Chapter 4. Results and Discussion

Transcription

1 Chapter 4 Results and Discussion The present investigation studies on removal of cyanogenic glycosides removal from flaxseed and development of flaxseed fortified rice flour based extruded product and cookies. The process technology of these products has been optimized. Further, storage stability of developed products in two different packaging materials were studied at ambient temperature. The results obtained during investigation and subsequent discussion is divided in the following topic and subtopics. 4.1 Optimization of microwave roasting of flaxseed Effect of microwave roasting on cyanogenic glycosides content Effect of microwave roasting on proximate composition and fatty acid profile of flaxseed 4.2 Development of flaxseed incorporated rice flour based extruded product Effect of flaxseed flour incorporation, moisture content, temperature and screw speed on expansion ratio of extruded product Effect of flaxseed flour incorporation, moisture content, temperature and screw speed on breaking strength (BS) of extruded product Effect of flaxseed flour incorporation, moisture content, temperature and screw speed on bulk density (BD) of extruded product Effect of flaxseed flour incorporation, moisture content, temperature and screw speed on overall acceptability of extruded product Optimization of Flaxseed incorporated rice flour based extruded product 4.3 Development of flaxseed incorporated cookies Effect of refined wheat flour replacement with roasted flaxseed flour on physical characteristics of cookies Effect of refined wheat flour replacement with roasted flaxseed flour on sensory attributes of cookies Effect of refined wheat flour replacement with roasted flaxseed flour on textural attributes of cookies 93

2 4.4 Proximate composition and fatty acid profile of optimized products Proximate composition of extruded product Fatty acid profile of control and flaxseed fortified extruded product Proximate composition of control and flaxseed fortified cookies Fatty acid profile of control and flaxseed fortified cookies 4.5 Storage stability studies on developed flaxseed fortified products in different packaging material Effect of different packaging material and storage period on characteristics of extruded product Effect of different packaging material and storage period on characteristics of cookies 4.1 Optimization of microwave roasting of flaxseed CG presents a major limitation in application of flaxseed. The reason for toxicity of CG lies in releasing of hydrogen cyanide (HCN) due to the action of β- glycosidase enzyme which acts as a potent respiratory inhibitor by complexing with metalloporphyrin-containing enzymes (Poulton, 1989). So their level in food or feed is expressed through content of HCN (Ivanov et al., 2012). Food Standards and Safety Authority of India (FSSAI) mentioned maximum permissible limit of hydrogen cyanide in food grains as 37.5 mg/kg (FSSAI Act, 2006). Microwave roasting of flaxseed was carried out to reduce CG content. Microwave roasting of flaxseed was optimized using RSM as a statistical tool. The effect of independent variables i.e. microwave power and time of treatment on flaxseed CG content (response) was discussed as follows Effect of microwave roasting on cyanogenic glycosides (CG) content The data presented in Table 4.1 showed that CG content is negatively related with the linear effect of microwave power and time of heating (p<0.01). As microwave power and time of heating increased, CG content reduced. The quadratic effects of microwave power and time of heating were also found positively related at p<

3 (Seconds) Results and Discussion Table 4.1 Results of regression analysis for microwave flaxseed roasting Parameters Estimated regression coefficient for CG content Intercept (b 0 ) Microwave Power (b 1 ) * Time of heating (b 2 ) * (Microwave power) 2 (b 11 ) 28.42* (Time of heating) 2 (b 22 ) 27.05* Interaction (b 12 ) -11 R P-value for quadratic model < P-value for model lack of fit Mean SD Subscripts 1= Microwave power, 2= time of heating *Significant at 0.01 level Fig. 1 Contour plot of microwave power and Heating time for reducing CG content 95

4 When microwave power of 60% (580 W) and 80% (790 W) for 360 seconds and longer was used, flaxseed samples were burned. Moreover, when 80% microwave power and 240 seconds used, reduction in CG content could not be achieved under allowed limits. Therefore, maximum limit of 60% microwave power and below 360 seconds was taken criteria for optimizing this treatment. Numerical optimization was carried out to find minimum time of heating at 60% (580 W) power, in order to avoid burning of samples and to reduce HCN content under permissible limit. For this purpose, dependence of HCN content with regards to heating time and microwave power is presented through contour plot graph (Fig. 1), generated by Design Expert software, where every contour define specific content of HCN. Minimal time of heating which would provide reduction of HCN content under 37.5 mg/kg was suggested by the software and it was 59.97% microwave power and seconds heating time. For targeted conditions in practice, 60% microwave power (580 W) and 300 seconds heating time was considered as optimized. By applying these, HCN content of flaxseed was reduced from 190±2.13 mg/kg to 36.4±0.9 mg/kg. HCN removal rate of 80.84% was achieved. These results are well in agreement with Feng et al., (2003) who reported that microwave roasting achieved the highest level of HCN reduction in linseed among autoclaving, pelleting and dry heating in oven with 750 W for 4 min. Yang et al. (2004) also reported hydrogen cyanide reductions of 89%, 27%, 82%, and 100% using solvent extraction, autoclaving, microwave roasting, and water boiling, respectively. Ivanov et al., (2012) reported that microwave roasting at 400 W for 290 seconds reduced CG content from 317 mg/kg to 250 mg/kg. Research has also shown that commercial processing of flaxseed could affect the cyanogenic glycosides content in flaxseed (Oomah and Mazza, 1998a) Effect of microwave roasting on proximate composition and fatty acid profile of flaxseed Proximate composition of raw flaxseed (Table 4.2) revealed that it is good source of oil (41.31±0.74%), protein (21.74±0.92%) and fibre (12.78±0.7%1). Table 4.3 showed that flaxseed is richest source of linolenic acid (54.28%). Several other researchers reported similar results. (Masoodi et al., 2012; Chetana et al., 2010b; Singh 96

