Protein Dispersibility Index and Trypsin Inhibitor Activity of Extruded Blends of Acha/Aoybean: A Response Surface Analysis
The effect of extrusion variables on the protein dispersibility
index and tyrpsin inhibitor activity of blends of acha and soybean were
studied using response surface analysis. Soybean flour was mixed with
acha flour at 0, 12.5, 25, 37.5 and 50% levels of substitution. Moisture
content of the mixtures was adjusted to 15, 20, 25, 30 and 35%. Extrusion
was carried out in a single screw Brabender extruder by adjusting the
screw speed from 90 to 120, 150, 180 and 210 rpm and barrel temperature
from 100 to 125, 150, 175 and 200°C following a 4-variable central
composite rotatable response surface design. Protein dispersibility index
and trypsin inhibitor activity of raw and extruded blends were evaluated.
Raw acha, soybean flour and raw acha/soybean flour blends had PDI
of 86.84, 91.84 and 74.27%, respectively compared to extrudate PDI which
ranged from 3.77-8.70%. Blending decreased the protein dispersibility
index of the raw flours while extrusion cooking significantly improved
the protein dispersibility. For TIA, the results showed that TIA of extrudates
ranged from 4.0-46.1 units, compared to the raw samples (64.5 units for
raw soybean). The results showed that extrusion cooking reduced TIA by
about (70.33-97.40%) with feed moisture and barrel temperature exerting
the greatest influence on extrudate TIA. The decrease in TIA corresponded
to increased protein dispersibility of extrudate samples.
Acha, Digitaria, exilis Skippis Stapf occupies about 300,000 ha
in West Africa and provides foods for about 4 million people (Kwon-Dung
and Misari, 2000). In Nigeria, acha is popularly grown in five states
(Bauchi, Kaduna, Kebbi, Plateau and Niger) and the Federal Capital Territory.
According to Kwon-Dung and Misari (2000), acha is one of the worlds
best tasting cereals. In recent times, comparison of dishes of acha and
rice showed that majority preferred acha dish. Traditionally, acha is
used in preparation of unfermented porridge food. It is also made into
gwette and acha-jollof. It is also used in dietary preparations for diabetic
patients (Victor and James, 1991). The protein content of acha grains
is rich in methionine and cysteine (above the recommended levels). These
levels are unusual for cereals. However, Victor and James (1991) reported
that with the exception of methionine and cysteine the essential amino
acid content of acha is lower than in other cereals. They therefore advocated
its complementation with protein rich foods to make a balance diet.
Most of the soybeans grown in Nigeria are used for human consumption
and are being promoted as nutritional supplement. The potential for incorporation
and utilization of soybeans in the local diet is enormous and serves as
a basis of the need for adequate processing. Soybeans have been processed
in a number of ways, but a more recent processing method is extrusion
cooking (Iwe, 2001).
A key problem associated with soybean utilization is the presence of
certain antinutritional factors, which may inhibit the availability of
the desirable nutrients such as proteins and minerals. Some of the prominent
antinutritional factors in soybean include trypsin inhibitor, hemaglutinin,
phytic acid, goitrogens, urease activity and flatulence causing factors
(starchyose and raffinose) (Osho and Dashiell, 1995). The significance
of soybean Trypsin Inhibitors (TI) lies in their implication in inhibiting
the pancreatic enzymes (trypsin and chymotrypsin) resulting in reduction
in protein digestibility both in humans and animals. It also causes hypertrophy
of the pancreas in smaller animals like cats and chicks (Liener, 1994).
Efforts have been made to inactivate or remove trypsin inhibitors from
soybean. Osho and Dashiell (1995), Keshun (1999) and Iwe and Ngoddy (2000)
reported that many approaches had been based largely on heat treatment.
According to Iwe and Ngoddy (2000) most commercially available soybean
products intended for human such as tofu, soybean milk, soybean-based
infant formula, soybean protein isolates and concentrates and textured
meat analogues have received sufficient heat treatment to cause inactivation
of at least 80% of the TIA present in raw soybeans. This level of TIA
destruction is well above the threshold of 50 to 60% inactivation found
to be necessary for eliminating significant growth inhibition and pancreatic
hypertrophy in rats (Rackis et al., 1976).
Iwe et al. (2001) have observed that protein solubility is an
important target parameter and in the animal and feed industry the protein
dispersibility index is often used to characterize the protein quality
of raw materials. Poel et al. (1990) and Camire et al. (1990)
reported that PDI could be used as a chemical indicator for inactivation
of antinutritional factors and effects on functional properties.
