P U B L I C A T I O N S CODON Italian Journal of Food Science, 2022; 34 (3): 105–123 ISSN 1120-1770 online, DOI 10.15586/ijfs.v34i3.2238 105 P U B L I C A T I O N S CODON Effect of extrusion processing conditions on the techno-functional, antioxidant, textural properties and storage stability of wholegrain-based breakfast cereal incorporated with Indian horse chestnut flour Farhana Mehraj Allai1,2, Z.R.A.A. Azad1*, B.N. Dar2*, Khalid Gul3* 1Department of Post-Harvest Engineering and Technology, Faculty of Agricultural Science, Aligarh Muslim University, Uttar Pradesh, India; 2Department of Food Technology, Islamic University of Science and Technology, Awantipora, India; 3Department of Food Process Engineering, National Institute of Technology, Rourkela, India *Corresponding Authors: Z.R.A.A. Azad, Department of Post-Harvest Engineering and Technology, Faculty of Agricultural Science, Aligarh Muslim University, Uttar Pradesh, India. Email: zrazad@gmail.com; B.N. Dar, Department of Food Technology, Islamic University of Science and Technology, Awantipora, India. Email: darnabi@gmail.com; and Khalid Gul, Department of Food Process Engineering, National Institute of Technology, Rourkela, India. Email: gulmk@nitrkl.ac.in Received: 3 June 2022; Accepted: 15 July 2022; Published: 27 October 2022 © 2022 Codon Publications OPEN ACCESS PAPER Abstract The central composite rotatable design was used to plan the experiments using feed moisture (FM) content, barrel temperature (BT), screw speed (SS), and concentration of Indian horse chestnut flour (IHCF) as process variables var- ied between 10–18%, 70–150°C, 290–380 rpm and 1.75–4.75%, respectively. Surface mechanical energy, water hold- ing capacity, swelling volume, piece density, longitudinal expansion, water activity and colour attributes were used as response variables. The whole grain flours added with 2.5% IHCF and extruded at 12% FM, 130°C BT and 380 rpm SS produced optimum quality breakfast cereals with 0.713 desirability. Extrudates packed in aluminum pouches were found to be suitable packaging materials and were shelf-stable for 6 months with better quality retention. Keywords: extrusion cooking, fibre, horse chestnut, packaging, shelf-life, whole grains Introduction Over the decades, people across the world have struggled to ensure a healthy and delicious diet. For this purpose, producers had to adopt different techniques aimed at making food products more appealing and tastier with increased nutritional value and organoleptic attributes (Nkhata et al., 2018). The traditional cooking method improves the bioavailability and bioaccessibility of nutri- ents but at the same time causes significant loss of macro- as well as micronutrients (Kamau et al., 2020). Nutrient utilization can be increased by processing methods such as extrusion cooking. Extrusion cooking is the most popular food process- ing technique in which the ingredients are subjected to amalgamation of temperature, internal pressure, mois- ture and shear forces, leading to the modification and disruption of molecular structure and texture resulting in the development of an ample range of food products such as breakfast cereals, pasta, snacks, baby foods and texturised meat substitutes (Espinosa-Ramirez et al., 2021). It is also used to overcome the limitations associ- ated with the processing of traditional cereal-based food products by enhancing its functional, physicochemical and shelf stability (Jabeen et al., 2021). The extrusion process improves the food quality by reducing the deteri- oration of essential nutrients by thermal treatment while increasing the palatability, digestibility by starch gelatini- zation, modifying the molecular structure of the protein, and inhibiting unacceptable compounds including anti- nutritional factors and enzymes (Pasqualone et al., 2020). mailto:zrazad@gmail.com mailto:darnabi@gmail.com mailto:gulmk@nitrkl.ac.in 106 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. IHCF, and so far, no research has been conducted on the use of WWF, WBF, WCF and IHCF for the development of breakfast cereal. Therefore, it is necessary to characterise the wholegrain flours and IHCF to analyse their possible utilisation in food industry and also to optimise the processing condi- tions, that is, feed moisture content, barrel temperature and screw speed and IHCF for the development of break- fast cereals. In this research, the physicochemical and functional properties, colour, texture and storage stability of the developed extrudates were studied. Materials and Methods Wholegrain flours Whole grain wheat (SW-2) and whole white corn (DT-2) were purchased from Sher-e-Kashmir University of Agricultural Sciences and Technology (SKUAST), Shalimar, Jammu and Kashmir, (J&K), and whole grain barley (PL-807) was obtained from Kargil, India. The whole grains were cleaned to eliminate the impurities. After removing the impurities, the whole grains were milled to obtain flour. The whole grain flours were then packed and stored at −21°C for further use. Indian horse chestnut seeds (Aesculus indica) were manually har- vested from local areas of Shalimar, J&K. Preparation of Indian horse chestnut flour The preparation of IHCF is done according to the method described in our previous research (Allai et al., 2022a). Conception of experimental design Response surface methodology (RSM) consists of different experimental designs based on statistical calculations and mathematical models on raw experimental data, to describe the empirical association between two or more independent variables, including design factors (xi) and design response (yi) that are required to be optimised through prediction approach (Moussaoui et al., 2021). Statistical Software Design-Expert-12 (Stat-Ease Inc. Minneapolis, MN, USA) was applied to determine the influence of process param- eters on response variables by using the second-order poly- nomial model as shown in the below equation. The adjusted and predicted R-square was in a reasonable agreement for all the models as presented in Table 1. = = = = = + + +∑ ∑ ∑∑ 4 4 4 4 2 i 0 i i ii i i ij i j i 1 i 1 i 1 i 1 y b b X b X b X X Ready-to-eat products such as breakfast cereals are most commonly consumed in the morning as the first meal of the day (Oliveira et al., 2017b). Traditionally, breakfast cereals are prepared from refined cereal flours due to their high availability, ease of processing, low cost, and their bland taste. Technically, refined flours are nutri- tionally poor in micro- and macronutrients and con- tain only the digestible carbohydrate, that is, starch in comparison to wholegrain flours (Oliveira et al., 2017a). In food industry, cereals are predominantly used as the base ingredient for the development of extruded prod- ucts, but the trend is now changing and shifting towards the healthy ingredients due to the increased incidences of chronic diseases (Naseer et al., 2021). As such, addi- tion of different whole grains such as whole wheat flour (WWF), whole corn flour (WCF) and whole barley flour (WBF) in breakfast cereals has a promising scope for enhancing the potential health benefits and market acceptability of cereal-based products. Wholegrain flours are rich sources of antioxidant activity, bioactive com- pounds, vitamins, minerals, and dietary fibres such as cellulose, hemi-cellulose, lignin, β-glucan