Internet Journal of Food Safety

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1 Internet Journal of Food Safety, Vol.13, 2011, p Internet Journal of Food Safety Copyrght 2011, Food haccp.com The Use Of Fungal Enzymes vz Protease, Cellulase, And Xylanase For Polshng Rce. Loveleen Kaur Sarao1*, Mannder Arora2, V.K Sehgal3, Surekha Bhata4 1,2 Department of Mcrobology, Punjab Agrcultural Unversty, Ludhana, , Punjab, Inda. 3,4 Department of Processng and Food Engneerng, Punjab Agrcultural Unversty, Ludhana, , Punjab, Inda. Abstract Ths study nvolves the use of three fungal enzymes vz; cellulase, xylanase and protease to treat brown rce varety Pusa-44 pror to mechancal polshng. The effect of enzymatc pretreatment on the cookng and mllng qualty of rce was nvestgated. Three enzyme solutons were used vz; 100% (undluted), 85% (85ml undluted enzyme + 15 ml buffer) and 70% concentraton (70ml undluted enzyme + ml buffer). The brown rce was treated wth 3 ml of the enzyme soluton for 1-3 mn at 27ºC, 37ºC and 47ºC. Accordng to response surface methodology the optmzed condtons were 85% concentraton of the enzyme, 2 mn of the treatment tme at a temperature of 42 ºC. As a result of the enzymatc pretreatment under the optmzed condtons the head rce yeld ncreased from 64.6% to 69%, percentage brokens decreased from 5% to 3.10%, polshng tme decreased from 95 seconds to 54 seconds and the optmal cookng tme reduced from 22.5 mn to mn. hence, the enzymatc pretreatment pror to polshng ncreased the mllng performance and cookng qualty of the rce. Keywords: Enzymatc pretreatment, Basmat rce, response surface methodology, fungal enzymes. Introducton Enzymes have been used for the processng of rapeseed to ncrease the dehullng effcency (Sarkar et al, 1995), enzymatc hydrolyss of soyflakes to ncrease ol recovery (Aggarwal et al, 1997) and for treatng Basmat-386 to mprove ts mllng and cookng characterstc (Arora et al, 2007). Cereals are the most mportant food stuff and domnate the food sector as these are a versatle and relable source of food. They are easy to store and can be used to produce a myrad of food products. Cereal processng thus forms a large and mportant part of food producton chan (Owens, 2001) and rce s the most mportant crop n Asa (Smth, 2000). Inda produces 93 mllon tonnes of rce (Anonymous, 2007). * Corrrespondng author. Malng Address :- 36-C Bha Randhr Sngh Nagar, Ludhana Tel : E- maladdress:loveleen_sarao84@yahoo.com The commercal value of rce s due to ts fne and slender grans; ths characterstc also favours hgh percentage breakage durng mechancal mllng whch s not desrable n the nternatonal market. Polshng rce up to an approprate level s mportant to mantan qualty of rce. The removal of percarp or the bran layer s the prmary step n the preparaton of rce grans for human consumpton (Hoseney, 1986). Loss of nutrents s observed f polshng s done more than requred. Ths dsadvantage of mechancal polshng can be overcome by enzymatc pretreatment pror to polshng to mprove the mllng characterstcs. Wth the applcaton of specfc fungal enzymes, the hydrolyss of bran layer s possble n a selectve manner, wth mnmum loss of gran mass. Ths technque can gve hgher head yeld of rce from paddy and helps n retanng mportant components of the bran layer, and also reduces tme of polshng (Morrs and Bryce, 2004). The major consttuents of the bran layer are Proten %,Fat %, Crude fber- 6-14%,Ash-7-26

