STUDY ON INTERACTION AMONG EXTRUSION-COOKING PROCESS VARIABLES AND ENZYME ACTIVITY: EVALUATION OF...

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STUDY ON INTERACTION AMONG EXTRUSION-COOKINGPROCESS VARIABLES AND ENZYME ACTIVITY: EVALUATION

OF EXTRUDATE STRUCTURE

TERESA DE PILLI1,3, JACK LEGRAND2, ANTONIO DEROSSI1

and CARLA SEVERINI1

1Department of Food ScienceUniversity of Foggia

Napoli Naples, Foggia, Italy

2Université de NantesCNRS, GEPEA, UMR 6144, CRTT

St. Nazaire, Cedex, France

Accepted for Publication January 20, 2008

ABSTRACT

A blend of wheat flour (moisture content 11.61%) and different amountsof two commercial enzymes like GRINDAMYL (GA) amylase 1000 (0–0.1 g/kg) and GA protease 41 (0–1.0 g/kg) were extruded through a co-rotatingtwin-screw extruder.

The effects of barrel temperature (60–120C), dough moisture (20–36%),screw speed (100–400 rpm) and different amounts of enzymes on structure ofextrudates (porosity and break strength) were studied using response surfacemethodology.

Results showed that the highest value of porosity (62.47% � 4.07) wasobtained at the highest value of barrel temperature (120C) and at the largestamounts of GA protease 41 (1.0 g/kg) or GA amylase 1000 (0.1 g/kg).However, the simultaneous addition of large amounts of both enzymes causeda negative effect on expansion degree (porosity < 30%). Large amounts ofmoisture dough and GA amylase 1000 resulted in a high value of breakstrength (25.7 N/mm2) and in stiff-textured extrudates.

PRACTICAL APPLICATIONS

This research could provide some information about the effects of and theinteractions between extrusion parameters and enzyme activities, which are

3 Corresponding author. TEL: +39-881-589245; FAX: +39-881-589308; EMAIL: [email protected]

Journal of Food Process Engineering 33 (2010) 65–82. All Rights Reserved.© Copyright the AuthorsJournal Compilation © 2008 Wiley Periodicals, Inc.DOI: 10.1111/j.1745-4530.2008.00260.x

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commonly used to produce bakery products, in order to improve the texture ofextrudates processed at low barrel temperatures, i.e., 60C, for example, extru-dates with thermolabile components like vitamins or extrudates with high lipidfraction like breakfast cereals, pet food, etc.

INTRODUCTION

Extrusion-cooking processing is used to produce direct expanded prod-ucts such as snacks, breakfast cereals and pet food (Miller 1990; Moore 1994;Rokey 1994).

Generally, breakfast food and snacks should have an expanded structureand a crispy texture. During extrusion-cooking of biopolymers, the passingof visco-elastic material through a die causes a sudden pressure drop with agiven water vaporization, resulting in an expanded and porous structure.Both elastic swelling and bubble growth effects contribute to the resultingexpansion (Padmanabhan and Bhattachayrya 1989). This characteristicstrongly depends on moisture content and extrusion temperature. Severaltheoretical considerations for extrudate expansion have been published(Alvarez-Martinez et al. 1988; Kumagai and Yano 1993). In particular, extru-sion temperature less than 100C gives a stiff structure to wheat flour-basedextrudates, as this treatment is not able to denature gluten (Sevryukova et al.1985). The lack of gluten denaturation reduces the dough deformation at theexit of the die, resulting in a nonexpanded product. On the contrary, the useof high extrusion temperature (130–200C) causes gluten denaturation, result-ing in an expanded product. However, low temperatures are needed in someextrusion processing because of the presence of thermolabile compounds(e.g., snacks enriched with vitamins) or ingredients with high lipid content.In the latter case, for example, high process temperature determines theleakage of oil, and subsequently, percolation from the extruder (De Pilliet al. 2000). On the one hand, this situation is unable to preserve both theextrudates and the hygienic conditions of plants. On the other hand, thestructural characteristics of wheat flour-based extrudates could be improvedby using enzymes like amylase and protease.