and resistant starch (Allai et al., 2021). Several researchers have pre- pared extruded products from non-conventional seeds and evaluated their antioxidant and phytochemical prop- erties (Beigh et al., 2019; Jabeen et al., 2021). Indian horse chestnut is an underutilised nut that has not been used previously with wholegrain flours and can, therefore, be explored for the development of healthy, sugar-free breakfast cereal. Indian horse chestnut (Aesculus indica) provides an excellent opportunity for the pharmaceutical and food industries for having unique medicinal proper- ties that include anti-obesity, anti-fever, antiviral, anti- inflammatory and anti-oedemic (Ahmad and Gani, 2021). In India, horse chestnut remains unrecognised and is mainly consumed by cattles and wild animals and considered as waste (Mishra et al., 2018). The seeds are poisonous and bitter in taste, if consumed without pro- cessing (raw), because of the presence of anti-nutrients such as tannins and saponins. So, the seeds must be kept under running water in order to eliminate the bitterness and grind to fine flour. Indian horse chestnut flour (IHCF) is simply added with other ingredients, partially replacing the wheat flour, to make wide range of products such as halwa (porridge), chapatti (flat bread), pasta and noodles (Rafiq et al., 2021). Horse chestnut could be uti- lised as a nutritional supplement due to their high levels of dietary fibre, starch, minerals, vitamins, polyphenols, flavonoids and other bioactive compounds such as triter- penoids, epicatechin and kaempferol (Idris et al., 2020). Due to its nutritional quality, IHCF may be considered as a potential non-conventional alternative to conventional flours. Very limited research has been done regarding Italian Journal of Food Science, 2022; 34 (3) 107 Wholegrain-based breakfast cereals Table 1. ANOVA for the fit of data to response surface models. Dependent variables Models R2 Adjusted R2 Predicted R2 Adequate precision CV (%) P Lack of fit SME SME = 129.88 + 1.99x 1 – 4.54x 2 – 9.17x 3 + 5.69 x 4 + 2.07x 1 2 + 1.18x 4 2 0.97 0.95 0.90 23.97 1.91 <0.05 NS PD (g/ml) PD = 6.34 + 0.0996x 1 + 0.803x 2 – 0.1388x 3 – 0.407x 4 – 0.210.6x 2 x 4 – 0.0594x 3 x 4 – 0.5430 x 1 2 – 0.186x 2 2 0.99 0.99 0.97 50.10 1.59 <0.05 NS LEI LEI = 0.286 – 0.011x 1 – 0.010x 2 + 0.0133x 3 + 0.0285x 4 0.79 0.76 0.72 18.67 5.58 <0.05 NS a w a w = 0.3033 + 0.042x 2 – 0.01x 3 0.93 0.87 0.79 16.27 4.77 <0.05 NS WHC (g/g) WHC = 5.57 + 0.182x 1 + 0.662x 2 + 0.464x 3 – 0.275x 4 – 0.266x 2 * x 3 + 0.11x 1 2 + 0.252x 2 2 + 0.412x 3 2 – 0.134x 4 2 0.96 0.93 0.83 22.87 4.00 <0.05 NS SV (g/ml) SV = 4.81 + 0.093x 1 + 0.264x 2 + 0.205x 3 – 0.154x 4 – 0.121x 2 x 3 – 0.045x 2 2 + 0.471x 3 2 – 0.215x 4 2 0.98 0.97 0.93 49.20 1.99 <0.05 NS L* L* = 66.94 – 0.623x 2 – 0.717x 3 + 1.40x 3 x 4 + 0.833x 1 2 + 0.905x 4 2 0.85 0.71 0.56 9.54 1.68 <0.05 NS a* a* = 4.26 – 0.124x 1 + 0.572x 2 + 0.227x 3 + 0.052x 4 + 0.045x 2 x 3 + 0.077x 1 2 + 0.166x 2 2 + 0.234x 3 2 + 0.036x 4 2 0.99 0.98 0.95 38.40 0.44 <0.05 NS b* b* = 22.64 + 1.15x 2 + 1.08x 3 0.77 0.74 0.65 15.83 3.75 <0.05 NS a*, redness; a w , water activity; b*, yellowness; CV, coefficient of variance; L*, luminosity; LEI, longitudinal expansion index; NS, non-significant; PD, piece density; SME, surface mechanical energy; SV, swelling volume; WHC, water holding capacity. Where yi is the design response, and xi is the independent factors, that is, X1, X2, X3 and X4 represent feed compo- sition (IHCF), feed moisture content, barrel tempera- ture and screw speed, respectively, and the coefficients of regression for constant, linear, quadratic and interac- tion regression terms were indicated by b0, bi, bii and bij, respectively. In this study, the percentage of WBF and WWF was kept constant (10%) throughout the experiment. In the control, WCF accounted for the remaining 80% of the feed formulation. In the feed mixture, IHCF was used to replace WCF. The central composite rotatable design was used to investigate the effect of the four independent variables. X1, X2, X3 and X4 at each five levels are shown in Table 2. The experiment runs along with actual and coded levels for the independent variables are presented in Table 3. Extrusion cooking The breakfast cereals were prepared in a co- rotating twin-screw extruder (Basic Technology Pvt. Ltd., Kolkata, India) with a die width of 3.0 mm and a length to diameter ratio 8:1. The temperatures of the first, second and third zones were maintained at 40°C, 60°C and 80°C, respectively, throughout the experiment, while the fourth zone varied according to the design of experiment. The extrusion process was carried out using different feed Table 2. Coded levels and experimental ranges of the independent variables generated using central composite rotatable design. Numerical variable Symbol Coded variable levels −2 −1 0 1 2 Indian horse chestnut flour (%) X 1 1.75 2.5 3.25 4 4.75 Feed moisture content (%) X 2 10 12 14 16 18 Temperature (°C) X 3 70 90 110 130 150 Screw speed (rpm) X 4 290 320 350 380 410 108 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. Table 3. Effect of independent variables on techno-functional characteristics of extrudates. Runs Independent variables Physical Properties Functional Colour IHCF (%) Feed moisture (%) Barrel temperature (ºC) Screw speed (rpm) SME (Wh/Kg) PD (g/ml) LEI (%) a w WHC (g/g) SV (mL/g) L* a* b* 1 2.5(−1) 12(−1) 90(−1) 320(−1) 142.2 4.86 0.28 0.27 5.04 4.48 71 4.1 20.17 2 4(+1) 12(−1) 90(−1) 320(−1) 145.56 5.24 0.22 0.29 5.56 4.92 71.64 3.8 19.64 3 2.5(−1) 16(+1) 90(−1) 320(−1) 130.5 7.09 0.25 0.36 7 5.23 69.75 5.3 23.13 4 4(+1) 16(+1) 90(−1) 320(−1) 137.4 7.23 0.23 0.37 7.11 5.46 70.33 4.7 23 5 2.5(−1) 12(−1) 130(+1) 320(−1) 120.7 4.72 0.32 0.24 6.41 5.06 64.71 4.47 22.67 6 4(+1) 12(−1) 130(+1) 320(−1) 124.3 5.01 0.3 0.25 6.78 5.35 66.92 4.22 22 7 2.5(−1) 16(+1) 130(+1) 320(−1) 110.6 6.83 0.28 0.34 7.34 5.37 67.41 5.74 25.19 8 4(+1) 16(+1) 130(+1) 320(−1) 114.5 7 0.25 0.36 7.55 5.55 66 5.53 24.31 9 2.5(−1) 12(−1) 90(−1) 380(+1) 149.8 4.57 0.33 0.27 4.22 4.3 68.74 4.22 20.69 10 4(+1) 12(−1) 90(−1) 380(+1) 152.6 4.83 0.32 0.3 5.04 4.36 69.32 4.13 18.02 11 2.5(−1) 16(+1) 90(−1) 380(+1) 142.4 5.95 0.3 0.36 6.34 5.08 66.23 5.37 23 12 4(+1) 16(+1) 90(−1) 380(+1) 145.3 6.11 0.28 0.35 6.88 5.17 67 5.05 23.15 13 2.5(−1) 12(−1) 130(+1) 380(+1) 133.4 4.24 0.35 0.24 6 4.96 69.7 4.53 23 14 4(+1) 12(−1) 130(+1) 380(+1) 139.5 4.44 0.33 0.26 6.12 5.11 70 4.44 22.78 15 2.5(−1) 16(+1) 130(+1) 380(+1) 121.5 5.41 0.33 0.34 6.66 5.21 68.36 5.78 25 16 4(+1) 16(+1) 130(+1) 380(+1) 127.4 5.56 0.31 0.36 6.97 5.29 68 5.51 24.82 17 1.75(−2) 14(0) 110(0) 350(0) 134.2 4.03 0.3 0.31 5.46 4.79 68.92 4.73 23.03 18 4.75(+2) 14(0) 110(0) 350(0) 140.3 4.35 0.26 0.31 6.15 5.15 70.65 4.3 22.4 19 3.25 (0) 10 (−2) 110(0) 350(0) 135.6 4.11 0.29 0.24 5.07 4 66.14 3.7 21.65 20 3.25(0) 18(+2) 110(0) 350(0) 120.3 7.12 0.27 0.39 7.68 5.26 63.13 6.04 24.12 21 3.25(0) 14(0) 70(−2) 350(0) 146.55 6.54 0.28 0.33 5.89 6.19 67.42 4.66 21.88 22 3.25(0) 14(0) 150(+2) 350(0) 113.4 6.21 0.31 0.3 8.14 7.2 65.26 5.62 25.33 23 3.25(0) 14(0) 110(0) 290 (−2) 121.06 6.94 0.23 0.31 5.34 4.39 70.15 4.33 23.21 24 3.25(0) 14(0) 110(0) 410 (+2) 146.3 5.49 0.32 0.33 4.31 3.51 70 4.37 20.01 25 3.25(0) 14(0) 110(0) 350(0) 132.5 6.33 0.29 0.32 5.57 4.88 69 4.3 23.43 26 3.25(0) 14(0) 110(0) 350(0) 