SECTION IV RESULTS AND DISCUSSION

Transcription

1 SECTION IV RESULTS AND DISCUSSION

2 CHAPTER 1 PHYSICO-CHEMICAL PROPERTIES, ANTINUTRIENT AND PROTEIN PROFILE OF PEARL MILLET AS INFLUENCED BY PROCESSING Pearl millet has significant potential as food and feed in addition to its current usage as forage. It is a drought-tolerant crop and can be grown under difficult ecological conditions. For this reason it is widely grown in tropical regions of world including Africa and Asia. It is comparable and even superior in some of the nutritional characteristics to major cereals, with respect to its energy value, protein, fat and minerals content (Abdalla et al. 1998). The use of pearl millet for human consumption is limited due to non-availability in convenience form. The millet is mostly used as whole flour for traditional food preparation and hence confined to traditional consumers and to people of lower economic strata. Pearl millet can be consumed raw after soaking and sprouting in form of salads but most of them require cooking to improve digestibility, palatability and keeping quality. Two commercially available pearl millet varieties such as Kalukombu (K) & Maharashtra Rabi Bajra (MRB) were subjected to various processing methods [milling, wet and dry heat treatments (Pressure cooking, boiling and roasting) and germination] commonly adopted practices in Indian households and was studied for its influence on the physico-chemical properties, nutrient composition, antinutrient profile as well as influence of cooking medium which is a potential source of iron/calcium contamination on total iron and calcium was explored. Total sugar and total soluble protein of the raw and processed flours were also analyzed. Buffer-soluble proteins were extracted from the raw and processed flours and separated by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE). Page 85

3 Table 4.1. Proximate Composition of the Two Pearl Millet Varieties (per 100g) Proximate composition Reported values* Analyzed values Kalukombu MRB Moisture (g) Proteins (Nx6.25) (g) Fat (g) Ash (g) Iron (mg) Calcium (mg) Phosphorus (mg) Zinc (mg) Copper (mg) Manganese (mg) Magnesium (mg) Sodium (mg) Potassium (mg) * National Institute of Nutrition (NIN), Hyderabad, MRB Maharashtra Rabi Bajra. The proximate compositions of the two pearl millet varieties (K and MRB) were determined and compared with the reported values (Table 4.1). K variety contained 9.3% protein, 4.8% fat and 2.0% ash. Comparatively, MRB contained higher protein (10.2%), fat (5.4%) and lower ash (1.5%) content. However, these Page 86

4 values were similar to the reported values. The highest content of iron (6.4%) was found in MRB while K had high phosphorus content (327.8%). Values for minerals like calcium, zinc, copper and manganese were found to be similar for both varieties. However, values for iron, zinc, manganese, magnesium, sodium and potassium content were lower than the reported values. Table 4.2. Bulk Density, Water and Oil Holding Capacity of Pearl Millet as Influenced by Processing Processing Bulk Density (ml/g) Oil Holding Capacity (g/g) Water Holding Capacity (g/g) K MRB K MRB K MRB WF (Raw) 1.68 a ± a ± a ± a ± a ± a ± 0.05 SRF 1.84 ab ± ab ± a ± a ± a ± a ± 0.08 BRF 1.93 b ± c ± b ± b ± d ± d ± 0.05 Boiling 1.75 a ± a ± a ± a ± b ± b ± 0.17 PC 1.75 a ± a ± a ± a ± c ± c ± 0.30 Roasting 1.98 bc ± bc ± a ± a ± a ± a ± 0.17 G 2.08 c ± c ± a ± a ± a ± a ± 0.11 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly (P 0.05) different based on Tukey s test. K Kalukombu, MRB Maharashtra Rabi Bajra, WF whole flour, SRF semi refined flour, BRF bran rich fraction, PC pressure cooking, G - germination Page 87

5 Knowledge of physical/functional properties and their dependence on the moisture content are useful for design and development of any processing methods and equipment. Functional properties such as bulk density, water and oil holding capacities of raw and processed pearl millet are presented in Table 4.2. Bulk density is an important parameter that determines the packaging requirement of a product (Chandi et al 2007). The bulk densities of processed pearl millet flours (WF < Boiling < PC < SRF < BRF < Roasting < Germination) were significantly (P 0:05) different from each other, with whole flour (WF) having the lowest density (about 1.66 ml/g). Germinated grains produced finer flours which led to its higher bulking capacity (2.08ml/g). Interactions of water and oil with proteins are very important in the food systems because of their effects on the flavor and texture of foods (Chandi et al 2007). The oil holding capacity (OHC) ranged from g/g for K and MRB. OHC of the flours were not significantly (P 0.05) affected by different heat treatments and germination respectively. However, bran rich fraction, a byproduct of milling, showed highest OHC (3.00 and 2.87g/g for K and MRB respectively). Water absorption capacities are related to the starch and protein contents and the particle size distribution of the ingredient. The water holding capacity (WHC) of a sample is an important determinant of stool-bulking effect, which is more related to the manner in which water is held, rather than to the absolute amount held. These properties are also important for stating the usefulness of the sample obtained as bulking, swelling and/or thickening agents in formulations or foods of relatively high water activity (Raghavendra et al 2004; De Escalada et al 2007). The water holding capacity (WHC) ranged from 0.88 to 3.13 g/g for both varieties. BRF (3.13 and 2.97 g/g for K and MRB respectively) and wet heat treatments such as boiling (2.25 and 1.70g/g for K Page 88