5 and Jood, 2009; Husain, 2008). Some variations were observed. This might be due to varietal and growing environmental conditions Microwave roasted flaxseed at 60% microwave power (580 W) and 300 seconds heating time was intended for the further study. Proximate analysis of microwave roasted flaxseed (Table 4.2) was carried out to investigate the change in composition. The moisture content of roasted flaxseed significantly reduced as compared with raw flaxseed. This can be attributed to the evaporation of moisture due to heat generation in microwave heating. Consequently, significant increase in the ash and lipid content can attributed to the increase in the total solids content of roasted flaxseed. Another explanation for increase in the lipid content of roasted flaxseed is roasting demolishes fat cells in flaxseed (Cammerer and Kroh, 2009) and eases the release of lipid. A small but insignificant reduction in the protein and carbohydrate content was observed. This can be attributed to the maillard reaction occurred due to microwave heating which involves amino acid and reducing sugar interaction. Insignificant reduction in the crude fiber was noted. Available polysaccharides are mainly insoluble fibres which are very stable and only insignificantly participate in maillard reactions (Cammerer and Kroh, 2009). Table 4.3 depicts the comparison of fatty acid profile in raw and microwave roasted flaxseed. Traces of palmitolic acid, arachidic acid and other unknown fatty acids had disappeared after roasting of flaxseed. Slight increase in palmitic acid and linolenic acid was observed. Overall there were no major changes in fatty acid profile caused by microwave treatment. Increase in linolenic acid content is controversial to the findings of Chetana et al. (2010b) who reported that decrease in the same from 55.41% to 50.76%. Variations in the findings can be attributed to type and time of heat treatment employed for roasting of flaxseed. Mentioned author roasted flaxseed at C for 15 minutes. Otherwise Chen et al. (1994) reported that at baking temperature of C for 2 hours, linolenic acid content remained unchanged. Results were furthermore consistent with the literature findings, as fatty acid profile of roasted flaxseed was stable during and after roasting process (Kozlowska, 1989). 97

6 Table 4.2 Proximate composition of raw and roasted flaxseed powder SN Parameter Flaxseed powder* Roasted flaxseed powder* 1 Moisture (%),wb 5.81±0.70 a 3.62±0.76 b 2 Ash (%) 3.60±0.22 c 4.10±0.31 c 3 Fat/Lipid Content (%) 41.31±0.74 d 44.33±0.73 e 4 Protein content (%N X 6.25) 21.74±0.92 f 21.10±0.75 f 5 Crude Fiber (%) 12.78±0.71 g 12.33±0.55 g 6 Carbohydrate** (%) 27.53±0.54 h 26.84±1.87 h Mean values followed by same superscript alphabet in the same row is not significantly different at p<0.05 * - Reported results are based on the average of three determinations ** - Carbohydrate content was calculated by the difference. SN Table 4.3 Fatty acid profile of raw and roasted flaxseed extracted oil Fatty acid Flaxseed oil (Relative %) Roasted Flaxseed oil (Relative %) 1 Palmitic acid Palmitolic acid Stearic acid Oleic acid Trans oleic acid Linoleic acid Linolenic acid Arachidic acid Others

7 4.2 Development of flaxseed fortified rice flour based extruded product Preliminary trials (Appendix IV A) were conducted to decide the levels of four independent variables: Roasted flaxseed flour (%), moisture content of extruder feed (%), temperature of extruder barrel ( 0 C) and extruder screw speed (rpm). Extruder feed material particle size was found in the range of 308µ to 345µ (Appendix-IV B). On the basis of preliminary trial results, the levels of each independent variable were decided accordingly for further experimental statistical design (Table 3.2). Total 30 experiments (run) were carried out to investigate the effect of independent variables on the selected responses. The effect of different variables like roasted flaxseed flour (%), moisture content of extruder feed (%), temperature of extruder barrel ( 0 C) and extruder screw speed are discussed on the responses considered for the optimization process. The data of responses are presented in Appendix-V Effect of flaxseed flour fortification, moisture content, temperature and screw speed on expansion ratio of extruded product The data presented in Table 4.4 showed that ER was negatively related to linear effects of roasted flaxseed flour (RFF) fortification level and positively related to linear effect of moisture content (p<0.01). Similarly it is positively related to quadratic effects of RFF level, moisture content and screw speed (p<0.01) and negatively related to linear effect of barrel temperature (p<0.05). It is evident from Fig. 2 that as RFF fortification increased ER of extruded product decreased. This could be due to high fibre, fat and protein content of flaxseed that may affect the starch gelatinization. Non starch polysaccharides in the fibre may bind water more tightly during extrusion than do protein and starch. This binding may inhibit water loss at the die and thus reduces the expansion. These findings are well in line with the results reported by Seth and Rajamanickam (2012), Mahasukhonthachat et al. (2010) and Pai et al. (2009). Moreover, from Fig. 2, it can be revealed that as the moisture content increased from 12 to 16%, ER increased. At 16% moisture content, highest ER was observed. This is attributed to the viscosity of the starch would be low at high moisture content, allowing for extensive internal mixing and uniform heating which would account 99