Several investigators (Leslie and Dale, 1990; Iwe, 2001; Tayeb et
al., 1992; Frazar et al., 1982; Iwe et al., 2000; Mullen
and Ennis, 1979; Meyer, 1976) had noted that the one-variable-at-a-time
method of experimentation is not good strategy in many situations. Response
Surface Methodology (RSM) basic principle of relating product properties
(mechanical, functional, nutritional and sensory) to process variables
(geometry, raw material, operating variables) (Iwe et al., 2000)
could eliminate most of the observed limitations. There is however dearth
of information on acha/soybean blending while there is no information
on acha extrusion cooking. The objective of present study was to assess
the effect of extrusion variables on the protein dispersibility index
and tyrpsin inhibitor activity of blends of acha and soybean using response
MATERIALS AND METHODS
This study was carried out in 2005 as follows: The extrusion was conducted
at the Federal Polytechnic Mubi Adamawa State, Nigeria.
Acha and soybean flour were prepared for extrusion as shown in Fig.
1. Feed moisture content was adjusted at five levels according to
Wilmot and Nelson (1998). Extrusion was carried out using a Branbender
Laboratory single screw extruder (DUISBURG DCE-330 Model). It was powered
by a decoder drive (Type 832, 500) and driven by a 5.94 kw motor. The
grooved band had a length/diameter ratio of 20:1. The extruder had variable
screws and heaters with a fixed die diameter of 2 mm and length of 40
mm. A feed hopper mounted vertically above the end of the extruder and
equipped with a screw that rotated at a constant speed of 80 rpm on a
vertical axis takes feed into the extruder. The wet flour was allowed
to equilibrate for 2-3 h before extrusion. The extruder runs was stabilized
using acha flour.
The experimental design was a Central Composite Rotatable Design (CCRD)
response surface analysis. Four independent variables including Feed Composition
(FC), Feed Moisture Content (FMC), Screw Speed (SS) and Barrel Temperature
(BT) were tested at 5 levels coded (-2 to +2) (Meyers, 1976; Iwe, 2003).
||Laboratory preparation of extruded acha-soybean flour
This experimental design required 36 experiments of which 16 were performed
at the factorial points, 8 at the axial point and twelve at the center
point. After steady state conditions were attained emerging extrudates
were collected and air dried at ambient temperature (24-27°C) for
about 12 h, then packed in cellophane packs and stored in the refrigerator
Protein dispersibility index was determined at the National Cereals
Research Institute Laboratory Badeggi Niger State Nigeria while the trypsin
inhibitor activity was assayed at the Soil Science Laboratory, University
of Nigeria Nssuka.
Protein solubility and protein dispersibility index were determined on
20 g sample of raw acha flour, soybean flour and 75:25% mixture of acha/soybean
flour along with extruded samples according to the methods described by
Iwe et al. (2001). Milled samples of raw and extruded blends that
passed a 1 mm sieve were blended with 300 mL of distilled water for 10
min in a warring blender operated at 8500 rpm and 21±1°C. The
slurry was allowed to settle for 10 min. After decanting, 50 mL of the
decantate was centrifuged at 2500 rpm (614x g) for 10 minutes. The supernatant
(20 mL) was pipetted into Kjedhal tubes for protein determination according
to AOAC (1984). Percentage solubility and protein dispersibility indices
were calculated as shown:
Protein solubility (%) = 6.25x14 (mL HCl-mL HClr) x
N/300 mL/20 mLx100/wt of sample
||Value of blank
||Normality of acid (0.1 N HCl)
||% protein solubility/Total protein content of sample x100
Trypsin Inhibitor Activity
This was evaluated following the procedures reported by Tanteeratarm
and Weingartner (1998): Finely ground samples (1.0 g) that passed 100
mesh were extracted with 50 mL of 0.01 N NaOH. Extracts were allowed to
stand for 1 h while the pH of the suspension was adjusted from 9.5 to
9.8 with NaOH. Portions (0, 0.6, 1, 1.4 and 1.8 mL) of the suspension
were pipetted into duplicate sets of test-tubes and adjusted to 2.0 mL
with distilled water.
Trypsin solution 2.0 mL was added to each test-tube; the tubes were placed
in a water bath at 37°C. To each tube, 5 mL solution of BAPA (Benzoyl-DL
arginine-p-nitroanilide) hydrate dissolved in dimethyl sulfoxide and diluted
to 100 mL) previously warmed to 37°C was added. Exactly, 10 min later,
the reaction was terminated by adding 1ml of 30% acetic acid. After thorough
mixing, the contents of each tube were filtered (Whatman Paper NO. 3)
and the absorbance was measured against blank at 410 nm.