6 and MRB respectively) and pressure cooking (2.38 and 2.23 g/g for K and MRB respectively) presented highest WHC, whilst no significant (P 0:05) differences were found between whole flour; semi refined flour, roasted or germinated flour. The highest WHC of wet heat treated millet (boiling and pressure cooking respectively) might be attributed to its lowest density among the processed samples. Flours with lower bulk densities have larger surface area, polar groups, and uronic acid groups to the surrounding water, leading to an increase in water absorption or swelling volume (Bao et al 1994). However, this trend was not followed in case of BRF, which exhibited highest bulk density, WHC and OHC. In this study, variations in the functional properties (bulk density, WHC and OHC) amongst the processed millet flours indicated that all these properties were affected by different preparation methods. Page 89

7 Table 4.3a. Swelling Power of Pearl Millet as Influenced by Processing (g/g) Processing WF (Raw) SRF BRF Boling PC Roasting G 55 o C 65 o C 75 o C 85 o C 95 o C K MRB K MRB K MRB K MRB K MRB 2.77 a3 ± 2.94 b2 ± 3.95 b2 ± 3.31 c3 ± 4.71 d4 ± 4.91 e f3 ± 6.05 g4 ± 8.01 h i ± ± 0.02 ± b4 ± 2.95 a2 ± 3.37 d4 ± 3.14 c2 ± 4.47 e2 ± 4.93 f4 ± 5.54 d2 ± 6.69 g3 ± 8.76 i i ± 0.03 ± b5 ± 3.90 bc3 ± 3.72 a5 ± 3.94 c5 ± 4.63 d3 ± 4.80 e3 ± 6.06 f6 ± 6.32 g5 ± 8.57 i h ± 0.06 ± b6 ± 4.56 a4 ± 5.31 d6 ± 5.24 c e5 ± 5.86 fg6 ± 5.83 g4 ± 6.06 h4 ± 6.48 i j ± ± 0.00 ± a6 ± 5.14 b5 ± 5.54 d7 ± 5.61 e7 ± 5.70 f6 ± 5.11 b5 ± 5.95 g5 ± 5.19 c2 ± 6.70 h h ± 0.00 ± a2 ± 2.88 b2 ± 3.17 c3 ± 2.62 a d2 ± 4.72 e2 ± 6.02 g6 ± 5.73 f3 ± 7.73 h i ± ± 0.00 ± b1 ± 2.55 c a f4 ± 3.39 e1 ± 3.28 d1 ± 3.29 d1 ± 3.53 f1 ± 3.63 g f ± 0.04 ± ± 0.02 ± 0.00 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the row and means with different superscripts (1, 2, 3, 4, 5, 6,7) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF whole flour, SRF Semi refined flour, BRF bran rich fraction, PC pressure cooking, G - germination Page 90

8 Processing WF (Raw) b12 ± 0.00 Table 4.3b. Solubility of Pearl Millet as Influenced by Processing (g/g) 55 o C 65 o C 75 o C 85 o C 95 o C K MRB K MRB K MRB K MRB K MRB b2 ± b2 ± b1 ± a1 ± a1 ± a1 ± b2 ± a1 ± b1 ± 0.05 SRF d23 ± cd2 ± d5 ± cd1 ± bcd2 ± cd34 ± a1 ± bcd1 ± bc3 ± b1 ± 0.00 BRF ab123 ± c34 ± ab4 ± b1 ± a2 ± a3 ± ab4 ± b2 ± a4 ± a1 ± 0.00 Boiling d3 ± e4 ± b3 ± c1 ± a12 ± c5 ± a2 ± c2 ± a2 ± b1 ± 0.00 PC c123 ± d23 ± a12 ± b1 ± b2 ± c45 ± ab3 ± c2 ± a3 ± ab1 ± 0.00 Roasting bc12 ± bc1 ± ab1 ± f1 ± de2 ± bc2 ± cde3 ± bcd1 ± a2 ± e1 ± 0.00 G bc4 ± c5 ± ab6 ± a1 ± d3 ± d6 ± d5 ± d3 ± b c5 ± 0.03 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the row and means with different superscripts (1, 2, 3, 4, 5, 6,7) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF whole flour, SRF semi refined flour, BRF bran rich fraction, PC pressure cooking, G - germination c2 ± 0.01 Page 91

9 Swelling is a desirable physicochemical property making it useful in a range of foods. The effect of temperature on swelling power and solubility of raw and processed pearl millet is presented in table 4.3a&b. The swelling index is the measure of the ability of the flour to absorb water and swell. Swelling power of raw and processed flours increased with increase in temperature (55 C 95 C). Swelling power of all the flours significantly increased with increase in temperature. Whole flour had the highest value (about 9g/g), while germinated flour had the lowest (3.5g/g) at 95 o C. Whole flour of K and MRB showed 8.5% and 11.2% solubility at 55 o C. The solubility of the millet after germination profoundly increased to 34.2 and 42.6% in K and MRB respectively. Contrasting to swelling property, solubility of all the processed flours decreased with the increasing temperature. For example, solubility of processed flours was highest at 55 o C and 65 o C, as the temperature increased from 75 o C onwards the solubility of the flours decreased. For example, solubility of WF of K and MRB was and g/g at 55 o C which dipped to and 0.079g/g at 95 o C. Similar decreases were found in all processed flours except for germinated flour. Germination altered the swelling and solubility of pearl millet. In germinated flours, the decrease was not consistent, for instance, its solubility decreased at 65 o C, increased at temperatures 75 o C 85 o C and again decreased at 95 o C. The solubilization of the flours progressively deceased with increasing in swelling power. This result is different from that reported for corn (Yung et al 2006). Page 92