130.2 6.4 0.26 0.29 5.61 4.8 67.22 4.28 23 27 3.25(0) 14(0) 110(0) 350(0) 127.3 6.32 0.25 0.33 5.5 4.71 65.22 4.32 22.65 28 3.25(0) 14(0) 110(0) 350(0) 125.2 6.41 0.27 0.31 5.46 4.79 68.42 4.25 22.13 29 3.25(0) 14(0) 110(0) 350(0) 130.5 6.3 0.28 0.29 5.87 4.85 66.7 4.2 22.87 30 3.25(0) 14(0) 110(0) 350(0) 133.6 6.29 0.29 0.28 5.41 4.81 65.1 4.22 23 a*, redness; a w , water activity; b*, yellowness; IHCF, Indian horse chestnut flour; coded values are in parenthesis; L*, luminosity; LEI, longitudinal expansion index; PD, piece density; SME, surface mechanical energy; SV, swelling volume; WHC, water holding capacity. compositions and process parameters as given in Table 3. The amount of water to be added before the extrusion was calculated in order to adjust the feed moisture content. A homogeneous flour mixture was obtained by mixing properly for 20 min, and water was added to the feed. The prepared feed mixtures were allowed to equilibrate for 12 h prior to extrusion to stabilise the moisture content. The prepared samples were then collected and cooled to ambient temperature, dried and packed in low-density polyethylene (LDPE) bags for further analysis. Surface mechanical energy Surface mechanical energy (SME) is the amount of energy given to extrudates for the conversion of starch. Italian Journal of Food Science, 2022; 34 (3) 109 Wholegrain-based breakfast cereals Colour analysis The CIELAB space parameters of extrudates were ana- lysed with a Hunter Lab colorimeter (CR 300, Konica Minolta, Japan). L*, a* and b* values represent lightness or darkness, redness and blueness or yellowness, respec- tively. Each value is an average of five different indepen- dent measurements. Functional parameters Swelling volume (SV) and water holding capacity (WHC) SV and WHC of extrudates were determined by follow- ing the method by Espinosa-Ramirez et al. (2021). WHC measures the amount of retained water in the extrudates without undergoing any stress, and SV was defined as the ratio of total volume of swollen extrudates to the weight of solids. Optimal point and validation Optimization of independent variables was done through the highest desirability function in order to validate the developed models, that is, by comparing the predicted and actual values. The optimal conditions considered for numerical optimisation was minimum piece density and maximum longitudinal expansion (LE), SME, swelling volume (SV) and WHC. The developed extrudates were evaluated for all the selected parameters, and percentage prediction error was measured as follows (Scheuer et al., 2016). Actual value predicted value Pr ediction error (%) 100 Pr edicted value − = × Texture analysis The texture of extrudates was investigated using a TA-HD Plus texture analyser (Stable Micro System, Godalming, Surrey, UK) equipped with five blade Kramer shear cell (Oliveira et al., 2017b) fitted with a 50 kg load cell. The compression test was done by arranging the sample on a single-layer bed, and the test was carried out at a pre-test speed of 1.0 mm/s, test speed of 2 mm/s and post-test speed of 10.00 mm/s. The trigger type used was button type, and the samples were compressed to 50% of the original height. The software recorded the number of peaks produced from the force deformation curve, resulting in the wall fracture of the sample, which represented the crispiness (Np) (Dogan and Kokini, 2007). Hardness was measured as the maximum force (N) required for rupturing the sample. Also, average crushing force (Cf) and crispiness work (Cw) were mea- sured using the below equations (Igual et al., 2020): n Np d = SME was calculated using the following formula (Oliveira et al., 2017a), the motor torque was recorded for each treatment displayed in the monitor panel. SME (Wh/Kg) Screws peed (rpm) motor power (kW) torque (%) kg Maximumscrewspeed (rpm) mass flow rate 100 h = × ×  × ×    Where motor power = 4000 W, and maximum screw speed = 682 rpm. Techno-functional parameters Physical properties Piece density (PD) Piece density of breakfast cereals was measured accord- ing to the protocol followed by Seker (2005). A 250 ml graduated cylinder was filled with 4 g of the sample. The cylinder was filled by adding mustard seeds. The extru- dates were then removed, and the volume of leftover mustard seeds was measured and noted. Piece density was calculated as follows: Mass of extrudates 100 Volume of extrudate PD s (g/ml) = × Longitudinal expansion index (LEI) The LEI of the extruded products was determined by the method explained in by Alvarez- Alvarez-Martinez et al. (1988). It is the ratio of the velocity of expanded extru- dates to the velocity in the die orifice, which is indicated as follows: d d e e 1 M1 LER (%) SER 1 M ρ ρ    −=    −   Where, ρd is the density of melt behind the die (ρd= 1400 kg/m3), and ρe is the extrudate density. Me is the mois- ture content of extrudates, and the moisture content of the melt (Md) was measured by drying 2–3 g of the samples in an oven at 105°C until constant weight was achieved. Water activity (a w ) Water activity is a dimensionless number representing the ratio of vapour pressure of the sample to that of pure water at a given temperature. aw of flour and extrudates was measured using a water activity meter (Novasina AG CH-8853, Lachen). 1.0 g of flour was kept in the sample cup of water activity meter. The lid was closed and the sample was allowed to equilibrate, read as aw. 110 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. et  al., 2020). BD was calculated by using mass/volume relationship as given below: g weight of sample (g) Bulk density mlL volume of sample after tapping (ml)   =    Swelling capacity (g/ml) Swelling capacity was determined by following the method of Okaka and Potter (1977) with some modifications. A 100 ml graduated cylinder was taken and filled with sam- ple to the 10 ml mark. Distilled water was added to adjust the total volume to 50 ml. The top of the measuring cyl- inder was covered tightly, and the solution was mixed by inverting the cylinder. After 120 s, the suspension was again inverted and allowed to rest for 30 min. The final vol- ume occupied by the sample was noted after 30 min. Shelf-life studies The optimised product was packed in aluminum-lam- inated (AL) pouches and LDPE, and stored at 25°C for 6 months. The stored samples were analysed for water activity, moisture, hardness, crispiness, free fatty acids (FFAs), peroxide value (PV), total plate count and overall acceptability. Moisture (%) Moisture content was determined by the Oven Method, as per the protocol described by AOAC (2005). Free fatty acid (%) FFA was estimated by the standard procedure of AOAC (1973), with some slight modifications. 5 g of sample was placed in a 250 ml flask, and to this 50 ml of benzene was added. The mixture was kept undisturbed for 30 min for extraction of FFA. Then, 5 ml of the extract was taken to which 10 ml of alcohol, 5 ml of benzene and two drops of phenolphthalein indicator were added. It was then titrated against 0.02 N KOH till the light pink colour dis- appeared. FFA was expressed as the percentage of oleic acid, and was calculated using the following formula, ( )FFA % of oleic acid 282 0.02 N KOH ml of alkali used dilution factor 100 1000 weight of sample taken = × × × × × Peroxide value (%) 30 ml of acetic acid chloroform solution and 0.5 ml of potassium iodide was added to 5 g of sample, and the mixture was allowed to stand for 60 s with occasional shaking. 