8 for enhanced starch gelatinization (Lawton and Handerson, 1972). Similar results are reported for extrusion of rice with pea grit by Singh et al. (2007). Parameters Table 4.4 Results of regression analysis of extruded product Expansion ratio Estimated regression coefficients Breaking Bulk strength density (kgf) (g.cm -3 ) OAA Intercept (b 0 ) b * * * * b * * ** b ** *** ** b b * * * b * ** b ** b * ** * b ** b b b b * b R P-value for quadratic model P-value for model lack of fit < Mean SD Subscripts 1= Roasted flaxseed flour, 2= Moisture content, 3= Temperature, 4= Screw speed *Significant at 0.01 level **Significant at 0.05 level ***Significant at 0.1 level 100

9 The final equation in terms of actual factors for ER is as follows Expansion Ratio = *x *x *x *x E-003*x *x E-004 *x E-004 * x E-003*x1 *x E-004 *x 1 *x E-004*x 1 *x E-003*x 2 *x E-03*x 2 *x E-004 * x 3 * x 4 Temperature C Screw Speed 315 RPM (%) (%) Fig 2. Response Surface for Expansion Ratio as a function of flaxseed fortification and moisture content at Barrel temperature C and screw speed of 315 RPM 101

10 4.2.2 Effect of flaxseed flour fortification, moisture content, temperature and screw speed on breaking strength (BS) of extruded product The data presented in Table 4.4 revealed that BS was positively significantly related to the linear and quadratic effect of RFF fortification (p<0.01). It is negatively related to the interaction effect of moisture content and screw speed (p<0.01). Quadratic effect of screw speed and interaction effect of flaxseed and moisture content were found significantly positively related (p<0.05). It is evident from Fig. 3 that BS was increased as the RFF fortification level increased. This might be due to high fiber and protein content of flaxseed which reduces the expansion; product became less porous and perhaps due to aggregation of protein molecules. Wu et al. (2007) also reported the increase in the hardness of the product as flaxseed increased from 5 to 15% in flaxseed-corn puff. Ilo et al. (1999) reported similar results for rice and amaranth blend extrusion. Increasing amaranth content caused an enormous decrease in expansion and an increase in the BS. Fig. 4 shows the interaction effect of moisture content and screw speed. This interaction effect is found significant (p<0.01). This interaction is negatively affected on the BS of extruded product. As the moisture content and screw speed increased, BS value also increased. This might be due to higher moisture content drops down the blend temperature (Liu et al., 2000). Higher screw speed reduces the retention time of the blend in the extruder cooking zone. Consequently, it affects on the gelatinization of starch. A more expanded product would take less force to shear (Wu et al., 2007) and hence had a lower BS and vice versa. The final equation in terms of actual factors for BS is as follows. Break Strength = *x * x *x *x E-003 * x E-003 * x E-004 * x E-004 *x E-003 *x 1 *x E-004 *x 1 * x E-004 *x 1 *x E-003 *x 2 *x E-003 *x 2 *x E-005 *x 3 *x 4 102

11 (kgf) (kgf) Results and Discussion Temperature C Screw Speed 315 RPM (%) (%) Fig. 3 Response Surface for Breaking strength as a function of flaxseed fortification and moisture content at Barrel temperature C and screw speed of 315 RPM Flaxseed 20% Temperature C D: Screw Speed (RPM) Fig. 4 Response Surface for Breaking strength as a function of moisture content and screw speed at Flaxseed fortification 20% & Barrel temperature C (%) 103

12 4.2.3 Effect of flaxseed flour incorporation, moisture content, temperature and screw speed on bulk density (BD) of extruded product Bulk density has been linked with the expansion ratio in describing the degree of puffing in extrudates. A high bulk density is associated with a low expansion index (Rayas-Duarte et al., 1998; Suknark et al., 1997) because more compact material is obtained after milling a less expanded product (Onyango et al., 2004). The effect of independent variables on bulk density is given in Table 4.4. It is evident from the data that bulk density was influenced by linear effect of RFF fortification and moisture content (p<0.01). It is also influenced by linear effect of barrel temperature (p<0.10). Negative significant quadratic effect of RFF fortification and screw speed were observed (p<0.01). Moreover, BD is also negatively affected by the quadratic effect of moisture content (p<0.05). As the RFF fortification level increased, BD increased (Fig. 5). RFF fortification was an important variable in the response surface model of product BD as its linear and quadratic terms was significant at p<0.01 level. The minimum BD (0.095 g.cm -3 ) of product at lowest flaxseed fortification level (10%) and the maximum BD (1.131 g.cm -3 ) of product was observed at highest flaxseed fortification level (30%). This is due to the negative impact on the starch gelatinization and also protein aggregation. These results are well in agreement with the results reported by Yagci and Gogus (2008) for extruded snack food products developed from fruit waste fibers. As the value of feed moisture content increased the value of bulk density decreased and vice versa. The dependence of product density on feed moisture reflects its influence on the rheological characteristics of the starch based material. This is attributed to the higher of amount of non starch polysaccharides tightly bounds the water and do not allow to vaporize at the exit of die. Similar results are reported by Kulkarni and Joshi (1992). As the barrel temperature increases, BD was found to increase marginally at 0.10 significant level (Fig. 5). Bulk density increase and porosity decrease at higher extruder temperatures can be attributed to increased dextrinization and weakening of structure (Mendonca et al., 2000). This can also be attributed to decrease in the elasticity of extrusion cooked melts with increasing temperature (Ilo et al., 1999). Results are well in line with the results reported 104

