PaPer 50 Ital. J. Food Sci., vol. 28 - 2016 - Keywords: pistachio nut flour, starch-lipid complexes, optimization response surface methodology, contour plot, breaking strength, bulk density - OPTMIZATION OF EXTRUSION PROCESS OF RICE FLOUR ENRICHED WITH PISTACHIO NUT FLOUR C. SEvERINI, T. DE PILLI* and A. DEROSSI University of Foggia, Department of Science of Agriculture, Food and Environment (S.A.F.E.), Via Napoli 25, 71100 Foggia, Italy *Corresponding author: Tel. +39 881 589245, email: teresa.depilli@unifg.it AbstrAct response surface methodology deriving by superimposing individual contour plots, was used to investigate the optimum operating conditions for extrusion-cooking of rice flour enriched with pistachio nut flour. the highest barrel temperature (128°c) produced a stiff extrudates (high val- ues of breaking strength i.e. 100 N/mm2 and bulk density i.e. 2.2 g/mL). However, graphical op- timization studies showed that the optimal operating conditions involved values of 16-17% water feed content and 70-95°c barrel temperature. this research points out the importance to study the biopolymer changes that occur during extrusion-cooking processing because of their huge ef- fect on quality characteristics of extrudates. mailto:teresa.depilli@unifg.it Ital. J. Food Sci., vol. 28 - 2016 51 1. INtroductIoN Nowadays, consumers prefer foods easy and convenient to eat (scHWArtZ, 2009). snacks and breakfast cereals are easy to carry, pur- chase and consume but they are essential- ly produced from starchy substances such as corn, rice, wheat (YAsEEN and sHouK, 2005) and therefore they could lack some important nutrients. Foods with poor nutritional value, lack in micronutrients such as vitamins, min- erals, amino acids, fibers and high content of calories can be considered unhealthy. For this reasons, researches are focused on the im- provement of nutritional characteristics by the addition of ingredients such as fruit, nuts, fi- bres, etc. Among nuts, pistachios could favour- ably be used thanks to their ability to lower the risk of cardiovascular diseases, to improve to- tal cholesterol to HdL-c ratios, LdL cholester- ol to HdL cholesterol ratios, and HdL choles- terol levels (KocYIGIt et al., 2006; sHErIdAN et al., 2007; GEbAuEr et al., 2008). consumption of pistachios was also found to increase anti- oxidant activity in the body (KocYGIt, 2006) and to improve blood glucose levels (sArI et al., 2010). A possible application of pistachio nut flour could be the production of extrudates in order to obtain snack foods with high nutri- tional and health value. dE PILLI et al., (2011; 2012) studied the processing conditions that lead to the formation of starch-lipid complex- es in a model system and in real food like ex- trudates made up of rice starch and pistachio nut flour, by differential scanning calorimeter (dsc). In addition, they evaluated the effects of starch-lipid complexes formation on system parameters, fat loss and the breaking strength of extrudates. the results of that work showed that the barrel temperature had a huge effect on system parameters in the real foods as the extraction of lipid fraction determined a de- crease of friction force and therefore a decrease of mechanical energy input of processing (dE PILLI et al., 2008a). Moreover, the formation of starch-lipid complexes in real food, was strong- ly dependent on water feed content, that con- sequently affected starch gelatinization. the highest fat loss and the hardest texture of ex- trudates made up of pistachio nut flour were obtained under processing conditions that fa- voured the maximum formation of starch lipid complexes. the main objective of those studies was to verify the efficacy of model system to de- scribe the biopolymers changes that occur dur- ing processing of real food. However, the rela- tionships between process variables and char- acteristics of the extrudates has not been stud- ied in detail. therefore, this study aimed to in- vestigate the optimum operating conditions of extrusion and the effects of extrusion process variables on the characteristics of rice extru- dates enriched with pistachio nut flour by us- ing the response surface methodology (rsM). Furthermore, the regression models to predict the characteristics of the extruded material as a function of the process variables were also established. 