30 ml of distilled water was added to the mix- ture. This was then titrated against 0.1 N sodium thio- sulphate solution with constant swirling till the yellow colour disappeared. 0.5 ml of starch solution as indicator Where, n = number of peaks D = distance travelled by the probe S Cw n = Where, S = Area under deformation curve (upto 50% deformation) S Cf d = Total phenolic content The procedure by Allai et al. (2022b) was used to cal- culate the total phenolic content in the extrudates. The extraction process was carried out using methanol as solvent.1 g of extrudate was homogenised in 30 ml of methanol. The mixture was placed into an ultrasonic water bath for 15 min. Then the homogenate was left undisturbed for 12 h. The resulting mixture was centri- fuged for 10 min at 3500 rpm. 0.5 ml of the extract was taken after centrifugation and blended with 2.5 ml of Folin–Ciocalteu reagent and incubated at room tempera- ture for 5 min. The mixture was then mixed with 2 ml of 7.5% Na2CO3 and kept aside for 1 h at 25°C. After 1-s reaction time, the absorbance at 750 nm was measured using a spectrophotometer. Gallic acid was used to make a calibration curve, and the total phenolic content was expressed as mg GAE/100 g of dry sample. Antioxidant activity The antioxidant activity of the samples was estimated by the DPPH radical scavenging assay according to the method by Zhang et al. (2018) with a slight modification. Methanol was used to prepare 0.1 mM of DPPH solution. Extraction of different concentration was made, followed by the addition of 5 ml of DPPH solution and was mixed properly. The mixture was left undisturbed for about 45 min in dark at 25°C, and absorbance of the sample at 517 nm was measured. DPPH radical scavenging activity was calculated using the following equation: control sample sample A A 100 % Inhibition A − × = Where, Acontrol and Asample are the absorbance values of the control and the sample, respectively. Techno-functional properties of flours Bulk density (BD) BD was measured by filling pre-weighed sample in a 100 ml graduated cylinder. The base of cylinder was gen- tly tapped till a constant volume was achieved (Adeloye Italian Journal of Food Science, 2022; 34 (3) 111 Wholegrain-based breakfast cereals Where, A and B represent the average score given to the product and the maximum score obtained for the prod- uct, respectively. A product that obtains an AI score of 70% is considered a good product (Gusmao et al., 2019). Statistical Analysis SPSS (Version 20) statistical software was used to anal- yse the data acquired throughout the studies. The results were expressed as mean ± standard deviation. Duncan’s multiple range test and analysis of variance (ANOVA) were used to find a significant difference at (P < 0.05), among the means of samples. Results and Discussion A fit of models ANOVA was used to select the appropriate models for different dependent responses. Statistics of fit sum- mary suggested quadratic models for SME, PD, WHC, SV, L*, and a*; and linear models for LEI, aw and b*. The regression models were significant (P < 0.05) with a high coefficient of determination (R2 = 0.99–0.77) for all the responses. These dependent variables were significantly influenced by feed composition, feed moisture content, barrel temperature and screw speed. The predicted and adjusted R2 were observed to be in reasonable agreement, and the regression models showed less than 1.91–7.84% of coefficient of variation (CV), suggesting high precision and reproducibility of obtained results. Surface mechanical energy SME is the key parameter that determines the molecu- lar degradation or breakdown of ingredients received during the extrusion process (Lee et al., 2022). Higher SME indicates a rapid conversion of starch, resulting in an increased puffing of extruded products (Jabeen et al., 2021). The values of SME varied between 110 and 152 Wh/kg for wholegrain breakfast cereals enriched with IHCF (Table 3). The fitted regression analysis exhibited quadratic coefficients of feed composition, feed moisture content, barrel temperature and screw speed in extru- dates (Table 1). The regression analysis showed that barrel temperature had the most dominant effect on SME (Table 1) among all parameters. Table 1 shows ANOVA results for the fit of data to response surface models. It was noticed that the feed moisture content (X2) and barrel temperature (X3) were inversely proportional to SME. With an increment in X2 and X3, a reduction in SME was recorded, whereas was added and titrated until the blue colour disappeared. Blank was also determined. PV was expressed as meq/kg and was calculated using the following formula (Tatledgis et al., 1960): Titre N 100 PV weight of the sample (g) × × = Where, titre = sample reading–blank reading N = normality of sodium thiosulphate solution Microbiological analysis Total plate count The total plate count of samples was carried out after every 1 month of storage interval, up to 6 months. Samples were evaluated for total fungi and bacteria by using the standard serial dilution method. 10 g of grounded sample was dissolved in 90 ml of water and stirred for 5 min. After stirring, the aliquots were serially diluted; 1 ml in 9 ml of sterile saline was prepared in test tubes, and 0.1 ml of dilution was transferred aseptically on sterile plates using nutrient agar as media. The plates were rotated gently for uniform spread of the inoculum before the media solidified. The plates were then incu- bated at 37°C for 3–7 days. The number of colonies devel- oped on each plate of different dilutions was counted using the digital colony counter and is calculated as: Number of colonies dilution factor CFU/g volume of sample used (ml) × = Sensory analysis Twenty-five consumers who take breakfast cereal daily (15 females and 10 males) were selected randomly. Samples were served to the consumers in a three-digit coded manner and were carried out on a nine-point hedonic scale (1 meant ‘dislike very much,’ and 9 denoted ‘like very much’). Consumers were guided to rinse their mouths after the consumption of every different treat- ment to determine different attributes (texture, taste and overall quality) of the extrudates. Overall acceptability of breakfast cereals was evaluated as the average score of sensory attributes determined. The purchase intent of each sample was questioned using a five-point scale (5 = certainly would buy, 3 = might or might not buy, 1 = definitely would not buy) to complement the acceptance results. The acceptability index (AI) was calculated using the fol- lowing formula A AI (%) 100 B = × 112 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. X1, X2, X3, X4, X2X4 X3X4, X1 2, and X2 2 were significant at P < 0.01, whereas the model terms X1X2, and X2X3 were non-significant. As evidenced in Table 3, the feed composition and feed moisture content showed a posi- tive linear effect, whereas the barrel temperature and the screw speed exhibited negative linear effect on the PD of extrudates. RSM (Figure 1) illustrates the effect of inde- pendent parameters on PD. . The increase in PD of extrudates with the increased feed composition might be due to the presence of fibre in IHCF that enhances the extrudate