton condton predcted by the polynomal equaton s extracton wth solvent to raw materal rato 10:1 (v/w) at 60 C for 15 mn. Under ths condton, the model gave predcted Y values (the non-reducng sugar content) of mg/g extract. The work done by other researchers (Lee et al., 1987; Nssreen and Mckenna, 1997) also reported that hgher extracton temperature ncreased the effectveness of olgosaccharde extracton. However, extracton at too hgh temperature mght cause denaturaton of soluble protens, whch thereafter entrapped soluble sugars and mpared the extracton (Km et al, 2003). 3.2 Contnuous extracton at plot scale Durng contnuous extracton, the jackfrut seed can be vewed as a statonary phase that was extracted cont-

6 640 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , 2010 Non-reducng sugar content (mg/g extracted) Resdent tme (mn) Temperature ( C) Non-reducng sugar content (mg/g extracted) L/S rato (v/w) Temperature ( C) Fgure 6. Response surface plots showng the effect of extracton temperature (X 1 ) and resdent tme (X 2 ) on non-reducng sugar content (Y) by extracton usng solvent to raw materal rato 8:1 (v/w). Fgure 8. Response surface plots showng the effect of extracton temperature (X 1 ) and solvent to raw materal rato (X 3 ) on non-reducng sugar content (Y) by extracton wth resdent tme 30 mn. Nonreducng sugar content (mg/g extracted) Temperature ( C) Nonreducng sugar content (mg/g extracted) Temperature ( C) Resdent tme (mn) L/S rato (v/w) Fgure 7. Response contour plots showng the effect of extracton temperature (X 1 ) and resdent tme (X 2 ) on non-reducng sugar content (Y) by extracton usng solvent to raw materal rato 8:1 (v/w). Fgure 9. Response contour plots showng the effect of extracton temperature (X 1 ) and solvent to raw materal rato (X 3 ) on non-reducng sugar content (Y) by extracton wth resdent tme 30 mn. nuously by the solvent flow. The extract yelds and nonreducng sugar contents obtaned from plot scale contnuous extracton are shown n Table 2. At Preprocess 1 the extracton was only carred out n Unt C. All unts (Unt A-C) were run snce Preprocess 2. At Preprocess 2 the frst extracton wth fresh solvent was carred out n Unt A. In Unt B the fresh jackfrut seed sample was extracted wth the extracted solvent from Unt C n the prevous process. In Unt C the extracted jackfrut seed sample was extracted wth fresh solvent. The three-stage extracton was frstly complete at Unt A n Process 2, whch the extracton yeld of 20.42% was obtaned. After that the three-stage extracton was complete at Unt B n Process 3 and at Unt C n Process 4, respectvely. It can be seen that the extracton yeld ncreased wth the number of stage. However, ncreasng number of stage also rases the captal and operaton cost. 4. Concluson The optmum condton of contnuous extracton of prebotcs from jackfrut seeds at laboratory scale was nvestgated by RSM to obtan the desred levels of non-reducng sugar content. The optmum condton was extracton temperature 60 C, extracton tme 15 mn, and L/S rato at 10:1 (v/w) usng 50% ethanol as a solvent. Ths condton was appled for plot scale contnuous extracton. The extract stage number of 3 gave the average extracton yeld of 20.25% and the average non-reducng sugar of 400 mg/g extract. The plot scale contnuous extracton unt and the extracton procedure developed n ths research can reduce the cost, whch s a strong bearng n the manufacturng of herbal extracton n Thaland. Acknowledgement The authors gratefully acknowledge the Thaland Natonal Scence and Technology Development Agency, the Reverse Bran Dran Program, the Thaland Research Fund Master Research Grants (TRF-MAG) and the Faculty of Engneerng, Prnce of Songkla Unversty for ther knd supports of ths work and the Department of Chemcal Engneerng for provdng the facltes and resources to complete ths study.

7 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , Table 2. Results of plot scale contnuous extracton. Process Results of contnuous extracton Preprocess 1 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) Preprocess 2 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) Process 1 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) Process 2 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) Process 3 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) Process 4 Unt A Unt B Unt C Extracton yeld (%) Non-reducng sugar (mg/g extract) References Chen, J.M., L, W.K., Shen, Y.H., Peng, H.M., and Xu, B.J Composton analyss and content detecton of polysacchardes from boat-fruted stercula seeds. Journal of Chnese Matera Medca. 17, Dubos, M., Glles, K.A., Hamlton, J.K., Rebers, P.A., and Smth, F Calormetrc method for determnaton of sugars and related substances. Analytcal Chemstry. 228, Gbson, G.R., and Roberfrod, M.B Detary modulaton of the human colonc mcrobota : ntroducton the concept of prebotcs. Journal of Nutrton, 125, Km, S., Km, W., and Hwang, I.K Optmzaton of the extracton and purfcaton of olgosacchardes from defatted soybean meal. Internatonal Journal of Food Scence and Technology. 38, Lee, W.C., Yusof, S., Hamd, N.S.A., and Baharn, B.S Optmzng condtons for enzymatc clarfcaton of banana juce usng response surface methodology (RSM). Journal of Food Engneerng. 73, Lee, Y.H., Jung, H.O., and Rhee, C.O Solds loss wth water uptake durng soakng of soybeans. Korean Journal of Food Scence and Technology. 19, Lngyun, W., Janhua, W., Xaodong, Z., Da, T., Yaln, Y., Chenggang, C., Tanhua, F., and Fan, Z Studes on the extractng techncal condtons of nuln from Jerusalem artchoke tubers. Journal of Food Engneerng. 79, Mannng, T.S., and Gbson, G.R Prebotcs. Best Practce & Research Clncal Gastroenterology. 18, Mller, G.L Use of dntrosalcylc acd reagent for determnaton of reducng sugar. Analytcal Chemstry. 31, Nssreen, A.G., and Mckenna, B Hydraton knetcs of red kdney beans (Phaseolus vugars L.). Journal of Food Scence. 62, Paboon, T Studes of some Tha crops as sources of prebotc ngredents project. Faculty of Agro-Industry, Prnce of Songkla Unversty, Songkhla, Thaland. Robertson, J.A., Ryden, P., Louse Botham, R., Readng, S., Gbson, G., and Rng, S.G Structural propertes of det-derved polysacchardes and ther nfluence on butyrate producton durng fermentaton. Lebensmttel-Wssenschaft und-technologe. 34, Shen, S.M Dscusson on the sngle-pot extracton and multstage count-current and contnual extracton. Pharm. Eng. Des. 22, 6 9. Shen, S.M., and Da, J.H Development of multstage count-current contnuous extractng machne. Pharm. Eng. Des. 18, 4 5. Trven, R., Shamala, T.R., and Rastor, N.K Optmsed producton and utlsaton of exopolysaccharde from Agrobacterum radobacter. Process Bochemstry. 36, Wang, Q.e., Ma, S., Fu, B., Lee, F.S.C., and Wang, X Development of mult-stage countercurrent extracton

8 642 V. Bhornsmthkun et al. / Songklanakarn J. Sc. Technol. 32 (6), , 2010 technology for the extracton of glycyrrhzc acd (GA) from lcorce (Glycyrrhza uralenss Fsch). Bochemcal Engneerng Journal. 21, Wu, Y., Cu, S.W., Tang, J., and Gu, X Optmzaton of extracton process of crude polysacchardes from boat-fruted stercula seeds by response surface methodology. Food Chemstry. 105, Report.pdf [Aprl 23, 2009] [Aprl 23, 2009] [Aprl 23, 2009]

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