10 Table Proximate Composition of Pearl Millet as Influenced by Processing (Dry basis g/100g) Processing treatments Moisture Protein Fat Ash Kalukombu WF (Raw) 10.1 b ± ab ± bc ± a ± 0.35 SRF 10.8 b ± ab ± bc ± a ± 0.15 BRF 10.2 b ± ab ± d ± c ± 0.36 Boiling 7.2 a ± a ± c ± a ± 0.29 PC 7.5 a ± a ± b ± a ± 0.11 Roasting 6.6 a ± a ± b ± b ± 0.08 Germination 7.4 a ± b ± a ± b ± 0.04 Maharashtra Rabi Bajra WF (Raw) 9.6 c ± d ± c ± a ± 0.06 SRF 9.5 c ± d ± bc ± a ± 0.17 BRF 9.5 c ± bc ± bc ± b ± 0.09 Boiling 8.4 b ± cd ± a ± a ± 0.08 PC 7.0 a ± a ± c ± a ± 0.41 Roasting 6.4 a ± ab ± a ± a ± 0.09 Germination 9.4 c ± ab ± b ± a ± 0.02 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction. Page 93

11 The proximate composition of processed pearl millet is presented in Table 4.4. The moisture content of the flours were not altered by the process of milling or germination however, heat treatments significantly (P 0.05) reduced the moisture content in both varieties. The drying effect due to roasting caused a significant (P 0.05) moisture loss in the millet. Reduction in moisture is favorable as high moisture could possibly affect the storability of the product. The decrease in moisture content upon roasting was in accordance with the earlier report (Komeine et al 2008). Nonetheless the moisture content of all the flours, both raw and processed was below the maximum moisture content limit (13%) for pearl millet flour recommended by FAO/WHO (1995). This is the maximum allowable moisture content acceptable for pearl millet flour meant for human consumption. The mean protein content of K and MRB was 10.3% & 11.3% respectively. Processing did not affect the protein content of K variety grains however; significant (P 0.05) reduction due to processing (heat treatments, germination and bran rich fraction) was noticed in MRB. Reduction in wet heat treatmented (boiling/pressure cooking) or germinated millet could be attributed to leaching of water soluble and low molecular weight proteins into cooking/soaking water (Alka et al 1997; Habiba 2002) while, decrease during roasting could be due to destruction of amino acids as result of high temperature (Mauron 1982). The fat content of K & MRB was 5.3% & 6.0% respectively. The BRF of K variety (7.8%) retained significant (P 0.05) amount of fat content. The fat content of the heat treated millet reduced significantly (P 0.05), could be attributed to its diffusion into the cooking water or due to conversion of fat to fatty acid and glycerol which was further hydrolyzed to acetate at high temperature (King et al 1987). Ash represents the non-combustible fraction of the sample, i.e. minerals. The % ash content of K and Page 94

12 MRB was 2.2% and 1.6% respectively. A significant (P 0.05) amount of ash content was found in the bran rich fraction (3.6% in K & 3.9% in MRB). Roasting and germination significantly (P 0.05) reduced ash content in K variety grains.the fat and ash content reduced upon germination which could be due to losses of total soluble solids during soaking prior to germination (Wang et al 1997). Table 4.5. Mineral Content of Pearl Millet as Influenced by Processing (Dry Basis, mg/100g) Processing treatments Iron Calcium Phosphorus Kalukombu WF (Raw) 5.9 c ± a ± b ± SRF 4.6 b ± abc ± b ± 7.75 BRF 3.6 ab ± d ± b ± Boiling 3.2 a ± bc ± a ± 27.5 PC 3.3 a ± ab ± a ± 17.0 Roasting 3.2 a ± a ± b ± 6.0 Germination 6.7 d ± cd ± a ± 11.4 MRB WF (Raw) 7.1 c ± ab ± bc ± 36.9 SRF 6.5 bc ± b ± c ± 18.8 BRF 5.2 ab ± c ± d ± 31.4 Boiling 6.6 bc ± b ± abc ± 10.1 PC 7.3 c ± b ± ab ± 14.2 Roasting 4.0 a ± a ± abc ± 7.7 Germination 4.5 a ± b ± a ± 21.1 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking. Page 95