density. The fibre tends to breakdown the air cells, which reduces exten- sibility and expansion, resulting in higher density of extrudates with less porous structure (Dos Santos et al., 2019). This suggests its suitability and applicability for the development of food products. High feed moisture content also decreases frictional force between screw and mixture leading to reduced expansion and enhanced density (Bisharat et al., 2013). The extrusion process does not evaporate whole moisture content at the exit die point. So, the retention of some of the water content makes the product denser with decreased puffing (Asare et al., 2004). An increase in barrel temperature and screw speed causes a reduction in the density of the extruded products. This might be because of the higher barrel temperature that enhances the degree of starch gelatini- zation and also the generated superheated vapours that produce a more expanded structure with lighter weight products (Samray et al., 2019). Additionally, an increased shear rate disintegrates the structure of macromolecules of proteins and starches that subsequently weaken the structure, leading to a reduced density with increased screw speed (Bisharat et al., 2013). Figure 1 showed the higher feed composition (X1) and screw speed (X4) showed increased SME values. An increase in feed com- position along with an increasing SME was mainly due to the increased starch content of IHCF (Meuser et al., 1990). Furthermore, the presence of fibre in wholegrain flours increases SME as high fibre content has more water binding affinity and could dilute the concentra- tion of starch in the mixture (Singha et al., 2018). Feng and Lee (2014) reported that an increase in feed mois- ture content causes a reduction in the viscosity of feed ingredients. Viscosity is temperature dependent, and reduction of barrel temperature can reduce the gelatini- zation of starch, resulting in enhanced viscosity (Karkle et al., 2012). Thus, increasing barrel temperature causes a reduction in viscosity leading to a decrease in torque and SME values (Kesre and Masatcioglu, 2022). Kaur et al. (2015) described that with an increment in screw speed, SME progressively enhanced due to the high shear force and less residence time that induces viscosity, starch con- version and high SME. The CV assessed the relative dispersion of the exper- imental points, that is, 1.91% for developed breakfast cereals (Table 1). R2, a coefficient of determination, indi- cated that the correlation among the selected process- ing conditions for extrudates was good, which showed a value of 0.90. Piece density (PD) The PD for developed extrudates was observed to be in the range of 4.03–7.23 g/ml (Table 3). The fitted model for PD is shown in Table 1. In Table 1, the model terms 8 7 6 5 4 380 P D ( g/ m L) 8 7 6 5 4 P D ( g/ m L) 370 360 350 340 D: Screw speed (RPM)C: Moisture (%) 330 320 380 370 360 350 340 D: Screw speed (RPM) 330 320 12 13 14 15 16 C: Temperature (˚C)90 100 110 120 130 Figure 1. Response surface plots of piece density as a function of whole wheat flour, corn flour, barley flour and Indian horse chestnut flour. Italian Journal of Food Science, 2022; 34 (3) 113 Wholegrain-based breakfast cereals and starch gelatinization, which may enhance the expan- sion of extrudates (Yadav et al., 2021). As a result of the increase in speed, less residence time for material was observed, leading to less degradation of the material and higher expansion of extruded products (Kaur et al., 2015). Water activity (a w ) The aw of the extrudates prepared from blend of wholegrain flours and IHCF varied from 0.24 to 0.39 (Table 3). The higher values of aw accelerated the rate of reaction in the food products (Shah et al., 2017) that determined the shelf life of foods. Fit statistics summary suggested a quadratic model for aw among all parame- ters. The feed moisture content had a significantly dom- inant effect (P < 0.01) on water activity. The impact of the feed moisture content on aw is presented by the 3-D surface plots in Figures 2A,B. Figure 2A showed a straight line with respect to temperature (110°C) along the axis of feed moisture content (14%). A similar obser- vation is shown in Figure 2B, where the variation of aw with respect to the feed moisture content (14%) also presents a straight line along the axis of screw speed (350 rpm). Generally, water activity above 0.7 promotes microbial load (Zhou et al., 2021). Thus, from our study, all the extruded samples prepared under different treat- ment conditions fell in the safe category of shelf-stable products. Colour analysis The colour values of extruded products are displayed in Table 3. The regression analysis for L*, a* and b* values of extrudates are presented in Table 1. The regression negative interaction effect of X2X4 and X3X4. Similar results for the interaction terms have been reported by Jabeen et al. (2021) in corn-water chestnut extrudates and by Pansawat et al. (2008) in fish rice–based snacks. Longitudinal expansion index LEI values of extrudates varied from 0.22–0.35 (Table 3). The regression analysis revealed that the feed composi- tion and feed moisture content had a significantly (P < 0.05) negative linear effect (Table 1). This might be due to the enhanced water content in the melt that would soften the molecular structure of amylopectin, reduc- ing its elasticity and thus decreasing the longitudinal expansion (Alvarez-Martinez et al., 1988). It can be well described by the fact that low moisture content promotes drag forces that enhance the die pressure, leading to more expansion of the developed products (Kaur et al., 2022). Higher feed moisture content induces a lubrica- tion effect, decreasing the internal barrel temperature as well as the shear rate of the extruder. As a result, a reduction in cooking of the ingredient and expansion can take place (da Silva et al., 2014). The addition of IHCF caused a negative effect on the product expansion rate as the presence of fibre in the feed composition binds with some of the water content in the matrix and acts as an interference factor, thus decreasing its availability for longitudinal expansion (Witczak et al., 2021). The LEI values increased significantly as the barrel temperature enhanced (Table 3). A similar trend was also observed for the screw speed response. The highest LEI value (3.5) was observed at the highest barrel temperature (130°C), screw speed (380 rpm) and at the lowest feed moisture content (12%) and feed composition (2.5%) (Table 3). The increased temperature inside the barrel causes super- heating, which implies a higher degree of protein cooking 0.4 (A) (B) 0.38 0.36 0.34 0.32 0.3 0.28 0.26 0.24 130 120 110 100 90 12 13 14 15 16 C: Temperature (˚C) B: Moisture (%) a w 0.4 0.38 0.36 0.34 0.32 0.3 0.28 0.26 0.24 a w 380 370 360 350 340 D: Screw speed (RPM) 330 320 12 13 14 15 16 B: Moisture (%) Figure 2. Response surface plots of water activity as a function of whole wheat flour, corn flour, barley flour and Indian horse chestnut flour. 