2. MAtErIALs ANd MEtHods 2.1 Raw materials rice starch (10.9% moisture) was provid- ed by A.d.E.A. (bursto Arsizio, Italy); pistachio nut flour was provided by cartellone (bronte, It- aly); oleic acid was provided by sigma-Aldrich (Milano, Italy). the used pistachio nut flour had a moisture content of 4.8±0.2% and the following chemical composition (dry basis): protein (18.1±0.1%); lipid (49±0.5%); starch (3.3 ±1.5%); soluble sugars (4.5±0.2%); fiber (10.6±2%) and ash (9.7±0.1%). the fat acid composition of lipid fraction of pis- tachio nut flour, determined according method proposed by rAtNAYAKE et al. (2006) was: C14:0 (0.09); C16:0 (9.45); C16:17 (0.86); C17:0 (0.04); C17:1 (0.07); C18:0 (2.12); C18:1 (70.17); C18:2 (15.5); C18:3 (0.32); C20:0 (0.18); C20:1 (0.48); C22:0 (0.09); C24:0 (0.04). the chemical characteristics of tap water used for extrusion trials was: pH 7.7 + 0.1, hard- ness (°f) 25.1±1.5, total dissolved solids dried at 180°c 645±38.5 mg/L and chloride content 54.6±0.4 mg/L. the content of moisture, ash, protein and fat of flours were determined according to the 44- 15A, 08-01, 46-10, 30-25 AAcc International Approved Methods (2003). 2.2. Extrusion experiments According to previous studies (dE PILLI et al., 2011), the formula containing 75% rice starch and 25% pistachio nut flour was used. the extrusion experiments were carried out using a thermo Prism PtW-24 (thermo Haake PolyLab system, Germany) co-rotating twin- screw extruder. the screw geometrical fea- tures were the following: diameter 24 mm and length 672 mm (L/d = 28:1) and distance be- tween shafts 19 mm. Fig. 1 reports the screw configuration used. during extrusion experi- ments, the screw speed was kept constant at 140 rpm, as well the flour feed rate was kept constant at 2.8 kg/h (dry weight). the flours were proportioned by volumetric gravity feeder. the extruder was divided into six zones, inde- pendent of each other for temperature control and adjustment. For all experiments, the first two zones were kept at 35 and 65°c respective- ly, whereas the last four zones were adjusted at the same temperature according to experi- mental plan (table 1). 52 Ital. J. Food Sci., vol. 28 - 2016 Fig. 1 - screw configuration used to extrude rice starch and pistachio nut flour. table 1 - coded and actual values of variables (A) and the arrangement and responses of factorial design (b). A) Coded Level Uncoded Barrel temperatures of last four zones Water feed content (X1) (X2) (°C)** (%) +1.4 128 21.8 +1 120 21.0 0 100 19.0 -1 80 17.0 -1.4 72 16.2 **The first two zones were kept at 35° and 65°C respectively, whereas the last four zones were adjusted according to experimental plan. B) Treatments Coded Level Processing variables Responses X1 X2 X1 (°C) X2 (%) Y1 (%) Y2 (nm) Y3 (N/mm2) Y4 (g/ml) 1 -1 -1 80 17.0 23.67±0.02 554±0.55 7.97±0.92 0,80±0.26 2 -1 1 80 21.0 23.00±0.06 556±0.80 14.21±3.12 0,81±0.45 3 1 -1 120 17.0 60.00±0.03 552±0.52 96.24±1.19 2,10±0.50 4 1 1 120 21.0 60.00±0.01 549±0.66 75.58±0.59 2,08±0.37 5 -1.4 0 72 19.0 20.00±0.07 559±1.17 7.58±1.40 0,80±0.26 6 1.4 0 128 19.0 70.00±0.01 552±0.41 99.04±0.19 2,11±0.11 7 0 -1.4 100 16.2 52.45±0.01 552±2.04 53.75±1.50 0,87±0.34 8 0 1.4 100 21.8 52.00±0.03 555±0.63 65.68±0.45 0,87±0.25 9 0 0 100 19.0 51.50±0.02 556±1.02 43.05±2.73 0,87±0.37 10 0 0 100 19.0 52.00±0.03 554±0.42 51.32±2.60 0,94±0.23 11 0 0 100 19.0 50.80±0.01 555±0.41 49.52±1.23 0,96±0.28 X1: barrel temperatures of last four zones (°C); X2: water feed content (%); Y1: complex index; Y2: λ max ; Y3: breaking strength; Y4: bulk density. the water was pumped to the first zone of the extruder and the delivery capacities of wa- ter pump were 7.5; 7; 6; 4.75; 4.25 L/h. these values were chosen to obtain the moisture feed content of dough indicated in the experimental plan (table 1). the die used had a spherical shape (diameter 300 mm) in which there was one circular hole with a diameter of 5 mm. At the exit of the die, the extrudates were manually cut into sticks (about 50 mm in length) using a knife. the ex- trudates were dried over night at 40°c in a vac- Ital. J. Food Sci., vol. 28 - 2016 53 uum oven salvis Vacucenter Vc 50 (salvis AG, reussbühl/Lucerne, switzerland). samples used to determine complexing index and iodine spec- trum of the soluble fractions of the extrudates were finely ground (particles < 300 microns) us- ing a buHLEr ML 1204 mill (Germany). All the ground samples were defatted in a soxtec fat-ex- tractor with petroleum ether at 37°c (bp 34.6°c) for 155 min to remove uncomplexed lipids before submit samples to chemical analyses (bHAtNA- GAr and HANNA, 1994). 