The use of enzymes in baking technology is a very common practice (VanOort and Hamer 1993). They are responsible for improving dough machin-ability and bakery product quality. In particular, a-amylases favor the produc-tion of fermentable sugars that increase fermentation rate; proteases favor thereduction of dough mixing time and enhance dough extensibility; oxidases andproteases increase dough stability (Bock 1991).

Historically, proteases were added to shorten the mixing time (Barrett1975) and to modify dough consistency. Some authors discussed the use of

66 T. DE PILLI ET AL.

proteases to regulate gluten strength in bread and roll flours and to solvebaking problems in special flours (e.g., biscuits and wafers) (Uhlig andSproessler 1971; Gaines and Finney 1989). Small amounts of proteases canhave large effects on gluten physical properties. Oka et al. (1965) demon-strated that breakage of a few peptide bonds resulted in a rapid decrease inviscosity of glutenin dispersion. Also, Redman (1971) reported evidence tosupport the hypothesis that gluten softening is the direct result of peptide bondscission catalyzed by proteases. Extensive softening was observed althoughvery few peptide bonds were broken.

In the field of extrusion, the possibility of using an extruder as a“bioreactor” – i.e., to optimize process parameters in order to favor enzymaticreactions – was studied. In particular, De Pilli et al. (2004) studied the effectsof two commercially available enzymes (protease and amylase) on the textureof products obtained from wheat and almond flours extruded at low tempera-ture (54C). Their results showed that the protease affected the rheologicalcharacteristics of dough, with a rapid drop in consistency during farinographicanalyses, and that it improved the structure of extrudates with high lipidcontent (less break strength, high porosity and high water solubility index).Nevertheless, this research did not consider the effect of process variables onenzymatic activity.

The aim of this research was to study the effects of both processingvariables (barrel temperature, dough moisture and screw speed) and activity oftwo commercial enzymes (GRINDAMYL [GA] amylase 1000 and GA pro-tease 41) on structure of extrudates (porosity and breaking strength).

MATERIALS AND METHODS

Flours

Wheat flours were supplied by Cereal Destrine (Cadelbosco Sopra,Reggio Emilia, Italy). The chemical composition of the flours is reported inTable 1.

TABLE 1.CHEMICAL COMPOSITION OF FLOUR USED FOR THE EXTRUSION EXPERIMENTS

Raw materials Moisture(%)

SD Ash(%)

SD Protein(%)

SD Driedgluten (%)

SD

Wheat flour 11.61 0.04 0.518 0.01 9.57 0.07 5.46 0.2

ENZYME ACTIVITY AND EXTRUSION 67

Enzymes

GA amylase 1000 from Aspergillus orizae (1000 fungal alpha amylaseunits (FAU)/g) and GA protease 41 from Bacillus subtilis (41 Units/mg/g)were both supplied by Danisco Cultor (Grindsted, Denmark).

Chemical Analysis of Flours

The moisture, ash, protein and fat contents were determined according tothe methods of the American Association of Cereal Chemists (2003). Eachanalysis was repeated at least three times.

Experimental Design

The factorial design of five variables (barrel temperature, screw speed,dough moisture expressed as dry basis percentage, amounts of GA amylaseand GA protease) and five levels, obtained by a Central Composite Design(CCD) (Box et al. 1978), are shown in Table 2; the factorial design was usedto evaluate the effect of each variable and their possible interactions. Theapplication of a CCD allowed reduction of the number of possible combina-tions. Thirty tests with different combinations of process variable values(barrel temperature, moisture dough, screw speed, amounts of GA amylase andGA protease) were obtained using the following equation:

n n n ntot c *= + + = + + =0 16 10 4 30 (1)

where n0 = 2n–1, n is the number of variables, nc is the number of central pointsand n* is the number of star points (Box et al. 1978). The 30 combinationsobtained for CCD are shown in Table 3.