114 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. structure (Wang et al., 2019a). The increased moisture content and temperature might increase the gelatiniza- tion of starch, where the granules of starch are disinte- grated and more water remains bound to it, resulting in enhanced WHC (Wang et al., 2019b). A high shear rate causes disruption of molecular structure, which leads to increase in solubility and decrease in WHC of extruded snacks (Ek et al., 2021). The regression model elucidates positive linear as well as quadratic terms for X1, X2, and X3, while X4 shows a negative effect on SV. Figure 3 shows that the variation of SV with respect to feed moisture content is curvilinear along the axis of barrel temperature. There was a posi- tive correlation between SV and WHC of extrudates (r = 0.98). The higher SV in extrudates has been ascribed to the changes in the fibre and starch fractions. This could lead to an increased change in the fibre integrity, result- ing in a higher SV for the prepared extrudates (Espinosa- Ramirez et al., 2021). The negative interactive effect (P < 0.05) of moisture content and barrel temperature (X2X3) suggested that the moisture content (X2) had a dominant effect over barrel temperature (X3). Optimization and model validation The highest desirability (0.713) was obtained on the basis of optimal solutions, suggesting that an IHCF substi- tution of 2.5%, a feed moisture content of 12%, a barrel temperature of 130°C, and a screw speed of 380 rpm were the optimum criteria to develop good quality sugar-free breakfast cereals with higher SME, LEI, WHC, SV, and low water activity and PD values (Figure 4). Table 4 depicts the optimum conditions applied for the optimi- sation of independent variables. The values for predicted responses were found to be similar to the actual values with less than 4.5% variation. equation for luminosity (L*) (Table 1) showed signifi- cantly negative(P < 0.01) linear and quadratic effects of feed moisture content (X2) and barrel temperature (X3). It was observed that the barrel temperature and the feed moisture content had the most predominant effect that influenced all the three colour coordinates. The L*, a* and b*values of extrudates developed by different pro- cessing conditions were in the range of 63.13–71.64, 3.7–6.04 and 18.02–25.33, respectively. Generally, extru- sion cooking, that is, increasing feed moisture content and barrel temperature caused reduction in the luminos- ity (L*) and increments in a* and b* values (Table 3). The presence of higher water content leads to the develop- ment of extrudates with dense air cells, packed together, which enhances the absorption of light and decreases the luminosity (Nakhon et al., 2018). Another parame- ter that affects the colour values is the increased barrel temperature that may cause loss of pigments, that is, non-enzymatic browning reactions such as carameliza- tion and Maillard reaction, which can occur during the extrusion process and change the colour of the ingredi- ents (Zhang et al., 2020), leading to a decline in the L* value and a subsequent increase in the a* and b* values (Jabeen et al., 2022). During the extrusion, the degrada- tion of pigment could also be used to measure the pro- cess intensity with regard to chemical and nutritional changes (Devrajan et al., 2018). It is also an essential quality parameter as it indicates the degree of cooking as well as the level of chemical reactions that occur during extrusion process. Water holding capacity and swelling volume The extrusion enhances the water holding and bind- ing capacity of the extrudates (Espinosa-Ramirez et al., 2021). WHC of extrudates varied between 4.22 and 8.14 g/g (Table 3). The impact of independent variables on WHC was measured using ANOVA. The fitted model showed a highly significant linear, quadratic as well as interaction effect on WHC at P ≤ 0.01, as shown in Table 1. The positive effect of the feed composition con- tent, moisture content and the barrel temperature indi- cates that WHC increases with an increase in IHCF, moisture content and temperature, while the negative coefficient for the screw speed showed that an increase in shear rate causes a decrease in WHC. The presence of fibre and starch in feed composition also led to a higher WHC. Fibre-rich flours have a higher WHC and can be utilised as functional ingredients to modify the texture and viscosity and also avoid syneresis in food products (Repo-Carrasco-Valencia et al., 2009). The proteins present in wholegrain flours and IHCF may be improved during extrusion treatment, by dissociation and unfolding of molecules that enhance the exposure of hydrophilic sites due to the variation in macromolecular 8 7 6 5 4 3 16 15 14 13 12 S V ( m L/ g dr y sa m pl e) B: Moisture (%) A: IHCF (%)2.5 2.8 3.1 3.4 3.7 4 Figure 3. Response surface plots of swelling volume as a function of whole wheat flour, corn flour, barley flour and Indian horse chestnut flour. Italian Journal of Food Science, 2022; 34 (3) 115 Wholegrain-based breakfast cereals Desirability 380 370 360 350 340 330 320 2.5 2.8 3.1 A: IHCF (%) D : S cr ew s pe ed ( R P M ) 3.4 3.7 4 Desirability 0.713 0.65 0.6 0.7 0.55 Figure 4. Desirability plot. Table 4. Numerical optimisation. Variable Goal Range Importance Values Variation level (%) Lower limit Upper limit Actual Predicted IHCF Is in range 2.5 4 3 - - - Feed moisture Is in range 12 16 3 - - - Barrel temperature Is in range 90 130 3 - - - Screw speed Is in range 320 380 3 - - - Responses SME Maximise 110.6 152.6 3 133.0 134.14 0.84 PD Minimise 4.03 7.23 3 4.17 4.24 1.65 LEI Maximise 0.22 0.35 3 0.33 0.34 2.9 a w Minimise 0.24 0.39 3 0.22 0.23 4.3 WHC Maximise 4.22 8.14 3 5.68 5.77 1.55 SV Is in range 3.51 7.2 3 4.8 5.0 4 L* Is in range 63.13 71.64 3 67.45 69.06 2.33 a* Is in range 3.7 6.04 3 4.33 4.5 3.7 b* Is in range 18.02 25.33 3 21.92 22.03 0.5 a*, redness; a w , water activity; b*, yellowness; L*, luminosity; LEI, longitudinal expansion index; PD, piece density; SME, surface mechanical energy; SV, swelling volume; WHC, water holding capacity. Techno-functional properties of wholegrain flours, IHCF and optimised extrudates The functional properties of wholegrain flours and IHCF are depicted in Table 5. The functional attributes define how a food material interacts with other food ingredi- ents. It also determines its suitability for end use. Thus, flour with good functional characteristics can be easily substituted for other foods and will yield good quality with acceptable end products. The BD of flour was similar in all flours, with a BD of 0.774 ± 0.52 in WWF, 0.722 ± 0.44 in WCF, 0.44 ± 0.26 in WBF and 0.6 ± 0.58 in IHCF. These results are similar to those reported by Tangariya and Srivastava (2022) for WWF, Adedeji and Tadawus (2019) for corn flour, Hamdani et al. (2014) for WBF and Shafi et al. (2016) for horse chestnut flour. The higher BD helps to enhance the weight of flour-supplemented foods without affecting the volume and the flours also favour their suitability in processing of different food products, while low BD helps in the preparation of complementary foods (Awuchi et al., 2019). WWF had slightly higher swelling power (0.85 ± 0.45 g/ml) than WCF (0.79 ± 0.43 g/ml), WBF (0.7 ± 0.33 g/ml) and IHCF (0.66 ± 0.27 g/ ml) (Adegunwa et al., 2014; Chaudhary et al., 2018). WWF was characterised by a higher hydration capacity and hydration index (6.72 g/g and 3.36 g/g, respectively) with relative to WCF (5.19 g/g and 2.5 g/g), WBF (4.6 g/g and 2.3 g/g) and IHCF (2.33 g/g and 1.16 g/g). These findings are consistent with the literature (Adegunwa et al., 2014; Boucheham et al., 2019; Rafiq et al., 