2 15%,Carbohydrates-26-27%,Cellulose %,Arabnoxylans -8.%,Mannans- 0.40%,Galactans- 1.00%, Pentosans -5-8%,Uronc acd -0.40% (Choct, 1997 and Houston, 1972). So the brown rce s treated wth protease, cellulase and xylanase enzymes that would degrade some porton of the bran layer. Materal and Methods The paddy varety Pusa-44 was procured from the local market to carry out the enzymatc pretreatment. Satake type rotary screen grade cleaner was used to grade the cleaned paddy. The mosture content of the paddy was adjusted to % on wet bass (AOAC, 1984). Ths s consdered to be the optmum level for mllng the paddy samples. Response surface methodology (RSM) was used to desgn the experments. To obtan the range of varables for mllng the paddy, a second order Box and Behnken desgn was used. Expermental desgn. Response surface methodology (RSM) has been employed n the present study to obtan the optmum values of the enzymatc pretreatment varables. It s a collecton of statstcal technques for desgnng an experment, buldng models, evaluatng the effects of factors, and searchng for optmum condtons of factors for desrable factors (L et al, 2006). These statstcal methods have proved to be powerful and useful tools as they allow optmzaton of the response whch s nfluenced by several varables. It gves nformaton about the nteracton between varables, process optmzaton and gves multple responses at the same tme. It s a powerful and effcent mathematcal tool appled for optmzaton of any process, e.g. meda components on fermentaton (Adnarayana and Ellaah, 2002; Park et al, 2002), for optmzaton of rosemary essental ol extracton by controlled pressure drop process (Rezzoug, Boutekedjret and Allaf, 2005) and for the optmzaton of enzymatc hydrolyss of fsh soluble concentrate by commercal proteases (Nlsang, Lertsr, Suphantharka and Assavang, 2005). A Box and Behnken desgn (Box and Behnken, 1960) of three varables and three levels each havng three centre pont combnatons was used. Most of the requrements of the enzymatc pretreatment optmzaton process are fulflled by ths method, so ths desgn was choosen. In ths desgn, X 1, X 2, and X 3 are the coded varables, whch are related to uncoded varables,.e. varables n actual unts lnearly by the relaton: X 2 ( ) / d Where, = Varable value n actual unts of the th observaton 1 = Mean of hghest and lowest varable value of. d = Dfference between the hghest and lowest varable value of. Based on ths relatonshp, the ndependent pretreatment varables and ther levels n the form of coded varables are gven below n Table 1. Table 1: Process varables and ther levels. Independent Varables Symbol Levels Coded Uncoded Coded Uncoded Temperature ( C) X 1 T Concentraton (%) X 2 C Tme (mns.) X 3 t Enzymes. Fungal enzymes protease produced from Rhzopus mcrosporus var olgosporus and cellulase and xylanase produced from Scopularopss acremonum va submerged fermentaton were used for the enzymatc pretreatment. Enzyme actvtes of the enzymes were expressed n Internatonal Unts (IU). One IU was defned as one mol of xylose (for xylanase actvty), glucose (for carboxy methyl cellulase actvty) or tyrosne (for protease actvty) equvalents released per mnute per ml under the followng assay condtons by usng xylose, glucose or tyrosne standard curve (Slva et al, 2005). Three dfferent concentratons of the enzymes were prepared 100% concentraton (undluted enzyme), 85% concentraton (85ml enzyme + 15 ml ctrate phosphate buffer ph 5.2) and 70% concentraton (70ml enzyme +ml ctrate phosphate buffer ph 5.2). Enzymatc pre-treatment of paddy. A sample of 250 g paddy was cleaned and then shelled n Satake rubber roll 27