2.3 Experimental plan the extrusion experiments were carried out at five temperature profiles (table 1a) and per- centages of water feed contents (21.8%, 21.0%, 19.0%, 17.0%, 16.2% expressed as percentage of dry basis). All extrusion experiments were per- formed at least in triplicate. coded and actual values of variables are shown in table 1a, the factorial design of two variables (temperature profile and feed water content) and five levels of values were used according to central composite design (ccd) (boX et al., 1978). this method was used to evaluate the single influences of the processing variables as well as their possible interactions. Eleven tests (table 1b) with different combina- tions of process variable values were obtained. 2.4 Complexing Index complexing Index (cI) was determined using the method described by GurAYA et al. (1997). the iodine solution used for analysis was pre- pared by dissolving overnight 2 g of potassium iodine and 1.3 g of I 2 in 50 mL distilled water. then the final volume was made to 100 mL using distilled water. A 5 g sample was mixed with 25 mL of distilled water in a test tube. the test tube was vortexed for 2 min and cen- trifuged for 15 min at 314.1 rad/s. the su- pernatant (500 ml) and distilled water (15 mL) were added to the iodine solution (2 mL). the tube was inverted several times and absorb- ance was measured at 690 nm through a uV/ VIs spectrophotometer (beckman du 640, cal- ifornia). cI was calculated from the following equation (2): (2) the analysis was carried out in triplicate. 2.5 Iodine Spectra of Starch Samples starch samples were solubilised in l N NaoH as recommended by scHocH (1964). the ab- sorbance spectra of starch-iodine complexes were measured using a spectrophotometer uV/ VIs Perkinelmer Lambda 25 (Milan, Italy) from 400-700 nm, and wavelength of maximum ab- sorption (λ max ) values were determined. 2.6. Breaking strength (N/mm2) A stable dynamometer Micro system tA- Hdi texture Analyser (ENco s.r.l., Venezia, It- aly) with a plunger was used for texture analy- sis. Extrudates were placed over two supports, 1.5 cm apart, and broken in the middle by a plunger that had a shape of a cone frustum (the thickness of contact surface with extrudate was 1 mm2 and the speed was kept constant to 0.5 mm/s). results were expressed as break- ing strength (N/mm2), i.e. the strength need- ed to break the extrudate. this index is related to microstructure of samples and it simulates the incisors impact at biting (VAN HEcKE et al., 1998). For each sample, at least ten repetitions were carried out. 2.7 Bulk Density (BD) bulk density was measured using a displace- ment method (Yu et al. 2012). Extrudates were cut into strands of about 25 mm long and about 10 g strands were weighed (M, grams) and put in a 100 mL cylinder; then yellow millet parti- cles were added to fill up the cylinder. the ex- trudates were taken out, and the volume of the yellow millet particles was measured (V, millilit- ers); ten measurements were performed to cal- culate the average. bulk density (bd) was cal- culated as equation (3): (3) 2.8 Statistical analysis data were submitted to statistical analysis using statsoft, vers. 5.1 (statsoft, tulsa, usA) software. the analysis was carried out in two steps. the first involved a stepwise regression analysis to identify the relevant variables, and the second used a multiple regression analysis (standard Least square Fitting) to fit a second order mathematical model, according to the fol- lowing polynomial equation: y = b 0 + Σbiχi + Σbiiχii2 + Σbijχiχj where y is the dependent variable (complex in- dex, iodine spectrum of the soluble fractions of the extrudates; breaking strength and bulk den- sity of extrudates), b 0 is a constant value, χi and χj are the independent variables (barrel tem- perature and water feed content) in coded val- ues and bi, bii and bij are the regression coef- ficients of the model. this model allowed the ef- fects of the linear (χi), quadratic (χi2) and com- bined (χiχj) terms of the independent variables to be assessed on the dependent variable. 