13 by Chakraborty and Banerjee (2009) for production of expanded product from green gram and rice. The final equation in terms of actual factors for BD is as follows. Bulk density = *x E-003 *x E-003 *x *x E-004 *x E-003 *x E-005 *x E-005 *x E-004 *x 1 *x E-006 *x 1 * x E-005 *x 1 *x E-004*x 2 * x E-005 *x 2 *x E-006 *x 3 *x 4 Temperature C Screw Speed 315 RPM (g.cm -3 ) (%) (%) Fig. 5 Response Surface for Bulk density as a function of flaxseed fortification and moisture content at Barrel temperature C and screw speed of 315 RPM 105

14 Moisture 14% Screw Speed 315 RPM (g.cm -3 ) ( 0 C) (%) Fig. 6 Response Surface for Bulk density as a function of flaxseed fortification and Barrel temperature at moisture content 14% and screw speed of 315 RPM Effect of flaxseed flour fortification, moisture content, temperature and screw speed on overall acceptability of extruded product Overall acceptability (OAA) scoring is subjective evaluation (sensory evaluation) for confirming the product acceptability by the consumer. The data pertaining to effect of various variables on OAA was presented in Table 4.4. OAA was negatively related to the linear effect of RFF fortification at 0.01 % significant level. It was positively related to the linear effect of moisture content (p<0.05). It is negatively related to the linear effect of the barrel temperature (p<0.05). It is also negatively related to the quadratic effect of barrel temperature (p<0.05). Fig. 7 showed that as the RFF fortification increased and moisture content decreased, OAA score decreased. The highest OAA score of 8.2 was found at lowest flaxseed incorporation (10%) while lowest OAA score of 5.8 at highest RFF fortification (30%). Porosity / expansion are the dominant quality attributes in extruded product. 106

15 Fortification of higher flaxseed and lowering moisture content reduced ER and hardened the product (already discussed in & 4.2.2) and consequently lowered OAA score. Other reason is nutty flavour of flaxseed which increased at its higher incorporation level. Moisture content was found significantly affecting OAA score at 0.05 significant level. From Fig. 8, it is evident that as the barrel temperature increased, OAA score decreased. This can be attributed to higher dextrinization and formation of mallard reaction end products which ultimately reduced OAA score. The final equation for OAA in terms of actual coded factors is as follows OAA = * x * x * x * x E-003 * x * x E-003 * x E-005 * x E-003 * x1 * x E-003 * x 1 * x E-004 * x 1 * x E-003 * x 2 * x E-004 * x 2 * x E-004 * x 3 * x 4 Temperature C Screw Speed 315 RPM (%) (%) Fig. 7 Response Surface for OAA as a function of flaxseed fortification and moisture content at Barrel temperature C and screw speed of 315 RPM 107

16 D: Screw Speed (RPM) ( 0 C) Fig. 8 Response Surface for OAA as a function of flaxseed fortification and moisture content at Barrel temperature C and screw speed of 315 RPM Optimization of Flaxseed fortified rice flour based extruded product Design expert program of the STAT-EASE software was used for simultaneous optimization of the responses. Desired goals were assigned for all the parameters for obtaining the numerical optimum values of the responses. The response parameters like expansion ratio and overall acceptability were kept maximum whereas breaking strength and bulk density were kept minimum. Figs show the optimum conditions for flaxseed fortified rice flour based extruded product to yield maximum expansion ratio and overall acceptability and optimum breaking strength and bulk density respectively. It is observed from Fig. 9 that as RFF fortification level increases, ER decreases. High fibre, fat and protein content of flaxseed that may affect the starch gelatinization negatively. Non polysaccharides in the fiber binds water more tightly and at exit end of extruder less moisture flashes off, which reduced ER. As moisture content increases from 12 to 16%, ER increases. Positive effect 108

17 is attributed to lower viscosity of the starch at high moisture content, allowing for extensive internal mixing and uniform heating which would account for enhanced starch gelatinization. At optimum conditions, the ER of 3.08 could be obtained. The contour plots of breaking strength as a function of process variables is depicted in Fig. 10. The results exhibited that as flaxseed incorporation level increases; the breaking strength also increases whereas as moisture content increases, breaking strength decreases. At optimum conditions, the BS of 0.53 kgf could be obtained. The contour plots of Bulk density as a function of process variables is presented in Fig. 11. It was noted that as RFF fortification level increases, the BD of extruded product also increases. This is due to negative impact of flaxseed on the starch gelatinization and also on protein aggregation. As the moisture content increases, the BD of extruded product decreases. This might be due to increased gelatinization at higher moisture. At optimum conditions, the BD of g.cm -3 could be obtained. Fig. 12 shows the contour plots of overall acceptability as a function of process variables. It was observed that as RFF fortification level increases, the OAA score decreased whereas as the moisture content increases, the OAA score increases. At optimum conditions, the OAA score of 7.86 could be obtained. Among 30 experiments (run) conducted, extruded product photographs of selected run are shown in Plate 8 and 9. The desirability graph for the most desirable flaxseed fortified rice flour based extruded product is presented in Fig. 13. The process variables for the best combination of the responses (Desirability 0.90) were 15 percent RFF fortification with rice flour, 16 percent moisture content of extruder feed, C extruder barrel temperature and 330 RPM of screw speed. The response functions were calculated from the final polynomial and the response at this optimized combination were expansion ratio (3.08), breaking strength (0.53 kgf), bulk density (0.106 g.cm -3 ) and overall acceptability score (7.86). 109

18 Plate 8. Flaxseed fortified extruded products obtained in run no. 1, 4, 6, 7, 11 and 12 Plate 9. Flaxseed fortified extruded products obtained in run no. 15, 17, 23, 24, 26 and