Expression of Activity
One Trypsin Unit (TU) is arbitrarily defined as an increase of 0.01,
absorbance units at 410 nm per 10 mL of the reaction mixture. Trypsin
Inhibitor Activity (TIA) was expressed in terms of Trypsin Units Inhibited
RESULTS AND DISCUSSION
Protein Dispersibility Index (PDI)
The results of extrudate PDI ranged from 3.77-87.08%. Raw acha flour,
raw soybean flour and raw acha/soybean flour blends had 86.84, 91.84 and
74.27%, respectively. The results showed that most extrudates had over
63% reduction in PDI. This level of (PDI) reduction is an indication of
adequate heat treatment and hence enhanced nutritional status. Soybean
flour had an increased dispersibility of 96.54%, while acha flour dispersibility
increased by 89.8%. Iwe et al. (2001) reported similar results
with potato flour extruded with defatted soybean flour. The results of
regression analysis of the effect of process variables Feed Composition
(FC), Feed Moisture Content (FMC), Barrel Temperature (BT) and Screw Speed
(SS) on protein dispersibility index of extrudate samples is shown in
The results showed that only the cross product effect of FC*FMC significantly
(p<0.05) affected the protein dispersibilty index (Fig.
2). Analysis of variance (Table 2) showed that there
was no significant (p>0.05) model difference. The coefficient of determination
R2 (0.54) also indicated no significant (p>0.05) fit of
the model to the linear regression. These results agreed with the observations
of Iwe et al. (2001) that increased starch percentage decreases
protein dispersibility probably due to reduced protein percentage. Decreasing
the FMC and level of acha flour in the blends led to decrease in PDI.
These results agreed with Camire et al. (1990) who reported that
products with higher protein contents had lower nitrogen solubilitys
attributable to greater denaturation and aggregation of protein molecules.
Aguilera and Luasas (1986) also reported decreased solubility for extrudated
corn/soybean blends compared to that of unextruded materials. According
to Iwe et al. (2001) PDI range of 25-28% was acceptable for products
containing soybean. The results of this study fell within this acceptable
Trypsin Inhibitor Activity
The results showed that TIA of extrudates ranged from 4.0-46.1units
compared to the raw samples (64.5 mg g-1) and acha/soybean
blend at 75:25 ratios (54.5 mg g-1). Iwe and Ngoddy (2000)
and Delvelle et al. (1983) reported TIA values of 58.8 and 40-50
mg g-1 for whole soybean, respectively.
|| Effect of feed composition on protein dispersibility
||Estimated regression coefficients for extrudate protein
|FMC = Feed Moisture Content; SS = Screw Speed; FC =
Feed Composition and BT = Barrel Temperature *Sign of combination
|| Anova for extrudate protein dispersibilty index
The difference in these reports might be due to differences in variety
of soybean used and other agronomic factors. The results indicated that
extrusion processing reduced TIA by about (70.33-97.40%).
The result of regression analysis on the effects of process variables
on Trypsin Inhibitor Activity (TIA) is shown in Table 3.
The analysis of variance (Table 4) showed a significant
(p<0.05) model difference indicating that the model fitted the experimental
data. The coefficient of determination of of R2 = 0.6 showed
that the model could be used for predictive purposes since over 60% of
the total variability in the data was explained by the model. Removing
the non-significant terms, the model equation for TIA inhibition became
TIA = 42.22 − 12.59 BT − 13.16 FMC
− 1.98 FC*FMC = 3.07 BT2 + 13.29 FMC*BT −
|| Estimated regression coefficients for extrudate tyrpsin
|FMC = Feed Moisture Content; SS = Screw Speed; FC =
Feed Composition; BT = Barrel Temperature; *Sign of combination
||Response surface plot of the effect of feed moisture
content and barrel temperature on extrudate trypsin inhibitor activity
||Response surface plot of the effect of screw speed and
feed moisture content on extrudate trypsin inhibitor activity
The response surface plot of FMC, BT and TIA units (Fig.
3) showed that increasing FMC and BT led to reduction of TIA units
up to 84.97% at 150-200°C and 25 to 32% moisture content. Tsen et
al. (1975) reported TIA inhibition or destruction from 50 to 100%
when extrusion temperature was raised from 115 to 140°C at approximately
25% moisture. Philip et al. (1983) reported TIA inactivation at
increased temperature. The results from this study are in agreement with
these reports. However, at 32% moisture content, even at 100°C TIA
inactivation began to reduce. The results showed that TIA reduction was
dependent more on the moisture content than on temperature. This phenomenon
may be due to the fact that increased moisture beyond the 32% exerted
pronounced shear lowering which in turn reduced extruder internal temperature
leading to decreased extrudate exit temperature.