54 Ital. J. Food Sci., vol. 28 - 2016 Variables with a significance lower than 95% (p>0.05) were left out of the equation. Iso-re- sponse surface were developed in order to de- scribe both individual and interactive effects of the independent variables of the extrusion-cook- ing process on complex index, iodine spectrum of the soluble fractions of the extrudates; breaking strength and bulk density of extrudates. Extrusion processing parameters were opti- mized by using the design-expert version 8.07.1 (stat-Ease Inc., Minneapolis, usA) through a conventional graphical method of rsM in or- der to obtain extrudates with acceptable prop- erties. All the processing variables were kept within a range while the responses were either minimized (breaking strength and bulk den- sity). contour plots of all the responses were then superimposed, and the optimum region appeared. the contour plots were obtained by superimposing of contour plots from which one could determine the optimum process variables range (barrel temperature and water feed con- tent) to obtain extrudates made up of rice and pistachio nut flour with specified properties. 3. rEsuLts ANd dIscussIoN Fig. 2a shows the complexing index values as a function of barrel temperature and water feed content. the barrel temperature was the only processing variable that had a significant effect on complexing index. In particular, values of the complexing index increased with increas- ing of barrel temperature (Fig. 2a). this means that, in this case, the highest barrel tempera- tures did not involve the melting of starch-li- pid complexes. It is possible to suppose that the presence of other components in the dough in- crease the melting temperature of starch-lipid complexes, that result then more protected by heating during processing. Moreover, the pres- ence in lipid fraction of triglycerides, di-glyc- erides and fatty acids involves an increase of characteristic melting temperature of starch- oleic complexes (dE PILLI et al., 2008b; 2011). to confirm the formation of starch-lipid com- plexes, values of λ max for native starch extrud- ed with and without pistachio nut flour were also determined. rice flour, extruded without nut flour and with an amylose contents of 89 %, showed λ max within 592-595 nm. Fig. 2b shows that the increase of barrel tem- perature shifted λ max from 595 nm towards the amylopectin side (520 nm), due to the decrease of available amylose that is bounded with li- pids. these results are in agreement with those of complex index and confirm the formation of starch-lipid complexes (Fig. 2a). In Fig. 3a is reported the break strength (bs) values as a function of barrel temperature and water feed content. Also in this case, the only variable that had a significant effect on mi- Fig. 2 - starch-lipid complex index (a) and λ max values (b) of samples made up of rice starch and pistachio nut flour blend as a function of barrel temperatures and water feed content. crostructure of the extrudates was the barrel temperature. In particular, the extrudates ob- tained at the highest values of barrel tempera- ture (128°c) opposed the highest resistance to break, while low values of break strength were obtained at the lowest barrel temperature (70°c) (Fig. 3a). the formation of starch-lipid complex- es obtained with the increase of barrel tempera- ture could explain the high compactness of ex- trudates (bHAtNAGAr and HANNA, 1994; dE PIL- LI et al., 2008a,b). data of bulk density (bd) are in agreement with those of the break strength. In fact, the ex- trudates showed high values of bulk density at the highest barrel temperature (Fig. 3b). the in- crease of bulk density and break strength val- ues of extrudates may be caused by an alter- ation in the ratio between free amylose and am- ylopectin. According to GuY and HorNE (1988), the elastic character of the molten extrudates creates a swell at the die that controls the over- all phenomenon of expansion of the extrudates. LAuNAY and LIscH (1983) suggested that amy- lose–lipid complex formation was the key factor influencing the flow properties of starch pastes. When starch is extruded, expansion is depen- dent on the formation of a starch matrix that