3 sheller to get brown rce. Ths brown rce obtaned was graded to get head brown rce. Three ml of the fungal enzymes was sprayed on the head brown rce spread as thn flm on a tray. The head brown rce was sprayed usng dfferent concentratons of the enzymes at dfferent temperatures and was kept for 1-3 mn. For polshng the pretreated samples, an abrasve type Satake laboratory polsher was used. The bran adherng to the polshed whte rce was removed by sevng. After weghng the whte rce and the bran ther percentages were calculated. The whole kernels were separated from the brokens by szng of length. Broken percentage. One of the promnent factors determnng the qualty of rce s the head rce. Head rce also ncreases the consumer acceptablty. Hgher value of percentage brokens lowers the qualty. For the selected degree of polsh.e. 5%, percentage broken was obtaned by takng the rato of whte rce obtaned (broken) to weght of polshed rce and t was expressed as percentage. The head rce yeld was obtaned by takng the rato of the weght of the head rce to the weght of the paddy sample. Ths was also expressed as percentage. Percentage broken = weght of whte rce (broken) X 100 weght of polshed rce Polshng tme. Polshng tme s defned as the tme requred to remove the bran layer to obtan the requred degree of polsh by the polsher. In ths study the pretreated samples were polshed to 5% degree of polsh. Optmal cookng tme. The process of preparaton of food stuff by the acton of heat s known as cookng. To calculate the optmum cookng tme the followng procedure was adopted. Two gram of whole kernel (whte rce) obtaned after gradng were taken n glass tubes contanng 20 ml dstlled water. The glass tubes were mmersed n bolng water bath. Each rce sample was cooked for fve dfferent perods of 10, 15, 20, 25 and mnutes. Thereafter, the tubes were taken out and the contents of each tube were poured on the flter paper to soak extra water. Manual rollng of the kernels was done on the flter paper to remove surface mosture, tll they lost the glstenng appearance assocated wth the surface flm of water. The rce kernels were weghed and the graph of mosture ganed (%) vs. cookng tme was plotted and from graph when the rce weght showed 250 percent ncrease that lmt was consdered as the optmum cookng tme (Thapar et al, 1998). Contour plottng. A contour plot has lnes or curves of constant response values drawn on a graph or plane whose coordnate axs represents the levels of the ndependent varables and the response s vsualzed perpendcular to the plane of the paper. Seres of the contour plots of equal responses were generated whch provded useful nformaton n nterpretng contrbuton of the response (Cox and Cochran, 1964; Montgomery, 2004). As three varables are nvolved n the pre-treatment process, the value of one varable was fxed to obtan the contours. The non varant parameters were set at optmum pont and a new relatonshp was developed and plotted between the dependent and ndependent varables. Results and Dscussons To develop a response surface model the data was analysed employng multple regresson technque. A lnear model and a second order model wth and wthout nteracton terms were tested for ther adequaces to descrbe the response surface and R 2 values were calculated. A second order polynomal of the followng form was ftted to the data of all the responses and the results are gven n Table 2 y' 0 n n n-1 n-1 2 x x j 1 where β 0, β, β j are the constant coeffcents usually determned by least squares method, x x j are coded ndependent varables and ε s the error nvolved n estmatng the coeffcents β from the expermental data. For optmzaton, the Analyss of Varance (ANOVA) for the overall effect of three factor varables on the response varable accordng to ftted model was done and the least sgnfcant factor affectng the response varable was selected Table 3. j x x j Table 2 Treatment factors and the response of the treatment on Pusa-44 S.NO Treatment Factors Responses Temperature (x 1 ) Concentraton (x 2 ) Tme (x 3 ) Broken percentage (%) Polshng tme(s) Optmal Cookng Tme (mn)

4 Control Table 3: Analyss of Varance (ANOVA) for Model Fttng Pusa-44 Sum of squares Source Degree of Broken percentage Polshng tme Optmal cookng freedom tme Model * 262.8* * Lnear * Quadratc * 146.3* Cross product * Resdual Total Correlaton coeffcent (R 2 ) * Sgnfcant (P<0.05) Effect of varables on percentage brokens. The enzymatc pretreatment reduced the number of percentage brokens. Ths decrease s due to the effect of cellulase, protease and xylanase enzymes whch breaks down the bran layer and facltates ts easy removal. The percentage brokens were found to decrease wth ncrease n concentraton of the enzyme from 70% concentraton to 85% concentraton. When the concentraton of the enzyme was ncreased to 100%, the percentage brokens were found to ncrease agan (Table 2, Fg ) whch may be due to the over softenng of the bran layer. The ncrease n treatment temperature from 27 C to 37 C caused a decrease n percentage brokens. The broken percentage ncreased agan when the temperature was ncreased to 47 C. The ncrease n treatment tme from 1 to 2 mn decreased the percentage brokens. A further ncrease n treatment tme upto 3 mn led to ncrease n percentage brokens. Ths may be due to ncrease n brttleness of the gran as a result of the prolonged treatment tme. Least percentage of brokens.e. 3.10% was obtaned wth a pretreatment tme of 2 mn, 85% concentraton of the enzyme and at 37 C temperature. Smlarly Arora et al (2007) also reported a reducton n the percentage brokens by commercal cellulase pretreatment brokens of Basmat-386. The least percentage brokens.e 3.23% were obtaned at 37 C temperature, wth 0.35g 100ml -1 concentraton of the enzyme at a treatment tme of 2 mn. The head rce yeld was found to ncrease as a result of the enzymatc pretreatment. The head rce yeld ncreased to 69% n case of Pusa