19 X2: Moisture content (%) X2: Moisture content (%) Results and Discussion Temperature C Screw Speed 330 RPM Prediction % CI Low % CI High X X X1: Flaxseed fortification (%) Fig. 9 Contour plots of expansion ratio of extruded product as function of flaxseed fortification, moisture content, temperature and screw speed Temperature C Screw Speed 330 RPM Prediction % CI Low % CI High X X X1: Flaxseed fortification (%) Fig. 10 Contour plot of breaking strength of extruded product as function of flaxseed fortification, moisture content, temperature and screw speed 111

20 X2: Moisture content (%) X2: Moisture content (%) Results and Discussion Temperature C Screw Speed 330 RPM Prediction % CI Low % CI High X X X1: Flaxseed fortification (%) Fig. 11 Contour plot of bulk density of extruded product as function of flaxseed fortification, moisture content, temperature and screw speed Temperature C Screw Speed 330 RPM Prediction % CI Low % CI High X X X1: Flaxseed fortification (%) Fig. 12 Contour plot of overall acceptability of extruded product as function of flaxseed fortification, moisture content, temperature and screw speed 112

21 X2: Moisture content (%) Results and Discussion Temperature C Screw Speed 330 RPM Prediction X X X1: Flaxseed fortification (%) Fig. 13 Desirability graph as function of flaxseed fortification, moisture content, temperature and screw speed 113

22 4.3 Development of flaxseed fortified cookies Roasted flaxseed flour (RFF) was fortified with the replacement of refined wheat flour at 5%, 10%, 15%, 20%, 25% and 30% in the development of cookies. The effect of flaxseed flour fortification on physical characteristics, sensory scores and textural attributes were investigated and discussed as follows Effect of refined wheat flour replacement with roasted flaxseed flour on physical characteristics of cookies The physical characteristics of seven types of cookies are shown in Table 4.5. Results of these studies indicated that there is significant difference (p<0.05) between control sample and treated samples except A (5% RFF flour) sample. As RFF level increased, thickness and diameter of cookies increased. Protein influences the dough viscosity. This is because the expansion of protein gluten is not resumed in the making of cookies. Inverse correlation was obtained between diameter and protein content (Leon et al., 1996). Protein gluten in flour will form a web in cookie dough when heated. During baking, the gluten goes through an apparent glass transition, thereby, gaining mobility that allows it to interact and form a web. The formation continuous web increases viscosity and stops the flow of cookie dough (Miller et al., 1997). Lowest thickness of 50 ±0.76 mm and diameter of ±0.96 at 5% RFF cookies while highest thickness of ±1.15 mm and diameter of ±0.96 at 30% RFF were observed cookies. No significant difference for thickness between B and C cookies sample was observed. As the RFF level increased, spread ratio for different treated cookies gradually decreased from 5.70 ± 0.07 to 4.63 ± This reduction in spread ratio might be due to increase in protein and dietary fiber percentage with increasing level of flaxseed flour because protein and dietary fiber has more water binding power. When more water is present in the dough, more sugar is dissolved during mixing. This lowers the initial dough viscosity and the cookie is able to spread at a faster rate during heating. The flour components that absorb large quantities of water reduce the amount of water that is available to dissolve the sugar in the formula. Thus, initial viscosity is higher and the cookies spread less during baking (Hoseney and Rodger, 1994). Cookie spread rate 114

23 Table 4.5. Physical characteristics of control and RFF cookies Treatment Thickness (mm) Diameter (mm) Spread ratio Control 50.33±0.76 a ±0.96 a 5.70±0.07 a A (5% flaxseed flour) 51.33±0.29 a 288.5±1.00 ab 5.62±0.03 a B (10% flaxseed flour) 54.33±0.58 b ±0.96 b 5.34±0.04 b C (15% flaxseed flour) 55.33±0.58 b ±0.50 c 5.35±0.05 b D (20% flaxseed flour) 62.00±1.32 c ±0.96 d 5.02±0.09 c E (25% flaxseed flour) 65.33±1.5 d ±0.96 e 4.83±0.10 d F (30% flaxseed flour) 69.33±1.15 e ±0.96 f 4.63±0.06 d Mean values in the same column which is not followed by the same superscript letter are significantly different (p<0.05). appears to be controlled by dough viscosity (Yamazaki, 1959, Miller et al., 1997). It is the reason due to which soft wheat varieties are recommended with low protein content to prepare cookies (Miller et al., 1997). Results obtained in the present study are in line with Bashir et al., 2006 and Hussain, Moreover, Hoojat and Zebik (1984) also showed that 20% and 30% replacement of navy bean, sesame flour reduced the spread factor of the wheat flour cookies. Rajiv et al. (2012) reported the similar result trends for spread ratio Effect of refined wheat flour replacement with roasted flaxseed flour on sensory attributes of cookies Mean score of sensory attributes (Color, Taste, Texture and Overall acceptability) for control and RFF cookies is presented in Table 4.6. Color score results for control and different treated samples indicate that the score decreased with increasing content of RFF. This can be observed in Plate 10. The probable reason for these results could be brown color of flaxseed which became dark brown at high baking temperature. Pigments such as leutin/zeaxanthin in flaxseed makes it dark brown (United States 115