entraps the water vapor, resulting in the for- mation of bubbles (GuY and HorNE, 1988). It Ital. J. Food Sci., vol. 28 - 2016 55 is reasonable to speculate that the addition of lipids might have affected the character of this matrix (i.e., the viscoelastic properties of mol- ten extrudate) so that it could no longer hold water vapor, resulting in lower expansion and higher break strength. the increase of bulk den- sity and break strength caused by decrease of swelling of starch can also be compared to that one of native starch upon gelatinization. swell- ing is generally considered a property of amy- lopectin while amylose is considered a diluent. the amylose and native lipids contained in ce- real starches may inhibit swelling under partic- ular conditions when amylose–lipid complexes are likely to be formed (tEstEr and MorrIsoN, 1990). According to KroG (1973), complex for- mation with the linear component of starches makes the structure more rigid and stabilizes the swollen granule against breakdown, result- ing in restricted swelling. these statements are in agreement with dE PILLI et al. (2008b). In this study, a conventional graphical method of multiresponse optimization technique was ap- plied to obtain the combination of optimum pro- cess variable for the production of extrudates en- riched with pistachio nut flour. to determine the extrudates with acceptable properties, main cri- teria of optimization constraints were related to bulk density (< 1.2 g/mL) and breaking strength (< 40 N/mm2). superimposing the individual con- tour plots for the product response variables re- sulted in the identification of a region (shown by the blank space area) that satisfied all constraints as shown in Fig. 4. superimposed contour plots indicated the ranges of variables that could be considered as the optimum range to obtain the best characteristics of extrudates in terms of bulk density and braking strength. the optimum rang- es of variables obtained from the superimposed contours were 16–17 % water feed content and 70-95°c barrel temperature. Extrusion-cooking Fig. 3 - breaking strength (a) and bulk density values (b) of samples made up of rice starch and oleic acid blend as a function of barrel temperatures and water feed content. Fig. 4 - superimposed contours for the product responses affect by water feed content and barrel temperature. *bd: bulk density and bs: breaking strength. 56 Ital. J. Food Sci., vol. 28 - 2016 was carried out for confirmation under the opti- mum process conditions and the responses were recorded (mean of five measurements). In par- ticular, the following operating conditions were chosen: 72 °c barrel temperature and 16% wa- ter feed content. the values predicted by the soft- ware were ≤ 0.8 g/mL for bulk density and ≤ 20 N/mm2 for breaking strength. the data obtained by extrusion experiments carried out at the same operating conditions were respectively 0.78 g/ mL and 7.38 N/mm2. the veracity of values of the responses predicted by the software was as- sessed with the help of a two-tailed, one-sample t-test. the results of the t-test indicated that the coefficient of variation was not greater than 5 %. therefore, the developed model was suitable in representing the optimum operating conditions for this particular application. 4. coNcLusIoNs the obtained results showed that the barrel temperature was the variable that has main- ly affected the formation of starch-lipid com- plexes and structure of the extrudates. In par- ticular, the worst characteristics of extrudates (hardness and bulk density of extruded prod- ucts) were obtained at the highest temperature that corresponds to the maximum formation of starch-lipid complex. Moreover, the model was found to be statis- tically valid and demonstrated adequate infor- mation regarding the behaviour of the respons- es upon variations of the process variables. op- timum process conditions and the correspond- ing predicted responses could be obtained with the help of the models. the predicted responses at the optimum conditions were not significantly different from the experimental values. Accord- ing to the optimum conditions given for the var- iables, the process could be referred to stand- ardization of industrial production of snack food made up of rice and pistachio nut flours with high qualitative characteristics. 