13 Table 4.5 depicts the mineral content of raw and processed pearl millet. The total iron content was 5.6 & 7.1 mg/100g for K & MRB respectively. Pearl millet subjected to milling and heat treatments significantly (P 0.05) lowered the iron content whilst, a significant (P 0.05) increase in the germinated K variety was noted which was in accordance with an earlier report on pearl millet (Sushma et al 2008). This increase could be attributed to the mineral contamination in the water used for soaking prior to germination. The total calcium content was 45.7 and 44.2 mg/100g in K and MRB respectively. The bran rich fraction contained significant (P 0.05) calcium levels. Pearl millet that was subjected to wet heat treatments or germination had high calcium content. The total phosphorus content was significantly (P 0.05) higher in K (364.0 mg %) compared to MRB (283.1 mg %). BRF of MRB contained significantly (P 0.05) higher phosphorus whilst, wet heat treated K variety had low phosphorus content. Similarly upon germination, the phosphorus content of the millet reduced significantly (P 0.05). Page 96

14 Table 4.6. Mineral Content of Processed Pearl Millet as Influence by Water Type (dry basis, mg/100g) Processing Iron Calcium Phosphorus TW DMW TW DMW TW DMW Kalukombu Boiling 3.8 a ± a ± a ± a ± a ± a ± 27.5 PC 4.6 b ± a ± a ± a ± b ± a ± 17.0 Germination 7.5 a ± a ± a ± a ± a ± a ±11.4 MRB Boiling 6.6 a ± a ± a ± a ± a ± a ± 10.1 PC 8.5 b ± a ± a ± a ± b ± a ± 14.2 Germination 5.8 b ± a ± a ± a ± a ± a ± 21.1 Values are mean ± SD (n = 4), means with different superscripts (a, b,) along the row are significantly (P 0.05) different, MRB Maharashtra rabi bajra, TW tap water, DMW demineralized water, PC Pressure cooking. Page 97

15 Mineral contamination has been reported during the postharvest treatments of cereals under uncontrolled conditions. Studies on the influence of iron pots and utensils on iron content in a variety of foods have consistently shown that foods cooked in iron pots and or with iron utensils have significantly higher total iron content than foods cooked with non-iron equipment (Prinsen et al 2003; Valerie et al 2011). Various food and water samples prepared in pots with and without an iron ingot revealed low bioavailability of contaminant iron, while, approximately 75% of the daily iron requirement was met by consuming 1L of lemon water prepared with an iron ingot. Its use may be a cheap and sustainable means of improving iron intake for those with iron-deficient diets (Christopher et al 2011). The introduction of iron pots for the preparation of food may be a promising innovative intervention for reducing iron deficiency and iron deficiency anemia (Prinsen et al 2003). Since this study was an approach towards household practice, it was interesting to find out the extent of contamination with extrinsic iron, calcium or potassium. Table 4.6 showed that pressure cooked or germinated K variety millet, prepared using tap water had 1.3 and 48.4 mg/100g more iron and phosphorus than those prepared using de-mineralized water. Similarly, pressure cooked MRB contained 1.2 and 45.7mg/100g more iron and phosphorus. However, germinated MRB contained 1.3 mg/100g of more iron. Millet cooked in tap water had around 1.3 mg/100 of iron and 47mg/100 of phosphorus; however calcium contamination due to use of tap water for processes like cooking or germination was not seen and also mineral transfer during the process of boiling the millet was not seen. Page 98

16 Table 4.7. Antinutrient Content of Pearl Millet as Influenced by Processing Processing treatments Phytin Phosphorus (mg/100g) Phytate (g/100g) Oxalate (mg/100g) Tannin (g/100g) Total Dietary fiber (g/100g) Kalukombu WF (Raw) c ± c ± b ± c ± a ± 1.80 SRF b ± b ± c ± b ± a ± 0.20 BRF d ± d ± d ± d ± b ± 5.00 Boiling b ± b ± a ± a ± a ± 0.30 PC b ± b ± b ± a ± a ± 0.70 Roasting a ± a ± b ± b ± a ± 0.30 Germination a ± a ± a ± e ± a ± 0.40 MRB WF (Raw) d ± cd ± b ± a ± a ± 1.80 SRF 93.2 ab ± ab ± c ± a ± a ± 0.40 BRF d ± d ± d ± b ± b ± 7.80 Boiling bc ± ab ± a ± a ± a ± 0.80 PC d ± d ± b ± a ± a ± 0.20 Roasting c ± bc ± b ± a ± a ± 1.00 Germination a ± a ± a ± a ± a ± 0.10 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB Maharashtra rabi bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking. Page 99

17 The antinutrient content of raw and processed pearl milled is presented in Table 4.7. The phytin P content of K variety was and mg/100g in MRB. The phytin P values were used to derive phytic acid content by assuming 28.20% of phosphorus is present in the molecule. Bran rich fraction (BRF) retained significant (P 0.05) phytin P and phytate content. As a result, semi refined flour contained lower values for phytin P and phytic acid. This result correlate well with an earlier report on pearl millet as well as wheat, stating that debranning to get refined flours considerably reduced the phytate content, signifying the distribution of phytate in the outer layers (Guansheng et al. 2005; Pawar et al. 2006). Pearl millet subjected to heat treatments and germination respectively decreased Phytin P and phytate content (P 0.05). The oxalate content was relatively low in pearl millet (31.6 and 36.0mg/g for K and MRB). Heat treatments (boiling) and germination respectively further reduced oxalate content to about 26% and 19% for K and MRB. Milling fractions such as SRF and BRF retained significant (P 0.05) amounts of oxalate (ranging from 41.8 to 65.8mg/100g). Nevertheless, the values for the processed millet (except for BRF) were below 50mg/100g, falling in the range of low oxalate foods. Similar amounts of oxalates were found to occur widely in many vegetables and fruits which do not pose a nutritional problem (Fasset, 1973; Ruth et al 2002). Tannins are polyphenolic compounds which bind to proteins, carbohydrates and minerals thereby reducing digestibility of these nutrients (Linda et al 2006). The tannin content of pearl millet varied between the two varieties (K-0.23% and MRB- 0.21% tannic acid equivalents). However, these values were within the range reported for millets (Pawar et al 1990; Ahmed et al 1996). The bran rich fraction of both varieties retained most of the tannin content (about 0.32%). This increase can be Page 100