5 Fgure 1.1 Surface plot for percentage brokens (Concentraton vs. Temperture) Pusa-44 Fgure 1.2 Surface plot for percentage brokens (Tme vs. Temperature) Pusa-44 Fgure 1.3 Surface plot for percentage brokens (Tme vs. Concentraton) Pusa-44 Effect of varables on polshng tme. In ths study the polshng tme decreased as a result of enzymatc pretreatment. The enzymatc pretreatment leads to the degradaton of the bran layer, thereby ts easy removal. The polshng tme ncreased wth ncrease n concentraton of the enzymes from 70% concentraton to 85% concentraton because the ncrease n concentraton saturated the actve stes of the substrate leadng to ts low actvty (Sarkar et al, 1998). It decreased agan wth further ncrease n enzyme concentraton upto 100%. The polshng tme also decreased wth ncrease n temperature from 27 C to 47 C. Wth the ncrease n treatment tme from 1mn to 2 mn the polshng tme showed a reducton. The polshng tme ncreased agan when the pretreatment tme was ncreased to 3 mn (Table 2, Fg ). The lowest value for polshng tme for Pusa-44 the treatment tme of 2 mn at 47 C wth an enzyme concentraton of 70% had the lowest polshng tme (54 seconds). Smlarly, Arora et al (2007) reported a decrease n polshng tme by treatng Basmat- 386 wth commercal cellulase. They reported that a treatment tme of 2 mn at 47 C temperature wth an enzyme concentraton of 0.15 g 100ml -1 gave the lowest polshng tme whch was 51 seconds.

6 Fgure 3.1 Surface plot for polshng tme (Temperature vs. Concentraton) - Pusa-44 Fgure 3.2 Surface plot for polshng tme (Temperature vs. Tme) - Pusa-44 Fgure 3.3 Surface plot for polshng tme (Concentraton vs. Tme) - Pusa-44 31

7 Effect of varables on optmal cookng tme. A reducton of the optmal cookng tme of rce s a desrable characterstc. A reducton n optmal cookng tme was observed as a result of enzymatc pretreatment. As the concentraton of the enzyme ncreased from 70% concentraton to 100% concentraton the optmal cookng tme was found to decrease. The ncrease n treatment temperature from 27 C to 47 C caused a lowerng of the optmal cookng tme. The treatment tme had no specfc effect on the cookng tme. The mnmum cookng tme was found at the treatment tme of 2 mn at 47 C wth an enzyme concentraton of 100% for Pusa-44 (16.54 mn) as presented n Table 2, Fg The reason for ths decrease may be attrbuted to the fact that the outer bran layer whch has an oly layer over t, acts as a barrer for the dffuson of water nto the kernel durng cookng whch was degraded by lpase enzyme Ths enzyme gets actvated along wth other enzymes at pretreatment condtons (Arora et al, 2007). Ths degradaton of the oly layer ncreased the rate of water dffuson nto the kernel hence decreasng the optmal cookng tme. Smlarly Arora et al (2007) used commercal cellulase for pretreatment of Basmat-386 and reported a reducton n cookng tme. The treatment tme of 3 mn, temperature 47 C and an enzyme concentraton of 0.35g 100 ml -1 concentraton gave an optmal cookng tme of 16 mn. Fgure 5.1 Surface plot for optmal cookng tme (Temperature vs. Concentraton) - Pusa-44 Fgure 5.2 Surface plot for optmal cookng tme (Temperature vs. Tme) -Pusa-44 32

8 Concentraton Fgure 5.3 Surface plot for Optmal Cookng Tme (Concentraton vs. Tme) - Pusa-44 OPTIMIZATION OF THE CONDITIONS FOR THE ENZYMATIC PRETREATMENT OF BROWN RICE PRIOR TO POLISHING Responses were optmzed usng Mntab 14 software (Tral Verson). The optmum value for each dependent varable had dfferent set of treatment condtons. In response surface analyss, the selected model was used to locate the statonary pont.e a pont at whch the slope of the response s zeroed n all drectons. The optmzaton was done wth the followng targets: Percentage brokens should be mnmum Polshng tme should be mnmum Optmal cookng tme should be mnmum To consder all the responses smultaneously for optmzaton contour plots were generated for each response for dfferent nteracton of any two varables whle holdng the thrd varable constant (Fg 2, 4 and 6 ). To get optmum enzymatc pretreatment condtons contour plots were overlad wth the targets mentoned above. Contour plot for Percentage brokens (Temperature Vs Concentraton) - Pusa Percentage brokens < > 4.25 Tme Temperatute 45 33