The appropriate range of processing parameters and amounts of enzymeswere chosen according to De Pilli et al. (2004).

Extrudates Formula

The dough was prepared using wheat flour, running water and differentamounts of enzymes (Table 2).

Extruder

A BC-21 Clextral (Firminy, France) co-rotating twin-screw extruder wasused. The screw’s geometrical features were as follows: diameter, 25 mm;


TABLE 2.CENTRAL COMPOSITE DESIGN

Codifiedvalues

Extrusiontemperature (C)

Doughmoisture (%)

Screwspeed (rpm)

GA amylase(g/kg)

GA protease(g/kg)

-2 60 20 100 0 0-1 75 24 175 0.025 0.25

0 90 28 250 0.05 0.5+1 105 32 325 0.075 0.75+2 120 36 400 0.1 1.0

TABLE 3.EXPERIMENTAL FACTORIAL DESIGN

Samples Extrusiontemperature (C)

Doughmoisture (%)

Screwspeed (rpm)

GA amylase(g/kg)

GA protease(g/kg)

1 75 24 175 0.025 0.752 75 24 175 0.075 0.253 75 24 325 0.025 0.254 75 24 325 0.075 0.755 75 32 175 0.025 0.256 75 32 175 0.075 0.757 75 32 325 0.025 0.758 75 32 325 0.075 0.259 105 24 175 0.025 0.25

10 105 24 175 0.075 0.7511 105 24 325 0.025 0.7512 105 24 325 0.075 0.2513 105 32 175 0.025 0.7514 105 32 175 0.075 0.2515 105 32 325 0.025 0.2516 105 32 325 0.075 0.7517 60 28 250 0.05 0.518 120 28 250 0.05 0.519 90 20 250 0.05 0.520 90 36 250 0.05 0.521 90 28 100 0.05 0.522 90 28 400 0.05 0.523 90 28 250 0 0.524 90 28 250 0.1 0.525 90 28 250 0.05 026 90 28 250 0.05 127 90 28 250 0.05 0.528 90 28 250 0.05 0.529 90 28 250 0.05 0.530 90 28 250 0.05 0.5


length, 900 mm; distance between shafts, 21 mm (length:diameter = 36:1).The screw configurations used (from inlet to the die) are shown in Table 4.

During extrusion experiments, the flour feed rate was kept constant at10 kg/h using a volumetric gravity feeder. The moisture content of dough wasadjusted using a water pump that injected the water into the first zone of theextruder, since the extruder was divided into nine zones independent of eachother for temperature control and adjustment.

The first five zones were kept at room temperature, whereas the tempera-ture of the last four zones was adjusted according to the experimental factorialplan temperatures shown in the experimental factorial design (Table 3).

The die, with a spherical shape and a diameter of 60 mm, was equippedwith two spherical holes (diameter of 5 mm).

The extrudates were cut at the exit of the die using a cutter set at 210 rpm;stick-shaped products were obtained (Fig. 1). Extrudates were put on alumi-num trays and dried at 80C for 10 min using a convection oven (DelonghiCombi & Functions Convection, Milan, Italy). After drying, extrudates with8 � 0.92% moisture content were air-cooled, stored in polyethylene bags andanalyzed.

Break Strength

A dynamometer Stable Micro System TA-HDi Texture Analyser (ENCOs.r.l., Venice, Italy) equipped with a small wedge was used. Extrudates wereplaced over two supports (1.5 cm apart) and broken in the middle by a conefrustum plunger (the thickness of contact surface with extrudate was 1 mm2

and the speed was kept constant at 0.5 mm/s).Results were expressed as breaking strength (N/mm2), i.e., the strength

applied to break the extrudate. This index is related to microstructure ofsamples and it simulates the incisors’ impact at biting (Hutchinson et al. 1987;Hayter and Smith 1988; Georget et al. 1995; Li et al. 1998; Van Hecke et al.1998). For each sample, at least 10 repetitions were carried out.