18 attributed to concentration of tannins in the seed coat of the grain (Shivani et al 2004). Tannin content significantly (P 0.05) reduced upon heat treatments but results varied between the two varieties studied. Germination has been reported to reduce the tannin content and improve in vitro digestibility of proteins in legumes (Maeda et al, 1991). In contrast, germination of pearl millet significantly (P 0.05) increased the level of tannin in both varieties (from 0.21 to 0.28g/100 in MRB and 0.23 to 0.36g/100g in K). The total dietary fiber content of the whole flour was higher in K variety (13.3%) than MRB (11.91%). BRF had high total dietary fiber content (about 28.5g/100g). Heat treatments and germination did not did not bring about any significant changes in the total dietary fiber content of pearl millet. Page 101

19 Table 4.8. Total and Soluble Amylose Content of Pearl Millet as Influenced by Processing (g/100g) Processing Soluble Amylose Total Amylose K MRB K MRB WF (raw) 1.06 b ± c ± d ± a ± 0.07 SRF 2.15 e ± c ± f ± f ± 0.08 BRF 1.60 c ± b ± a ± d ± 0.03 Boiling 0.86 b ± a ± b ± e ± 0.02 PC 0.53 a ± a ± a ± f ± 0.14 Roasting 1.46 c ± d ± c ± c ± 0.05 Germination 1.92 d ± c ± e ± b ± 0.06 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d, f) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking. Starch consists of two polydispersed α -D-glucan components, amylose and amylopectin. Amylose is linear (α-d-[1-4]) or slightly branched and when dispersed in water forms gels. The formation of a gel or paste, which is a determinant of food texture, depends not only on the starch concentration but also the amount of amylose and amylopectin leached from the granule and the heating conditions such as temperature, time, heating velocity and shear stress (Miles et al 1985). Starch is further classified based on the amylose content, as nonwaxy (17.0 to 31.9%), low amylose (7.8 to 16.0%) and waxy type (0 to 3.5%) (Nakamura et al 1995). The total Page 102

20 amylose content of defatted K (3.47g/100g) was higher than MRB (2.89g/100g) (Table 4.8). Of this, 1.72% amylose was soluble in MRB while only 1.02% was soluble in K. Based on the classification of starch as reported by Nakamura et al. the starch in pearl millet can be classified as waxy. All the processing treatments increased total amylose content in K variety with highest increase seen in pressure cooked (4.93%) millet followed by boiled (4.52%) millet. However, in MRB, only semi refined flour (4.29%) had the highest total amylose content followed by germination (4.12%). All the processing treatments, except for wet heat treatment, increased solubility of amylose content. Highest soluble amylose content was seen in SRF (2.15%) of K variety followed by roasted MRB (1.91%). Table 4.9. The Total Dietary Fiber Content and its Fractions of Pearl Millet as Influenced by Processing (g/100g) Processing Insoluble Dietary Fiber Soluble Dietary Fiber Total Dietary Fiber K MRB K MRB K MRB WF (raw) 12.6 a ± a ± a ± a ± a ± a ± 1.8 SRF 8.6 a ± a ± a ± a ± a ± a ± 0.4 BRF 27.1 b ± b ± a ± b ± b ± b ± 7.8 Boiling 11.9 a ± a ± a ± a ± a ± a ± 0.8 PC 12.0 a ± a ± a ± a ± a ± a ± 0.2 Roasting 11.6 a ± a ± a ± a ± a ± a ± 1.0 Germination 12.2 a ± a ± a ± a ± a ± a ± 0.1 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking. Page 103

21 The influence of common household processing methods on the dietary fiber composition of pearl millet is depicted in Table 4.9. The total dietary fiber content of the whole flour was higher in K variety (13.3%) than MRB (11.91%). The process of partial removal of bran by sieving to obtain semi refined flour resulted in a significant amount of dietary fiber (10.6% - MRB & 9.2% - K). Dehusking has been reported to decrease dietary fiber content in pulses (Ramulu et al 1997). The bran rich fraction, a byproduct of flour milling contained around 29% of total dietary fiber of which around 1.5% was soluble and 27% was insoluble fraction. By virtue of its high fiber content, the bran rich fraction can be used as a novel source of dietary fiber. Semi refined flour of pearl millet can also be used in bakery products as it will contribute to both the texture and fiber content of the products. From the nutritional point of view, data on dietary fiber content of processed millet is of importance, because millets are never eaten raw. In this study, wet and dry heat treatment of the millets did not considerably change the insoluble and soluble dietary fiber content. Although, boiling, pressure cooking and roasting increased the SDF in MRB, it was statistically not significant. Changes in dietary fiber composition of processed cereal and pulses have been reported where increase in TDF content could be due to formation of resistant starch (Ramulu et al 1997). Germination is an inexpensive technique for improving the nutritional quality of millet seeds. The total dietary fiber content of germinated millet was 9.68% in MRB and 13.4% in K variety. The results indicate that germination did not alter the total dietary fiber and its fractions. Studies have indicated that germination has a significant impact on the dietary fiber content. In legumes, germination increased the dietary fiber content, while another study reported a decrease in dietary fiber content Page 104