24 Department of Agriculture, 2007). Maillard reaction may have also contributed to the darker color of bakery products due to the high protein content of flaxseed (Borrelli et al., Sample Code Table 4.6 Sensory score of control and RFF cookies Color Taste Texture Overall Acceptability Control 8.1 a 7.9 a 8.3 a 8 a A 7.9 ab 7.7 a 8.1 ab 7.8 a B 7.3 bc 7.5 a 7.6 bc 7.7 a C 7 cd 7.4 a 7.2 c 7.5 a D 6.3 de 6.4 b 6.4 d 6.5 b E 5.6 e 4.5 c 5.6 e 5.1 c F 4.3 f 3.9 c 4.6 f 4.3 d Mean values in the same column which is not followed by the same superscript letter are significantly different (p<0.05). Plate 10. Flaxseed fortified cookies 116

25 2003). Taste of the cookies was considerably influenced due to flaxseed incorporation. Results reveals that there was no significant difference (p<0.05) in taste up to 15% incorporation of flaxseed cookies and control sample cookies. Beyond 15% RFF level, taste score significantly reduced. At first three RFF cookies (A, B and C), flaxseed pleasant nutty flavor is liked by judges. But, in high quantities of RFF, different taste was easily distinguished and was disliked by judges. Texture score values also showed decrease trend as RFF in cookies increased. This might be due to decrease in crispiness. A sample texture was not significantly differed from control sample. But, afterwards significant difference was found. Overall acceptability score suggested that up to 15% RFF incorporated cookies were not significantly different than that of control. Overall acceptability is governed by all dominant sensory quality attributes. At 20% and above RFF incorporation level, overall acceptability score decreased significantly (p<0.05). This might be due to high nutty flaxseed flavor and grittiness of flaxseed flour which gave unpleasurable aftertaste. As the amount of RFF in product increases, the intensified flavor, dark brownish color, rough surface, less crisp and gritty mouthfeel, making them to score low in sensory evaluation. From the above discussion, RFF incorporation at moderate levels (up to 15%) has resulted in acceptable products which were comparable with the control treatment. Similar results are reported by Cole et al. (2002) for flaxseed substitution in cinnamon bread without negatively affecting the appearance, texture, flavor and overall acceptability. Bashir et al. (2006) also reported that 15% flaxseed incorporated cookies and cakes were acceptable. Chetana et al. (2010b) reported that muffins incorporated with 20% roasted flaxseed had better overall acceptable quality. Tyagi et al. (2007) studied the nutritional, sensory and textural characteristics of biscuits prepared by incorporating defatted mustard flour at 5, 10, 15 and 20% levels. They reported that the textural characteristics of biscuits up to 15% of defatted mustard flour were almost similar, but at 20% level the values were significantly different and they recommended use of 15% defatted mustard flour to achieve desirable results. Husain (2006) also reported that cookies containing 20% and lower levels of flaxseed flour were acceptable. Variations in the results can be attributed to the flaxseed growing environmental conditions and variety used. 117

26 4.3.3 Effect of refined wheat flour replacement with roasted flaxseed flour on textural attributes of cookies The textural parameters are one of the most important quality attributes which affects the overall quality of cookies. The instrumental textural attribute analysis was conducted to confirm the textural score as obtained from subjective evaluation of texture. The effect of incorporation of RFF on the textural attributes i.e. hardness (g) and fracturability (mm) are represented in Figure 14 and 15 respectively. An increasing trend was observed for both the textural parameters up to 15% level of RFF flour incorporation and after that the textural parameters were found to decrease with increase in RFF incorporation. Since RFF has both gum mucilage (fiber) and protein, high water absorbing capacity components as well as there was a significant level of fat (around 41%) found in RFF, hence both these factors contributed in a sticky dough thus reducing extensibility of dough. The extensible and cohesive structure is contributed by sugar or water interaction with wheat protein thus forming gluten but with an increase in fat content the flour gets coated and this network gets interrupted thus properties of cookies are changed and a less harder. At very high fat content the lubricating function is high thus less water is required and a softer texture is obtained. Hence the hardness and fracturability gradually decreased forming a softer cookies with an increased level of RFF flour. The values of hardness (g) and fracturability (mm) varied from 1217±43 to 1364±64 and 0.54±0.01 to 0.67±0.04 respectively. Sensory texture score might be decreased due to lower hardness and fracturability values which confirmed more softness at higher RFF incorporation level. Rajiv et al. (2012) reported increased in breaking strength of cookies as roasted flaxseed flour increased from 5 to 20%. Variations in the result might be due to raw material composition and flaxseed varietal difference. Barnwal et al. (2013) reported similar results for partially de-oiled maize germ cake (DMGC) flour incorporated biscuits. They reported that the textural properties such as fracture force (N), hardness (N), breaking strength (N), breaking energy (N-mm), cutting strength (N) and cutting energy (N-mm) increased with DMGC flour incorporation level up to 30% but above 30%, decreased the values for textural attributes. 118

27 Fracturability (mm) Hardness (g) Results and Discussion % RFF Incorporation Level Fig. 14 Effect of RFF incorporation level on hardness of cookies % RFF Incorporation Level Fig. 15. Effect of RFF incorporation level on fracturability of cookies 119