Image Analysis

Air cell structures of extrudates were measured using the methoddescribed by Barrett and Ross (1990). Fifteen extrudates were selected fromall 30 samples. Each extrudate was cross-sectioned out. The sections werepressed against a desk pad in order to blacken cell structures. Sections ofextrudates were photographed using a dv-camera (Canon, Japan) equippedwith a 275 mm lens (0.8 megapixel, Micro Nikon, Turin, Italy). Images wereanalyzed using the Optimas ver. 6.1 software (Bioscan, Washington, DC). Thissoftware allowed evaluation of the surface porosity by application of a


gray-level value (i.e., position between black and white) to each pixel in theimage. Threshold levels (black and white) were selected to construct a binaryimage in which pixels having gray levels above the thresholds were black, andpixels having gray levels below these values were white. The area of the blackobject within the binary image was measured.

Results were expressed as percentage ratio between surface of pores andsurface of all extrudate section (porosity).

TABLE 4.SCREW PROFILE USED FOR EXTRUSION EXPERIMENTS

Type of screw element Screw element details(pitch/length)

Total length (mm)

Forward pitch 50/50 150

Kneading block

50/33 10050/25 20050/33 10050/25 100

Decreased pitch 50/16 250

FIG. 1. EXTRUDED PRODUCTS


Statistical Analysis

All data were statistically analyzed using Winstat version 6.0 (Statsoft,Tulsa, OK) software. The mathematical modeling was carried out in two steps.The first step involved a stepwise regression to identify the relevant variables;the second one consisted of a multiple regression (standard least square fitting)to fit a second order mathematical model, according to the following polyno-mial equation:

y B B B= + + +∑ ∑ ∑B0 i i ii ii ij i jχ χ χ χ2 (2)

where y is the dependent variable (porosity and break strength), B0 is aconstant value, ci and cj are the independent variables in coded values andBi, Bii, Bij are the regression coefficients of the model. Variables having asignificance lower than 99% (P > 0.01) were left out of the equation. Thismodel assessed the effects of the linear (ci), quadratic (χ i

2) and combined(cicj) terms of the independent variables (barrel temperature, dough mois-ture, screw speed, GA amylase and GA protease amounts) on the dependentvariable.

In order to describe the individual and interactive effects of the indepen-dent variables on the modification of extrudate structure (in terms of porosityand break strength), iso-response surfaces were developed and kept constantwith three of the other independent variables at the central level of CCD.

RESULTS AND DISCUSSION

Values of correlation coefficient, standard error of estimation (SE),Fisher test, P-level and general equations of each considered dependent vari-able are shown in Table 5. The values of r showed that each consideredequation results were significant since they were always greater than 0.81(Box et al. 1978).

Figure 2 shows the porosity of extrudates as a function of barrel tempera-ture and dough moisture. From iso-response surface and its partial equation(presence of positive angular coefficient), a considerable increase of porositywas observed at the increase of barrel temperature compared with moisturedough. The increase of barrel temperature probably caused a decrease in meltviscosity, which favored the bubble growth during extrusion processing. Thesedata support the report of Mercier and Feillet (1975). Moreover, high process-ing temperature had probably increased superheating of water, allowinggreater expansion of products. These results are in agreement with Ding et al.(2005).


TABLE 5.GENERAL EQUATIONS OBTAINED BY FITTING MULTIPLE REGRESSION

(BACKWARDS STEPWISE) TO VALUES OF DEPENDENT VARIABLES

Dependent variables General equation r SE F P-level

Porosity (%) Y = -20.34 + 0.48 * [SS] - 0.02 * [M]2 -0.001 * [SS]2 + 0.92 * [T] * [P] + 15.06 *[M] * [A] - 0.21 * [SS] * [P] - 692.12 *[A] * [P]

0.81 8.05 6.16 <0.00045

Break strength(N/mm2)

Y = -60.09 + 1.57 * [T] - 0.21 * [SS] +1,160.74 * [A] - 0.01 * [T]2 + 0.01 *[T] * [M]

0.82 3.84 3.5 <0.009

Alpha for highlighting effects: 0.05.T, barrel temperature; M, dough moisture; SS, screw speed; P, protease amount; A, amylase amount;r, correlation coefficent; SE, standard error; F, the Fisher test F value.