The response surface plot of TIA to changes in the independent variables
of FMC and SS (Fig. 4) showed that increasing FMC and
SS led to reduction of TIA units. It was observed that FMC played more
significant role than SS and had a quadratic effect.
|| ANOVA for extrudate tyrpsin inhibitor activity
||Response Surface Plot of the Effect of Screw Speed and
Barrel Temperature on Extrudate Trypsin Inhibitor Activity
|| Response Surface Plot of the Effect of Feed Composition
and Feed Moisture Content on
The results showed similar pattern with that of FMC*BT. Similar results
have been reported by Harper (1980) and Philip et al. (1983). Increased
TIA destruction at decreased moisture content may be due to the increase
in extruder shear (which would favour more inactivation) moisture content
(Cheftel, 1984; Camire et al., 1990; Liener, 1994; Iwe and Ngoddy,
The relationship between the extruder SS and BT and TIA (Fig.
5) showed that BT had both a linear and quadratic effect on TIA retention.
The results indicated that the linear effect was greater than the quadratic
hence decreasing BT and SS recorded increased TIA retention which was
expected. Below 120 rpm and 160°C, TIA units were as high as 26 TIA.
At higher SS and BT, TIA inactivation (reduction) rose. Several authors,
Tsen et al. (1975) Philips et al. (1983), Harper and Jansen
(1985) Lorenz et al. (1980), Iwe and Ngoddy (2000) had reported
the reduction of TIA at extrusion temperature of 121-150°C. Operating
conditions for increased TIA inactivation was located at 120 SS and 175°C
giving over 50.93% inactivation at 30% FMC and 12.5% soybean flour substitution.
The response of TIA to the FC and FMC (Fig.6) showed
that decreasing the level of substitution of soybean and the FMC resulted
in reduction of TIA. This was expected as the analysis showed that raw
soybean contained more than 89.38% of the total TIA content of the raw
blend. Iwe et al. (2001) showed a similar occurrence in the extrusion
of sweet potato/soybean blends. The results indicated that addition of
soybeans up to 60% in the feedstock would lead to rise in TIA ratio of
37 units of the original FC. This takes place at about 32% moisture content.
||TIA predicted using developed model equation
The results from this study showed that the range of heat treatment applied
in this study provided satisfactory inactivation of antinutrients and
produced acceptable functional products with good protein dispersibility
indices. However, because of low R2 and lack of model significance
(p>0.05) the model for protein dispersibility was not used for predictive
purposes. The predicted results of trypsin inhibitor activity (Table
5) approximated experimental results. It was therefore concluded that
the second order polynomial was adequate in predicting the dependence
of TIA on the process variables.
1: Aguilera, J.M. and E.W. Lusas, 1986. Laboratory and pilot extraction of extruded high-oil corn. J. Am. Chem. Soc., 63: 239-243.
2: AOAC., 1984. Official Methods of Analysis. 14th Edn., Association of Official Analytical Chemists, Washington, DC., USA., pp: 522-533
3: Camire, M.E., A. Camire and K. Krumhar, 1990. Chemical and nutritional changes in foods during extrusion. Critical Rev. Food Sci. Nutr., 29: 35-57.
CrossRef | PubMed | Direct Link |
4: Cheftel, J.C., 1984. Extrusion Cooking and Food Safety. In: Extrusion Cooking, Mercier, C., P. Linko and Harper (Eds.). AACC Inc., St. Paul. Minnesota, USA, pp: 1-15
5: Del Valle, F.R., M. Escobedo, M.J. Munoz, R. Ortega and H. Bourges, 1983. Chemical and nutritional studies on Mesquite beans (Prosopis juliftora). J. Food Sci., 48: 914-919.
CrossRef | Direct Link |
6: FAO, 1985. Technical Compendium on Composite Flours. United Nation's Economic Commission for Africa, Addis Ababa
7: Harper, J.M., 1980. Extrusion of Foods. Vol. 1, CRC, Press Inc., Boca Raton, Florida, pp: 1-3
8: Harper, J.M. and G.R. Jasen, 1985. Production of nutritional precooked foods in developing countries by low-cost extrusion technology. Food Rev. Int., 1: 27-97.
9: Iwe, M.O. and P.O. Ngoddy, 2000. Effects of extrusion on trypsin inhibitor contents of soy-sweet potato blends. J. Food Pro. Preservat., 24: 453-463.
Direct Link |
10: Iwe, M.O., 2000. Effects of extrusion cooking on some functional properties of soy-sweet potato mixtures: A response surface analysis. Plant Foods Hum. Nutr., 55: 169-184.