9 Concentraton Tme Tme Contour plot for Percentage brokens (Temperature Vs Tme) - Pusa Percentage brokens < > 4.2 Concentraton Temperatute 45 Contour plot For Percentage brokens (Tme Vs Concentraton) - Pusa Percentage brokens < > 4.2 Temperatute Concentraton Fgure 2 Contour plots for percentage brokens - Pusa-44 Contour Plot of Polshng tme (Temperature vs Concentraton) - Pusa Polshng tme < > Tme Temperature 45 34

10 Concentraton Tme Tme Contour plot of Polshng tme vs (Temperature Vs Tme) Pusa Polshng tme < > 70 Concentraton Temperature 45 Contour plot of Polshng tme (Concentraton vs Tme)- Pusa Polshng tme < > Temperature Concentraton Fgure 4 Contour plots for polshng tme - Pusa-44 Contour Plot of optmal cookng tme (Temperature vs Concentraton) - Pusa Optmal cookng tme < > 19.5 Tme Temperature 45 35

11 Tme Tme Contour plot of Optmal cookng tme (Temperature vs Tme) Optmal cookng tme < > Concentraton Temperature 45 Contour plot of Optmal cookng tme (Concentraton vs Tme) Optmal cookng tme < > Temperature Concentraton Fgure 6 Contour plots for optmal cookng tme - Pusa-44 References Adnarayana K, Ellaah P Response surface optmzaton of the crtcal medum components for producton of alkalne protease by a newly solated Bacllus sp. J Pharmaceut Sc 5: Aggarwal YC, Kashyap MC, Sarkar BC, Sngh BPN Response surface analyss of enzyme aded extracton of soybean. J Food Sc Technol 34 (5): Anonymous Statstcal abstracts of Inda, Agrculture. Webste: htpp:// Arora G, Sehgal VK, Arora M Optmzaton of process parameters for mllng of enzymatcally pretreated Basmat rce. J Food Engg 82 (2): AOAC Offcal methods of analyss. 10th Ed. USA. pp. 191, 431. Box GE, Behnken DW Some new three level desgns for the study of quanttatve varables. Technometrcs 2 (4): Cox GM, Cochran WG Expermental desgns. John Wley and Sons, Inc. New York. pp: Choct M Feed non starch polysacchardes. Chemcal structure and nutrtonal sgnfcance. Feed Mllng Int (June): Hoseney RC Prncples of Cereal Scence and Technology. Amercan Assocaton of Cereal Chemcals,Inc., U.S.A. pp Houston DF Rce Chemstry and Technology. Pub. AACC, U.S.A., Vol.ΙV. pp L Y, Ln J, Meng D, Lu J, Gu G, Mao Z Effect of ph, cultvaton tme and substrate concentraton on the Endoxylanase producton by Aspergllus awamor ZH-26 under submerged fermentaton usng central composte rotary desgn. Food Technol Botechnol 44: Mahndru SN Manual of Basmat Rce. 1st edn. Metropoltan Publshers, New Delh, pp Mntab 14 Software Tral Verson Avalable at Webste: Montgomery DC Desgns and analyss of experments. New York: John Wley and Sons. 36

12 Morrs PC, Bryce JH Cereal Botechnology. Woodhead Publshng Lmted, CRC Press, England. Owens G Cereal Processng Technology. Woodhead Publshng Lmted, CRC Press, England. pp Park YS, Kang SW, Lee JS, Hong SI, Km SW Xylanase producton n sold state fermentaton by Aspergllus nger mutant usng statstcal desgns. App Mcrobol Botechnol 58: Nlsang S, Lertsr S, Suphantharka M, Assavanng A Optmzaton of enzymatc hydrolyss of fsh soluble concentrate by commercal protease. J Food Engg 70 (4): Rezzoug S A, Boutekedjret C, Allaf K Optmzaton of operatng condtons of rosemary essental ol extracton by a fast controlled pressure drop process usng response surface methodology.j Food Engg 71(1):9-17. Sarkar BC, Sngh BPN, Agarwal YC Effect of enzymatc pretreatment on dehullng of rapeseed. J Food Sc Technol 32: Slva RD, Lago ES, Merheb CW, Macchone MM, Park YK, Gomes E Producton of xylanase and CMCase on sold state fermentaton n dfferent resdues by Thermoascus aurantacus MIEHE. Brazlan J Mcrobol 36: Smth WC Sorghum: Orgn, Hstory, Technology, and Producton.Publshed. John Wley and Sons.Inc New York. Thapar VK, Sehgal VK, Chhuneja PK, Arora M, Paul S Techncal Bulletn. Department of Processng and Agrcultural Structures, PAU, pp