FIG. 2. POROSITY (%) OF EXTRUDATES AS A FUNCTION OF BARREL TEMPERATUREAND DOUGH MOISTURE

Porosity (%) = -7.26 + 0.46 ¥ [barrel temperature] + 0.75 ¥ [dough moisture] -0.23 ¥ [dough moisture]2


The highest values of porosity were obtained at high values of barreltemperature and large amounts of both enzymes (Figs. 3 and 4). In particular,iso-response surfaces relating to GA amylase and GA protease show a positiveinteraction between barrel temperature and enzymatic activity on porosity.Likimani et al. (1991) studied enzymatic extrusion of corn and observed thatthe extent of starch hydrolysis increased with temperature, ranging from 80 to160C, and moisture contents ranging from 18 to 26%. The effect of tempera-ture seemed to be consistent with heat denaturation of enzymes, which shouldhave been enhanced by the high moisture contents used. Therefore, theincrease in water content had destabilizing effects on the enzyme, leading tofaster inactivation, while improving enzymatic activity (Tomás et al. 1997). Inthis research, the percentage of moisture content ranged from 20 to 36%;therefore, it can be supposed that the increase in temperature enhanced theactivity of both enzymes. In particular, the highest porosity obtained at thehighest value of barrel temperature and at the largest amount of GA amylaseand GA protease could be due to the increase in enzymatic activity thatdetermined the production of low molecular weight compounds, which gave a

FIG. 3. POROSITY (%) AS A FUNCTION OF GRINDAMYL AMYLASE AMOUNT ANDBARREL TEMPERATURE

Porosity (%) = -7.99 + 0.46 ¥ [barrel temperature] + 91.58 ¥ [GA amylase]


higher plasticity to dough. Chen and Yeh (2000) observed that the addition ofdextrin (the principal product of amylase activity) in rice-based extrusionresulted in the decrease of glass transition (Tg) as well as of expansion tem-perature (Te) due to the reduction in average molecular weight indicated by theintrinsic viscosity. In addition, dextrin could also act as a plasticizer. The Tg

was a good reference point for the pellet expansion. Both increasing equilib-rium moisture and adding dextrin resulted in an increase in (Te - Tg) and adecrease in Tg. At high moisture content, water-plasticized material should beeasier to expand. Nevertheless, the expansion was delayed. The expansionratio was reduced by the softening effect of moisture. The addition of dextrincaused the formation of a crust, which can hold more water vapor, and resultedin a higher (Te - Tg) and in more expansion. Several authors proposed the useof proteases as an alternative to chemical dough plasticizers in biscuit orcookie manufacture (De la Gueriviere 1972; Gaines and Finney 1989; Toshioet al. 1991). Ishida and Nagasaki (1989) investigated the use of proteases toreplace dough-slackening chemicals. They observed that proteases broke long

FIG. 4. POROSITY (%) AS A FUNCTION OF GRINDAMYL PROTEASE AMOUNT ANDBARREL TEMPERATURE

Porosity (%) = 39.97 - 88.35 ¥ [GA protease] + 0.923 ¥ [barrel temperature] ¥ [GA protease]


chains of proteins into smaller units, thus increasing gluten elasticity, andconsequently, resulting in dough that is more deformable.

The porosity values as a function of GA amylase and GA proteaseamounts are shown in Fig. 5. The iso-response surface and its partial equationshow a negative interaction between two independent variables. In particular,the smallest porosity values were obtained at the highest amounts of enzymeconcentrations. This phenomenon could be attributed to the lowest gelatiniza-tion degree that occurs at these operating conditions (data not shown). Theincrease in GA amylase amount caused a high dextrination of starch and aconsequent reduction of its gelatinization. The increase in GA protease,causing the degradation of gluten in hydrosoluble compounds with lowmolecular weight, could compete with starch for water absorption (De Pilliet al. 2004).