2021). The differences in functional properties might be due to the variation in the particle size of the flour, their variety and the milling process (Das et al., 2019). Table 5 depicts the functional properties of optimised product (extrudates). A significantly higher (P < 0.05) BD (4.95), hydration capacity (6.03), hydration index (3.015) and swelling power (0.91) were observed in the devel- oped extrudates than native wholegrain flours and IHCF. The increased capacity of extruded flours to hold water 116 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. Table 5. Functional, colour and textural attributes of individual flour, their blends and optimised extrudates. Parameters WWF WBF WCF IHCF Blend Optimised extrudate Techno-functional characteristics Bulk density (g/ml) 0.77 ± 0.52b 0.44 ± 0.58f 0.722 ± 0.26c 0.6 ± 0.58e 0.69 ± 0.37d 4.95 ± 0.32a Swelling power (g/ml) 0.85 ± 0.45c 0.7 ± 0.33e 0.79 ± 0.43d 0.66 ± 0.27f 0.85 ± 0.31b 0.91 ± 0.52a Hydration capacity (g/g) 6.72 ± 0.25a 4.6 ± 0.55e 5.19 ± 0.18d 2.33 ± 0.10f 5.24 ± 0.05c 6.03 ± 0.44b Hydration index 3.36 ± 0.03a 2.3 ± 0.05e 2.5 ± 0.06d 1.16 ± 0.11f 2.62 ± 0.06c 3.01± 0.13b Colour attributes L* 79.14 ± 0.22c 72.35 ± 0.47e 83.83 ± 0.1b 90.27 ± 0.06a 77.34 ± 0.35d 69.7 ± 0.32f a* 3.09 ± 0.04d 4.29 ± 0.07c 1.09 ± 0.1e 3.09 ± 0.06d 4.33 ± 0.17b 4.53 ± 0.23a b* 15.65 ± 0.20c 16.04 ± 0.17b 12.70 ± 0.3f 12.97 ± 0.06e 13.21 ± 0.65d 23 ± 0.17a Textural properties Hardness (N) - - - - - 199.29 ± 0.34 Crispiness - - - - - 56 ± 0.27 Crispiness work - - - - - 1.86 ± 0.47 Average crushing force - - - - - 5.1 ± 0.31 Total phenolic and antioxidant content Total phenolic content (mg GAE/g) 29.38 ± 0.33b 20.02 ± 0.46c 39.78 ± 0.21a 10.66 ± 0.17e 17.24 ± 0.02d 5.87 ± 0.14f Antioxidant activity (%) 25.72 ± 2.11f 68.21 ± 1.77b 70.31 ± 2.52a 63.55 ± 0.32c 42.77 ± 2.53d 18.33 ± 0.42e WWF, whole wheat flour; WBF, whole barley flour; WCF, whole corn flour; IHCF, Indian horse chestnut flour; WWF:WBF:IHCF:WCF 10:10:2.5:77.5, blended flour. Values are mean ± SD. Values with different superscripts within same column differ significantly (P < 0.05). content upon rehydration as compared to raw flours might be due to the disintegration of the starch granules or due to the molecular disruption as a result of the shear stress and the thermal extrusion process (Martínez et al., 2014). The phenolic compounds act as antioxidants and perform an essential role in stabilising the free radicals. The total phenolic content and the antioxidant activity (DPPH rad- ical scavenging) of raw flours, their blends and optimised extrudates are presented in Table 5. WCF had the highest total phenolic content and antioxidant activity among the flours, with a total phenolic and antioxidant contents of 39.78 mg GAE/g and 70.31, respectively (Lopez-Martinez et al., 2009; Oboh et al., 2010). The total phenolic content of WWF, WBF, IHCF and their blends was 29.38, 20.02, 10.66 and 17.24 mg GAE/g, respectively (Abozed et al., 2014; Baba et al., 2016; Shafi et al., 2016; Zengin et al., 2017). The antioxidant activities of WWF, WBF, IHCF and their blend were 25.72 (%), 68.21 (%), 63.55 (%) and 42.77 (%), respectively (Abozed et al., 2014; Horvat et al., 2020). After extrusion, the total phenolic and antioxidant content of optimised extrudates were reduced as com- pared to raw flours (Table 5). The reduction in the total phenolic content and antioxidant activity after extru- sion might be attributed to the changes in the molecular structure of bioactive compounds, leading to the reduc- tion in extraction efficiency and chemical reactivity due to the development of polymerised products (Pandey et al., 2021). The colour characteristics of WWF, corn flour, WBF, IHCF and optimised extrudates are presented in Table 5. Generally, extrusion reduces the luminosity (L*) of flour with an increase in a* and b* attributes. The results of this work are in accordance with the previous literature reports and can be mostly ascribed to the degradation of pigments and Maillard reaction during extrusion pro- cesses (Brahma et al., 2016; Jafari et al., 2017). For exam- ple, carotenoids present in whole grains are heat unstable pigments that are lost into colourless compounds during extrusion (Kadian et al., 2013). Thus, after extrusion, the colour developed may influence the extrudate acceptabil- ity of these flours. Textural analysis Textural parameters of extruded products depend largely on the ingredient composition and their blend. It is an essential physical property of extrudates. The tex- ture parameters for optimised extrudates are shown in Table 5. The hardness, crispiness, crispiness work (Cw) and average crushing force (Cf) values of optimised IHCF blended with wholegrain flours produced at min- imum feed moisture content (12%) and highest barrel Italian Journal of Food Science, 2022; 34 (3) 117 Wholegrain-based breakfast cereals Food Safety and Standards Authority of India (FSSAI) for dehydrated snacks. The increase in FFA of extrudates packed in AL pouches was non-significant for 120 days. The samples packed in LDPE had a significant effect that might be due to the property of AL pouches that acts a barrier for the transfer of light, which is mainly responsible for the rancidity of product. Furthermore, the lipid hydrolysis leads to the disintegration of long- chain FFA into single fatty acids (Syed et al., 2019) and also higher permeability to oxygen and water vapour resulting in higher values of FFA in LDPE as compared to AL pouches. Also, mold growth was absent during the entire period of storage for all the samples either packed in LDPE or AL pouches. The reason for low total plate count could be attributed to the low moisture level. The total plate count was too low to count (less than 25 colonies/plate) up to 180 days of storage period. Jabeen et  al. (2021) also reported similar results in total plate count of corn-based extrudates enriched with water chestnut during storage period. Peroxide value indicates the amount of rancidity because of the oxidation process during storage. It can be observed from Figure 5D that the PV of the break- fast cereals increased significantly (P < 0.05) from 0.28 to 0.59 meq/100 g in extrudates packed in LDPE and 0.47  meq/100 g for those packed in AL pouches after 180 days of storage. The increase in PV is due to the poor oxygen and moisture barrier property of LDPE in com- parison to that of AL pouches (Raleng et al., 2019). The PV in this study was found to be under the permissible range (<10 meq/kg) given by FAO. The hardness (Figure 5E) of the breakfast cereals increased from 199.29 to 217.56 N for the extruded prod- uct packed in LDPE and 199.29 to 208.56N for the extru- dates packed in AL pouches. The packaging materials significantly affected (P < 0.05) the hardness of the extru- dates during the storage period of 180 days. The hardness values increased due to the increment in water content of the extruded products, thereby modifying the balance in bonds in starch granules. However, there was a non-sig- nificant change during the initial months of storage. A similar trend was reported by Badding-Smithey et  al. (1995) for beef-based extrudates enriched with carrot pomace powder. The crispiness of the extrudates packed in LDPE and AL pouches reduced from 56 to 51.32 and 56 to 55.32, respectively (Figure 5F). The