22 due to germination, which could be partly attributed to the conversion of complex carbohydrates into simpler molecules by the action of hydrolyzing enzymes (Mahadevamma et al 2003; Mathers 1989). Table Total Sugar Content of Pearl Millet as Influenced by Processing Processing Kalukombu Total sugars (g/100g) MRB WF (Raw) 0.10 ab ± a ± 0.00 Semi Refined Flour 0.11 abc ± c ± 0.01 Bran Rich Fraction 0.19 d ± d ± 0.02 Boiling 0.09 a ± b ± 0.01 Pressure Cooking 0.14 c ± b ± 0.00 Roasting 0.12 bc ± bc ± 0.00 Germination 0.35 e ± e ± 0.01 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05); MRB Maharashtra Rabi Bajra, Page 105

23 The total sugar content in the whole flour of K and MRB was 0.10% and 0.06% (Table 4.10). Overall, the milling fractions showed an increase in the total sugar content for both varieties. Semi refining of MRB (0.15%) showed a substantial increase in the total sugar content while; the same did not alter the sugar content of K variety (0.11%). A three-fold increase in the total sugar content was noticed in the germinated sample (about 0.37%) followed by 0.20% in BRF of both varieties. Degradation of starch in grains during germination led to the increase in small dextrin and fermentable sugar (Wijngaard et al 2005). Wet and heat treatments such as boiling, pressure cooking and roasting respectively caused a marginal increase in the total sugar content of both varieties compared to whole flour (raw). Table Total Soluble Protein Content of Pearl Millet as Influenced by Processing Processing Total soluble proteins (g/100g) K MRB WF (Raw) 2.63 b ± c ± 0.30 Semi Refined Flour 2.48 b ± c ± 0.41 Bran Rich Fraction 2.83 b ± b ± 0.41 Boiling 0.92 a ± a ± 0.35 Pressure Cooking 1.03 a ± a ± 0.10 Roasting 1.29 a ± a ± 0.39 Germination 2.76 b ± c ± 0.34 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05); K Kalukombu, MRB Maharashtra Rabi Bajra. Page 106

24 Soluble protein content of raw and processed pearl millet ranged from 0.92 to 2.83% in K variety and 1.48 to 3.75% in MRB (Table 4.11). Overall, both varieties showed similar response to milling, heat treatments and germination. All the heat treated samples (boiled, pressure cooked and roasted respectively) showed a significant and sharp decline in the total soluble protein content however, semi refining or germination did not alter the total soluble protein content in both varieties. MWM Molecular weight marker, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction. Figure 4.1. Effect of Milling on Protein Profile of Pearl Millet Page 107

25 MWM Molecular weight marker, PC Pressure cooking Figure 4.2: Effect of Heat Treatment on Protein Profile of Pearl Millet MWM Molecular weight marker. Figure 4.3: Effect of Germination on Protein Profile of Pearl Millet Page 108

26 The SDS PAGE was performed on the soluble protein extracts of two pearl millet varieties (K and MRB) that were subjected to various processing treatments. Both varieties showed similar electrophoretic patterns. The milling fractions were classified as whole flour (WF), semi refined flour (SRF) and bran rich fraction (BRF). The main protein bands at 38, 30 and 23 kda along with some minor sub units of 66.2 and 45.0 kda were found in the milling fractions of K and MRB (Fig. 4.1). Protein bands of 66.2 and 45.0 kda were prominent in all the heat treated millet samples and low molecular weight protein bands were sparse in these samples. Further wet heat treatment resulted in an enhanced appearance of 45, 42 and 36 kda in K and 35 to 38 kda in MRB that was subjected to boiling (Fig. 4.2). Pearl millet subjected to pressure cooking showed three characteristic bands of 40, 27 and 19kDa, While, expression of only low molecular weight bands were found in MRB grains. In roasted grains, the most prominent band was seen at 36 kda. Further, expression of low molecular weight bands was more prominent in K grains compared to MRB. In general heat treatments caused shearing of protein, identified as a streak in the gel. Upon germination a number of minor bands were concentrated between and kda (Fig. 4.3). Biochemical as well as physical changes occur in millets during germination. Functional proteins are synthesized in millet under a stress circumstance, such as disease, physical or chemical stress. The stress plays an important role in germination in the course of physiological response and functional protein synthesis (Jingjun et al 2008). Summing up, the results of the present investigation demonstrated a wide variation in the nutrient composition of raw and processed pearl millet. MRB contained higher protein and fat, while K variety exhibited higher ash reflecting its Page 109