CrossRef | Direct Link |
11: Iwe, M.O., D.J. van Zuilichem, P.O. Ngoddy and D.J. Winlarmmers, 2001. Amino acid and protein dispersibility index of mixtures of extruded soy and sweet potato flours. Lebensm-wiss u Technol., 34: 71-75.
CrossRef | Direct Link |
12: Iwe, M.O., 2003. The Science and Technology of Soybeans. 1st Edn., Chemistry, Processing and Utilization, Rejoint Communication Services Ltd., Enugu, Nigeria
13: Iwe, M.O., D.J. Van Zuilichem, W. Stolp and P.O. Ngoddy, 2004. Effect of extrusion cooking of soy-sweet potato mixtures on available lysine content and browning index of extrudates. J. Food Eng., 62: 143-150.
CrossRef | Direct Link |
14: Keshun, L., 1999. Current constraints in soybean food utilization and efforts to overcome them. Proceedings of the World Soybean Research Conference VI, August 4-7, 1999, Chicago, Illinois, USA., pp: 409-418
15: Kwon-Ndung, E.H. and S.M. Misari, 2000. Over View of Research and Development of Acha Digitaria Exilis Kippis Stapf and Prospects of Genetic Improvement in Nigeria. Genetics and Food Security in Nigeria, Genet, Soc. of Nigeria, Nigeria, pp: 71-76
16: Leslie, L.R. and A.W. Dale, 1990. Response Surface Characterization of a Twin-screw Extruder. Vol. 3, Engineering and Food, Advanced Processes, Spiess, WEL
17: Liener, I.E., 1994. Implications of antinutritional components in soya bean foods. Crit. Rev. Food Sci. Nutr., 34: 31-67.
PubMed | Direct Link |
18: Lorenz, K., G.R. Jansen and J.M. Harper, 1980. Nutrient stability of full-fat soy flour and corn-soy blends produced by low-cost extrusion. Cereal Foods World, 25: 161-162.
19: Meyers, H.R., 1976. Response Surface Methodology. Allyn and Bacon, Boston MA. USA
20: Mullen, K. and D.M. Ennis, 1979. Rotatable designs in product development. Food Technol., 33: 74-81.
21: Osho, S.M. and K. Dashiell, 1995. Expanding the soybean production, processing and utilization in Africa: Post harvest technology and commodity marketing. Proceedings of the Post Harvest Conference, November 1-2, 1995, Accra, Ghana, RSB. IITA, Ibadan, pp: 151-156
22: Philips, R.D., M.S. Chinnan and L.G. Mendoza, 1983. Effect of temperature and moisture content on the kinetics of trypsin inhibitor activity, protein in vitro digestibility and nitrogen solubility of cowpea flour. J. Food Sci., 48: 1863-1867.
23: Poel, A.F., J. Blonk, D.J. van Zuilichem and M.G. van Oort, 1990. Thermal inactivation of lectins and trypsin inhibitor activity during steam processing of dry beans (Phaseolus vulgaris L.) and effects on protein quality. J. Sci. Food Agric., 53: 215-228.
Direct Link |
24: Rackis, J.J., J.E. McGhee and A.N. Booth, 1976. Biological threshold levels of soybean trypsin inhibitors by rat assay. Cereal Chem., 52: 85-91.
25: Tanteeratarm, K. and K.E. Weingartner, 1998. Trypsin Inhibitor Assay. In: Soybean Processing for Food Uses. A Training Manual, Sheldon, W.W. (Ed.). International Soybean Programme (INTSOY), Department of Food Science and Human Nutrition, USA, pp: 419-422
26: Tayeb, J., G. del Valle, C. Barres and V. Bruno, 1992. Simulation of Transport Phenomena in Twin-screw Extruders. In: Food Extrusion Science and Technology, Kokini, J.L., C. Ho and M.V. Karwe (Eds.). Food Extrusion Science and Technology, USA, pp: 41-70
27: Tsen, C.C., E.P. Farrel, W.J. Hoover and P.R. Crowdley, 1975. Extruded soy products from whole and dehulled soybeans cooked at various temperatures for bread and cookie fortification. Cereal Food World, 20: 413-418.
28: Victor, J.T. and D.B. James, 1991. Proximate chemical composition of Acha (Digitaria exilis) grain. J. Sci. Food Agric., 56: 561-563.
CrossRef | Direct Link |
29: Wilmot, B.W. and A.I. Nelson, 1998. Extrusion and Oil Expelting. In: Soybean Processing for Food Uses. A Training Manual, Sheldon, W.W. (Ed.). International Soybean Programme (INTSOY), Dep. Food Sci. Hum. Nutr., New York, pp: 149-157