crispiness of dehydrated foods like snacks, breakfast cereals, chips and crackers is desirable, but the excess absorption of mois- ture in samples causes sogginess and finally the rejection of extrudates (Dar et al., 2014). During storage, absorp- tion of moisture could be due to the packaging material and storage conditions (Badding-Smithey et al., 1995). Moisture gain reduces the storage shelf life and stability of the product. temperature (130°C) and screw speed (380 rpm) were found to be 199.29 N, 56, 1.86 and 5.1, respectively. Crispiness work (Cw) can be inferred as the work that is needed to break one pore or group of pores, and it is also the sensory attribute of fracturability. Average crushing force (Cf) is defined as the force required to compress a solid substance and depends mostly on the sensory per- ception such as hardness during chewing (Igual et al., 2020). Lower moisture content (12%) had a positive effect on the hardness and crispiness of the extrudates as low water content increases the SME and viscosity, producing soft extrudates with higher crispiness. Kesre et al. (2022) reported that higher screw speed and barrel tempera- ture exhibited a inverse relationship with hardness and direct relationship with expansion and crispiness. The expanded extrudates are more puffed with thinner cell walls that results in the easy crushing of extrudates under compression (Yao et al., 2006). Furthermore, the avail- ability of low residual moisture content during extrusion cooking and the increment in the degree of gelatinization and superheating of water might favour more expan- sion with softer texture and also improves the crispiness of extruded products (Guha and Ali, 2006; Wang et al., 2005). The results of our work are in agreement with the published literature, which explains that the crispiness of extrudates has a positive and strong correlation with expansion. Storage studies The shelf-life stability of breakfast cereals indicates that the storage days and the packaging materials had a con- siderable influence on the FFA, PV, moisture content, water activity, hardness, crispiness and overall accept- ability. The moisture content of the samples packed in LDPE and AL pouches was initially 3.04% that enhanced to 5.12 and 3.7%, respectively, during 180 days of storage (Figure 5A). The temperature, relative humidity (storage environment) and hygroscopic nature of extruded prod- ucts, as well as the nature of packaging material, increase the moisture content during storage (Sharma et al., 2004). The extrudates packed in LDPE showed increased mois- ture content compared to AL pouches due to its lower barrier characteristic. Jan et al. (2017) also reported the quick gain of moisture content in gluten-free extrudates packed in AL and LDPE packages. Water activity of extrudates was observed to be increased from 0.3 to 0.55 and 0.42 when packed in LDPE and AL pouches, respec- tively (Figure 5B) (Hussain et al., 2017). The FFA change was noticed in the extrudates with stor- age period in both the packaging materials. The change in LDPE was higher (0.04–0.32%) as compared to AL pouches (0.04–0.21%) (Figure 5C). These values were well within the acceptable range of 0.5% as reported by 118 Italian Journal of Food Science, 2022; 34 (3) Allai FM et al. 3 3.5 4 4.5 5 5.5 (A) 0 2 4 6 M oi st ur e (% w b) Storage (Months) LDPE AL 0.3 0.35 0.4 0.45 0.5 0.55 (B) (C) (D) 0 2 4 6 8 W at er a ct iv ity Storage (Months) LDPE AL 0.04 0.09 0.14 0.19 0.24 0.29 0.34 0 2 4 6 Fr ee F at ty A ci d (% ) Storage (Months) LDPE AL 0.25 0.35 0.45 0.55 0 2 4 6 P er ox id e va lu e (m eq /1 00 g) Storage (Months) LDPE AL 199 204 209 214 219 (E) (F) (G) 0 2 4 6 H ar dn es s (N ) Storage (Months) LDPE 49 50 51 52 53 54 55 56 57 0 1 2 3 4 5 6 C ri sp in es s Storage (Months) LDPE AL 7 72 3.5 8.2 78.58 4 0 20 40 60 80 Overall acceptability Acceptability Index (%) Intention to purchase Averages of acceptance of extrudates packed in LDPE and AL pouches LDPE AL Figure 5. Effect of storage period and packaging material on (A) Moisture content, (B) Water activity, (C) Free fatty acids, (D) Peroxide value, (E) Hardness, (F) Crispiness and (G) Over all acceptability of optimised extrudates packed in LDPE and AL pouches. Italian Journal of Food Science, 2022; 34 (3) 119 Wholegrain-based breakfast cereals Technology, SKUAST–Kashmir and Post-harvest Engineering and Technology, AMU, India for providing the necessary facilities to conduct the study. References Abozed, S.S., El-Kalyoubi, M., Abdelrashid, A. and Salama, M.F., 2014. Total phenolic contents and antioxidant activities of var- ious solvent extracts from whole wheat and bran.  Annals of Agricultural Sciences.  59(1): 63–67. https://doi.org/10.1016/j. aoas.2014.06.009 Adedeji, O. and Tadawus, N., 2019. 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The optimised samples packed in LDPE and AL pouches showed good scores in colour, texture, taste and overall acceptability. The overall acceptability for the samples packed in LDPE and AL pouches ranged from 7.0 to 8.2, representing ‘like slightly’ to ‘like very much’ in terms of the hedonic scale. The test for an index of pur- chase indicates the probable buying of a product. Samples packed in LDPE and AL pouches showed non-significant variations, and the panelist suggested that the products are recommended to buy (4 scores). The acceptability index (AI) was evaluated based on the mean scores given by the judges, where the samples packed in AL pouches reported the highest AI (78.58%), while samples packed in LDPE reported AI above 70% (Figure 5G). A prod- uct having at least 70% of approval is considered to be acceptable (Dutcosky, 2011). Conclusion Extrusion cooking is a versatile technology used to pro- duce ingredients with improved functional and textural properties from whole grains and non-conventional flour. This study exhibited that RSM was used to optimise the process conditions and feed composition for the devel- opment of fibre-rich wholegrain-based extrudates using IHCF. Furthermore, the incorporation of IHCF into wholegrain breakfast cereals could broaden consumer acceptability for a better phytochemical profile and hydra- tion rate. Results obtained from this study revealed that independent variables such as feed composition, mois- ture content, barrel temperature and screw speed had a considerable effect on all the dependent responses. The optimal conditions for the preparation of IHCF incorpo- rated wholegrain-based breakfast cereals were 2.5% IHCF, 12% feed moisture content, 130°C temperature and screw speed of 380 rpm. The hardness and crispiness of the opti- mised extrudates were found to be 199.29 (N) and 56, respectively, which reported that extrudates enriched with IHCF at 2.5% level improves textural attributes. The anti- oxidant and total phenolic contents of optimised breakfast cereal were 30.36 (%) and 5.03 (mg GAE/g), respectively, which indicated that breakfast cereal enriched with WWF (10%), WBF (10%), WCF (77.5%) and IHCF (2.5%) level improve the phytochemical profile for the consumer’s daily diet. The shelf-life studies depicted that the quality and overall acceptability of extruded products could be preserved safely up to 6 months in aluminum pouches under room temperature (25°C) without any deterioration. 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