27 high mineral content. The bran rich fraction (BRF), a by product of milling, retained significant amounts of fat, ash as well as antinutrients. Semi refining of pearl millet flour displayed desirable nutritional qualities of both whole and refined flour. Heat treatments and germination respectively reduced proteins, fat and ash content. Functional properties varied with the processing methods with germination showing higher bulk density. Variations in the total mineral content due to processing were seen. For example, milling and heat treatment lowered iron, while the same was high in the germinated millet. Calcium increased with the processing methods applied while phosphorus reduced due to heat treatments and germination respectively. Page 110

28 CHAPTER 2 STUDIES ON ISOLATED STARCH FROM PEARL MILLET Starch is the chief dietary source of carbohydrates and is the most abundant storage polysaccharide in plants. It is present in high amounts in roots, tubers, cereal grains and legumes (Eerlingen et al 1995). Starches have been used in the food industry for variety of applications. The range of food products utilizing starch in one form or another are soup, stew, gravy, pie filling, sauce or custard. Starch contributes greatly to the textural properties of various foods and has many industrial applications as a thickener, colloidal, stabilizer, gelling agent, bulking agent, water retention agent and adhesive (Singh et al 2003). Starch is a major component of pearl millet. In different pearl millet genotypes the starch content of the grain varied from 62.8 to 70.5 % (Taylor 2004). Attempts to isolate starch from pearl millet have been previously reported (Hadimani et al 2001; Adelaide et al 1980; Hoover et al 1996). Most of the researchers used random mating bulk populations of pearl millet or cultivars grown in crop research institutes for their research work. However, reports on isolated starch from local or commercial varieties are scanty. Starch from the two commercially available pearl millet varieties were isolated (K and MRB) and evaluate for its functional properties, proximate composition, pasting and X-ray diffraction. The gelatinized pearl millet starch was analyzed for nutritionally important starch fractions [rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS)] as described by Englyst et al (1992). Page 111

29 Table Chemical Composition of Corn and Pearl Millet Starches (g/100g) Corn Kalukombu MRB Yield a ± b ± 0.45 Moisture 9.62 a ± b ± b ± 0.20 Proteins 0.19 a ± b ± b ± 0.01 Fat 0.00 a ± b ± b ± 0.02 Ash a ± a ± a ± 0.00 Total Amylose 16.8 c ± a ± b ± 0.16 The value represents the mean of three determinations, on whole flour basis Nitrogen x 6.25 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra rabi bajra. Corn starch (control) K starch MRB starch Figure 4.4. Pictures of Corn and Pearl Millet Starches Page 112

30 The chemical composition of isolated starch from pearl millet is presented in Table The results revealed that MRB variety contained 29.4% starch which was higher than K variety (24.5%) on the whole flour basis. However these values were low when compared with the reported value (Wankhede et al 1990; Hoover et al 1996). The variations in the yield of starch from pearl millet may be due to isolation and purification methods adopted by the researcher. The moisture content of pearl millet starch was approximately 12% which was higher than corn starch (9.62%). The amount and distribution of water within starch granules is important in relation to the physical properties and chemical reaction of starch (Kokini et al 1992). The reported moisture content of pearl millet starch was 10.18% while for cocoyam Starch; it ranged from 9.4 to 17.3% which was considered to be within the acceptable range (Wankhede et al 1990; Horsfall et al 2009). The protein content of pearl millet starches (0.55 and 0.53% in K and MRB) was higher than corn starch (0.19%) and were in the range reported by researchers (Wankhede et al 1990; Adelaide et al 1980). The fat content of pearl millet starches was 0.37 and 0.38% for K and MRB. These values were less than that reported ( %) (Hoover et al 1996). The amylose content of the starch was approximately 14.5%. According to reported values (Badi et al 1976) pearl millet starch contains 17% amylose. Page 113

31 Table 4.13a. Water Holding Capacity, Oil Holding Capacity and Bulk Density of Pearl Millet and Corn Starches. Isolated Starch WHC (g/g) OHC (g/g) Bulk Density (ml/g) Corn (C) 0.41 a ± c ± a ± 0.05 Kalukombu 0.48 b ± a ± a ± 0.05 MRB 0.44 ab ± b ± a ± 0.05 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra Rabi Bajra. 4.13b. Swelling Power and Solubility of Pearl Millet and Corn Starches (g/g). Starch 55 o C 65 o C 75 o C 85 o C 95 o C Corn (Control) K MRB SP 1.80 a ± b ± c ± d ± d ± 0.04 Solubility a ± e ± b ± c ± d ± 0.0 SP 2.02 a ± b ± c ± d ± e ± 0.03 Solubility a ± c ± b ± b ± b ± 0.0 SP 2.77 a ± b ± c ± d ± e ± 0.04 Solubility a ± d ± e ± b ± c ± 0.0 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), K Kalukombu, MRB Maharashtra rabi bajra, SP Swelling power. Page 114

32 K-Kalukombu, MRB- Maharashtra rabi bajra Figure 4.5. Swelling Power and Solubility of Pearl Millet and Corn Starches The ability to hold water is an important functional attribute of all flours and starches used in food preparations such as custard, dough etc. The observed water holding capacity (WHC) of the starches was lower than that reported for pearl millet starch (Adelaide et al 1980). The ability of food materials to absorb water is sometimes attributed to its proteins content (Kinsella 1976). The observed WHC of starches studied cannot, however, be attributed to the protein content since pearl millet starch in particular has low protein. Oil holding capacity (OHC) is useful in structure interaction in food especially in flavor retention, improvement of palatability and extension of shelf life particularly in bakery or meat products (Adebewale et al 2004). The range of OHC of the starch samples ( ml/g) showed that corn Page 115