5. rEFErENcEs AAcc International. Approved methods of analysis, 10th 1 ed. Methods 44-15a, 08-01, 46-10, 30-25 Approved 2003. AAcc International: st. Paul, Mn. bhatnagar s. and Hanna M.A. 1994. Extrusion processing conditions for amylose lipid complexing. cereal chem. 71(6): 587. box G.E.P., Hunter W.G. and Hunter J.s. 1978. statistics for experiments. An introduction to design data analysis and model building. Wiley, New York. de Pilli t., carbone b.F., derossi A., Fiore A.G. and sever- ini c. 2008a. Effects of operating conditions on oil loss and structure of almond snacks. Int. J. Food sci. tech- nol. 43(3): 430. Paper Received October 29, 2014 Accepted March 27, 2015 de Pilli t., Jouppila K., Ikonen J., Kansikas J., derossi A. and severini c. 2008b. study on formation of starch-li- pid complexes during extrusion-cooking of almond flour. J. Food Eng. 87: 495. de Pilli t., derossi A., talja r.A., Jouppila K. and severini c. 2011. study of starch-lipid complexes in model system and real food produced using extrusion-cooking technol- ogy. Innov. Food sci. Emerg. technol. 12(4): 610. de Pilli t., derossi A., talja r.A., Jouppila K. and severini c. 2012. starch-lipid complex formation during extru- sion-cooking of model system (rice starch and oleic acid) and real food (rice starch and pistachio nut flour). Eur. Food res. technol. 234(3): 517. Gebauer s.K., West s.G., Kay c.d., Alaupovic P., bagshaw d. and Etherton P.M.K. 2008. Effects of pistachios on cardiovascular disease risk factors and potential mecha- nisms of action: a dose-response study. Am. J. clin. Nu- tritional 88(3): 651. Guraya H.s., Kadam r.s. and champane E.t. 1997. Ef- fect of rice starch-Lipid complexes on in vitro digest- ibility, complexing index and viscosity. cereal chem. 74: 561. Guy r.c.E. and Horne A. 1988. Extrusion and co-extrusion of cereals. In “Food structure-Its creation And Evalua- tion”. J.M.V. blanshard and J.r. Mitchell (Eds.), p. 331- 349, London. Kocyigit A., Koylu A.A. and Keles H. 2006. Effects of pista- chio nuts consumption on plasma lipid profile and oxi- dative status in healthy volunteers. Nutritional Metabo- lism cardiovasc. dis. 16: 202. Krog N. 1973. Influence of food emulsifiers on pasting tem- perature and viscosity of various starches. starch/stae- rke, 25: 22. Launay b. and Lisch J.M. 1983. twin screw extrusion cooking of starches: behaviour of starch pastes, expan- sion and mechanical properties of extrudates. J. Food Eng. 2: 259. ratnayake W.M.N., Hansen s.L. and Kennedy M.P. 2006. Evaluation of the cp-sil 88 and sp-2560 Gc columns used in the recently approved Aocs official Method ce 1h-05: determination of cis-, trans-, saturated, mono- unsaturated, and polyunsaturated fatty acids in vegeta- ble or non-ruminant animal oils and fats by capillary GLc Method. J. Am. oil chem. soc. 83: 475. sari I., baltaci Y., bagci c., davutoglu V., Erel o., celik H., ozer o., Aksoy N. and Aksoy M. 2010. Effect of pista- chio diet on lipid parameters, endothelial function, in- flammation, and oxidative status: a prospective study. Nutrition 26(4): 399. schoch t.J. 1964. Iodimetric determination of amylose. po- tentiometric titration: standard Method. In: “Methods In carbohydrate chemistry”. r. Whistler (Ed.), p. 157-160. Academic Press, New York. schwartz c. 2009. the impact of removing snacks of low nutritional value from middle schools. Health Education and behav. 36 (36): 999. sheridan M.J., cooper J.N., Erario M. and cheifetz c.E. 2007. Pistachio nut consumption and serum lipid lev- els. J. Am. coll. Nutrition 26(2): 141. tester r.F. and Morrison W.r. 1990. swelling and gelatini- zation of cereal starches. I. Effects of amylopectin, amyl- ose, and lipids. cereal chem. 67: 551. Van Hecke E., Allaf K. and bouvier J.M. 1998. texture and structure of crispy-puffed food products II: Mechanical Properties in puncture. J. texture stud. 29: 617. Yaseen A.A.E. and shouk A.A. 2005. Effect of extrusion var- iables on physical, structure and sensory properties of wheat germ-corn grits extrudates. Egypt. J. Food sci. 33 (1): 57. Yu L., ramaswamy H.s. and boye, J. 2012. twin-screw ex- trusion of corn flour and soy protein isolate (sPI) blends: a response surface analysis. Food and bioprocess tech- nol. 2(5): 485. http://www.springerlink.com/content/0003-021x/