28 4.4 Proximate composition of optimized products Proximate composition and fatty acid profile of optimized products (flaxseed fortified extruded product and cookies) were carried out to confirm the enhancement of desirable nutrients (i.e. protein, fiber and ALA). The results are discussed as follows Proximate composition of flaxseed fortified extruded product To compare composition of optimized extruded product, control extruded product (without flaxseed) was produced at the same extrusion parameter. In place of flaxseed flour, rice flour quantity was increased. Proximate compositions of optimized flaxseed flour fortified and control extruded products are presented in Table 4.7. Moisture content of flaxseed fortified extruded product increased as compared with control extruded product. This can be attributed to fiber (gum mucilage) present in flaxseed which has higher moisture retention property (Cui and Mazza, 1996). Ash content of flaxseed fortified extruded product increased as compared with control products. This might be due to higher mineral content of flaxseed flour (Morris, 2003). Moreover, fat content, protein content and crude fiber of optimized flaxseed fortified extruded products increased as compared with control products. This could be accounted by the fact that flaxseed is far higher in fat, protein and crude fiber content as its proximate composition represented previously in Table 4.2. Approximately three fold increase in crude fiber and 1.3 times increase in the protein content of flaxseed fortified extruded product as compared with control extruded product were observed. Similar results are reported by Jaysena and Nasar- Abbas (2012) for lupin flour incorporated pasta. Lupin grain has a unique nutritional composition with high protein and high dietary fiber contents. Lupin flour incorporation would improve the nutritional value of pasta, which is otherwise low in protein and dietary fiber contents. Therefore, flaxseed fortification can also enhance the protein and fiber content of extruded product without significant effect on the sensory quality. 120

29 Table 4.7 Proximate composition of extruded products SN Parameter Extruded control* Flaxseed fortified extruded product* 1 Moisture (wb), % 5.92±0.33 a 7.26±0.37 b 2 Ash, % 2.97±0.18 c 4.54±0.22 d 3 Protein (N x 6.25), % 7.61±0.52 e 9.86±0.64 f 4 Fat, % 0.76±0.07 g 3.50±0.13 h 5 Crude fibre, % 1.63±0.30 i 4.71±0.42 j Carbohydrate**, ±0.24 k 74.85±0.49 l % Mean values followed by the different superscript alphabet are significantly different at p<0.05 *Reported values are the average of three determinations **Carbohydrate is determined by difference Fatty acid profile of extruded product Oil was extracted from flaxseed fortified and control extruded product and subjected for fatty acid profile analysis by gas chromatography. Results obtained are depicted in Table 4.8. Saturated fatty acids i.e. mainly palmitic and oleic acid content decreased while unsaturated fatty acids i.e. linolenic acid (omega-3 fatty acid) increased from 24.28% to 46.51% in flaxseed incorporated extruded product. This increase can attributed due to the addition of roasted flaxseed powder which contains 55.37% linolenic acid (Table 4.3). Decrease in the saturated fatty acids can be attributed to dilution of fat with flaxseed oil. ALA (omega-3 fatty acid) content have almost doubled in the flaxseed fortified extruded product as compared with the control product. Malcolmson et al. (2000) also reported that macaroni containing ground flaxseed and dried at high temperature had very good lipid and SDG stability, which could be used as a means to enhance dietary ALA and SDG consumption. Lee et al. (2004) also reported that the extrusion and drying processes did not affect lipid extraction or stability of ground whole 121

30 flaxseed and flaxseed hull in pasta. Villeneuve et al. (2013) also reported the enhancement and stability of ALA in pasta by incorporating flaxseed flour. Table 4.8 Fatty acid profiles of extruded product SN Fatty acid Extruded product oil Control (%) RFF Extruded (%) 1 Myristic acid Palmitic acid Palmitolic acid Stearic acid Oleic acid Trans oleic acid Linoleic acid Linolenic acid Other fatty acids Proximate composition of control and flaxseed fortified cookies The refined wheat flour used in cookies preparation had 9.32% moisture, 0.48% ash, 8.1% dry gluten, 8.7% protein, 1.8% fat and 0.26% crude fiber. Proximate analysis of control cookies and RFF incorporated cookies are presented in Table 4.9. Moisture content of RFF cookies significantly increased (5.02%) as compared with control cookies (6.33%). This can be attributed to fiber (gum mucilage) present in flaxseed which has higher moisture retention property. The dietary fiber of flaxseed hull is about evenly split between an insoluble fiber fraction and highly soluble, periodic mucilaginous fiber fraction (Cui and Maza, 1996) which gives the flaxseed hull a high water absorption, moisture binding capacity as well as lubricity. 122

31 Ash content of RFF cookies (0.82%) increased significantly as compared with control cookies (0.43%). This might be due to higher mineral content of flaxseed flour (Morris, 2003). A small but insignificant increase in the fat content of optimized cookies was observed. Moreover, protein content (8.66%) and crude fiber (1.72%) of RFF cookies increased significantly as compared with control cookies. This could be accounted by the fact that flaxseed is far higher in protein and crude fiber content compared with refined wheat flour as their proximate analysis is mentioned previously. Chetana et al. (2010b) reported similar results for flaxseed incorporated muffins. Gambus et al. (2004) also observed that bread with 10 and 13% share of flaxseeds was characterized by higher amounts of protein, fat, dietary fiber, macro- and microelements in comparison to standard one. This confirmed that the nutritional enhancement can be done with the fortification of flaxseed flour in cookies without affecting its sensory quality. Table 4.9 Proximate composition of control and flaxseed fortified cookies SN Parameter Control Cookies* Flaxseed fortified cookies* 1 Moisture (wb), % 5.02±0.42 a 6.33±0.54 b 2 Ash, % 0.43±0.10 c 0.82±0.11 d 3 Protein (N x 6.25), 5.86±0.63 e 8.66±0.74 f % 4 Fat, % 31.13±1.15 g 33.73±1.18 g 5 Crude fibre, % 0.19±0.05 h 1.72±0.22 i 6 Carbohydrate**, % 57.55±1.30 j 50.47±2.17 k Mean values followed by the same superscript alphabet in the row are not significantly different at p<0.05 *-Reported values are the average of three determinations **- Carbohydrate is determined by difference Fatty acid profile of control and flaxseed fortified cookies Extracted oil from control and flaxseed fortified cookies were analyzed for fatty acid profile and the results obtained are presented in Table Saturated fatty 123