Break strength values as a function of barrel temperature and doughmoisture are shown in Fig. 6. Hard and stiff extrudates were obtained withincreasing barrel temperature and dough moisture, resulting from break

FIG. 5. POROSITY (%) AS A FUNCTION OF GRINDAMYL PROTEASE AND GRINDAMYLAMYLASE AMOUNTS

Porosity (%) = 18.881 + 421.764 ¥ [GA amylase] - 692.118 ¥ [GA amylase] ¥[GA protease] + 29.32 ¥ [GA protease]


strength values that increased with the increase in these processing variables.These results are in agreement with Faubion and Hoseney (1982). Theseauthors observed that both cell wall setting time and air bubble coalescence(which is responsible for the microporous structure) increased in productsextruded at high moisture and low temperature. Higher extrusion temperatureresulted in faster water evaporation, instantly fixing cell walls. In this way,time for air bubble coalescence was not sufficient to cause the stiffening ofproducts. On the contrary, the increase in screw speed led to a decrease inbreak strength: the obtained extrudates were more fragile than those extrudedat low screw speed values (Fig. 7). An increase in porosity, which correspondsto a lower hardness, was determined by higher incorporation of air insidedough extruded at elevated screw speed. In fact, the Pareto diagram shows apositive effect (B/SE = 3.42) of screw speed on porosity (Fig. 8).

Moreover, a negative correlation was found from regression resultsobtained correlating break strength and porosity (Table 6). GA amylase had apositive effect on break strength as shown in Fig. 9. In particular, an increasein GA amylase amount resulted in an increase in break strength (i.e., increase

FIG. 6. BREAK STRENGTH AS A FUNCTION OF BARREL TEMPERATURE ANDDOUGH MOISTURE

Break strength (N/mm2) = - 54.553 - 0.01 ¥ [barrel temperature]2 + 0.01 ¥ [barrel temperature] ¥[dough moisture] + 1.57 ¥ [barrel temperature]


in product hardness), likely caused by the lowest gelatinization degree thatoccurs at these operating conditions (data not shown).

CONCLUSIONS

Results showed that both processing parameters and enzymatic activitydetermined significant modification on extrudate structure. In particular,increasing barrel temperature increased porosity percentage. Moreover, it ispossible to improve the structure of wheat flour-based extrudates at tempera-tures less than 100C by using enzymes. Nevertheless, the concurrent additionof both enzymes (GA amylase and GA protease) caused a negative effect onexpansion degree. GA protease was more suitable to improve extrudate struc-ture since it determined an increase of porosity, and unlike GA amylase, didnot cause stiffness of texture of extrudates (highest values of break strength).The increase in screw speed showed a positive effect on extrudate structure; in

FIG. 7. BREAK STRENGTH AS A FUNCTION OF BARREL TEMPERATURE ANDSCREW SPEED

Break strength (N/mm2) = - 2.053 - 0.21 ¥ [dough moisture] + 1.85 ¥ [barreltemperature] - 0.01 ¥ [barrel temperature]


fact, the lowest values of break strength and the highest values of porosity wereobtained at the highest values of this processing variable.

ACKNOWLEDGMENTS

We would like to thank Dr. Farid Fodil Pacha and Dr. Yasser Kerhaili fortheir important contribution to this research.

FIG. 8. PARETO DIAGRAMS RELATING INDEPENDENT VARIABLES TOEXTRUDATE POROSITY (%)

TABLE 6.REGRESSION RESULTS OBTAINED FROM CORRELATION OF DEPENDENT VARIABLES

(BREAK STRENGTH AND POROSITY)

Dependent variables Regression equation r P-level

Break strength (N/mm2):Porosity (%)

y = 20.957 - 0.2520 ¥ [x] -0.57 0.0011*

* Significant at P < 0.005.r, correlation coefficient.


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