32 acids, Palmitic and oleic acid content, were dominating than the stearic acid. Similar results are reported by Vicario and Viviana (2003) for butter cookies. Among the polyunsaturated fatty acids, linoleic acid content was higher and linolenic acid content of control cookies was very less (0.19%). Saturated fatty acids i.e. mainly palmitic and oleic acid content decreased while unsaturated fatty acids i.e. linolenic acid (omega-3 fatty acid) increased from 0.19% to 4.76% in flaxseed fortified cookies. This increase can attributed due to the addition of roasted flaxseed powder which contains 55.37% linolenic acid (Table 4.3). Decrease in the saturated fatty acids can be attributed to dilution of fat with flaxseed oil. Significant increase in linolenic acid content of flaxseed incorporated cookies (i.e. 25 times higher as compared with control) was observed. Chen et al. (1994) also reported that flaxseed lipid was stable during baking of flaxseed muffins. They also reported that ground flaxseed alone readily absorbs oxygen under typical baking conditions, but this does not markedly affect its fatty acid composition. As a baking ingredient, ground flaxseed does not lose significant amounts of ALA during baking. These results are well in agreement with the results reported by Rajiv et al. (2012) for flaxseed cookies. Bilek and Turhan (2009) reported that flaxseed flour can be used as functional ingredient in beef patties as ALA content of raw and cooked beef patties increased with the increase in flaxseed flour addition. Table 4.10 Fatty acid profile of control and flaxseed fortified cookies SN Fatty acid Control Cookies oil RFF cookies 1 Myristic acid Palmitic acid Palmitolic acid Stearic acid Oleic acid Trans oleic acid Linoleic acid Linolenic acid Other fatty acids

33 4.5 Storage stability studies of developed flaxseed fortified products in different packaging material Proximate composition of flaxseed fortified extruded product confirmed that fiber increased by approximately 2.8 times as compared with the control product. As previously mentioned, fiber have more water binding capacity. Moreover, ALA (essential omega-3 fatty acid) increased approximately twice as compared with the control product. ALA, being one of the polyunsaturated fatty acid, might induce a higher susceptibility to lipid oxidation (Morrissey et al., 1998). Higher moisture and ALA content can affect the acceptability of products during storage negatively. Different strategies like proper packaging and use of antioxidants can be adopted to minimize moisture absorption and lipid oxidation. Therefore, the objective of the present investigation was to follow the changes occurred in the flaxseed fortified and control extruded product during 90 days of storage at ambient temperature in two different packaging material i.e. METPET/Al/LD laminate and PP/LD laminate. It will be helpful to predict the most suitable packaging material and also shelf life of optimized product. Analysis of two laminates are presented in Table Table 4.11 Packaging material laminates analysis SN Parameter METPET/Al/LD PP/LD 1 Thickness (micron) 52±3 61±2 2 WVTR (g/m 2 /24h at 38 0 C, 90% RH) OTR (cc/m 2 /24h at 28 0 C) WVTR Water vapor transmission rate, OTR Oxygen transmission rate Moisture content, breaking strength and sensory score were considered as response parameters for extruded product while moisture content, peroxide value and sensory score were considered as response parameters for cookies during storage stability study. During 90 days of storage, at every 15 days of interval, samples of control and flaxseed fortified product were analyzed for their respective response parameters and results are discussed as follows. 125

34 4.5.1 Effect of packaging material and storage on characteristics of extruded product Effects of two packaging material (METPET/Al/LD laminate and PP/LD) on the optimized flaxseed fortified and control extruded product during 90 days of storage at ambient temperature are presented in Table It was observed that two laminates significantly affected moisture content as compared to their respective control. The highest average moisture value of 8.22 per cent was shown by flaxseed fortified extruded product packed in PP/LD laminate while the lowest average moisture value of per cent was shown by control extruded product packed in METPET/Al/LD laminate. However, increase in the moisture content of products packed in PP/LD laminate was higher as compared with the products packed in METPET/Al/LD laminate due to better moisture barrier property of METPET/Al/LD (Table 4.11). Moreover the products packed in PP/LD laminate found to be soggy on 90 days of storage. It is also evident from Table 4.12 that significant reduction in breaking strength was observed for the products packed in METPET/Al/LD laminate. However, no significant change in breaking strength was observed among the products packed in PP/LD laminate. Significant effect of packaging material was observed when compared among A and C, B and D. The highest average breaking strength value of 0.42 kgf was observed for flaxseed fortified extruded product packed in METPET/Al/LD laminate while the lowest average breaking strength value of 0.3 kgf was observed for control extruded packed in PP/LD laminate. Table 4.12 Effect of packaging material on characteristics of extruded product during storage Packaging Material - Product Moisture content (%) Breaking strength (kgf) Sensory Score METPET Control (A) 6.31 d 0.35 b 7.81 a METPET Flaxseed extruded (B) 7.76 c 0.42 a 7.54 b PP/LD Control (C) 6.57 b 0.3 c 7.4 bc PP/LD Flaxseed extruded (D) 8.22 a 0.34 bc 7.19 c LSD at p< Means followed by same alphabet in a column do not differ significantly (p<0.05) 126