33 starch (1.90ml/g) had highest OHC followed by MRB (1.27ml/g) and K (0.73ml/g) (Table 4.13a). The higher OHC of the millet flour could be due to its higher fat contents, which can entrap more oil. Basically, the mechanism of OAC is mainly due to the physical entrapment of oil by capillary attraction (Kinsella 1976). Swelling and solubility of starches provides indication of noncovalent bonding between molecules within starch molecule (Horsfall et al 2009). Swelling and solubility of pearl millet and corn starches were determined over a range of temperatures (55 95 o C). Swelling power of pearl millet and corn starches (Table 4.13b & Fig 4.5) showed the amount of water absorbed significantly (P 0.05) increased with increase in temperature (55 95 o C). Swelling behavior of starch is mainly due to swelling of amylopectin (Tsai et al 1997). Starch from K and MRB had significantly (P 0.05) higher swelling power than corn starch. The swelling power has been shown to be influenced by the amylose/amylopectin ratio and by the characteristics of amylose and amylopectin in terms of molecular weight distribution, degree of branching, length of branches and conformation of the molecules (Hoover et al 2002). The solubility pattern was determined in conjunction with swelling power (Table 4.13b & Fig 4.5). Solubility patterns did not basically follow swelling patterns. Pearl millet and corn starches had higher solubility at 65 o C. From 65 to 95 o C, all the starch samples were less soluble in water. As the swelling of starch increased with increase in temperature, its solubility decreased. Similar trend was also seen in pearl millet raw and processed flours. Page 116

34 Page 117

35 Figure 4.6. X Ray Deffractograms of Pearl Millet and Corn Starches and Pearl Millet Flours Starch is a semi crystalline polymer with low and imperfect crystallinity. The crystal structures in native starches are formed by packing of hexagonal arrays of amylopectin in helical coils. The amylopectin side chains, organized in double helices and constituting the crystalline fraction of starch, are considered as mesogens, attached to the backbone through amorphous and flexible spacer units. The length of these amylopectin double helices are related to the crystalline morphology of starch (short double helices related to A type and long double helices to B type) (Zobel et al 1988; Amparo et al 2007). Native starch granules display quite a complex structure with several levels of organization. Starch constituting two main polymers (essentially linear amylose and branched amylopectin) is distributed in amorphous Page 118

36 and semi crystalline concentric shells. (Amparo et al 2007). Heat and humidity involved in many processing conditions results in the disruption of starch s structure, a phenomenon known as gelatinization. Gelatinization involves loss of granular and crystalline structures by heating with water and often including other plasticizers or modifying polymers (Vermeylen et al 2006). Information regarding starch granule s crystalline properties can be acquired by X ray diffraction studies by exposing the starch samples to X ray beam. Starch can be classified as A, B and C forms. In the native granular forms, the A pattern is associated mainly with cereal starches, while the B form is usually obtained from tuber starches. The C pattern is a mixture of both A and B types, but also occurs naturally, e.g. smooth-seeded pea starch and various bean starches (Norman et al 1998). The X ray diffraction patterns of corn and isolated pearl millet starches as well as whole flour of pearl millet are presented in Fig The results revealed that pearl millet starches showed X ray patterns similar to corn starch (control). Corn starch exhibited sharper peaks at 2θ values of 14.69, and with an unresolved doublet at 2θ of Similarly, K variety showed peaks at 2θ values 14.92, 17.8 and while MRB showed peaks at 15.3, and The diffraction pattern of corn and pearl millet starches was similar to any other unprocessed cereal starch indicating its semi crystalline nature. The whole flour of K exhibited diffraction patterns similar to its isolated starch but for a slight shift in the 2θ values (from to 14.92, to and 18.2 to 17.8 respectively). Whole flour and isolated starch from MRB exhibited similar diffraction patters. However, the intensity of the peaks for the starch was comparatively higher than that of the respective whole flour samples. The X ray diffractograms mainly represent the type of the starch present in the sample. Apart from starch which is the Page 119

37 major component of the whole flour, it also contains other components such as proteins, dietary fiber, fat etc. The presence of these components may slightly interfere with the diffraction pattern of the samples. Hence, slightly sharper peaks for the starch samples may be expected. It was observed from the diffractograms that these samples exhibited A type diffraction pattern similar to any other cereal starches (Norman et al 1998). The sharp peaks in an X ray diffractogram are directly correlated to crystalline region and the diffused peaks to amorphous region of the samples (Usha et al 2011). In the present study, all the three starches exhibited semi crystalline type. Table Nutritionally Important Starch Fractions of Corn and Pearl Millet Starches (g/100g). Starch TS RDS SDS RS SDI RAG Corn (C) 20.2 a ± a ± a ± b ± a ± a ± 0.30 K 22.5 b ± b ± b ± ab ± a ± b ± 0.34 MRB 21.5 ab ± c ± a ± a ± b ± c ± 0.20 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05). K Kalukombu, MRB Maharashtra rabi bajra, TS total starch, RDS rapidly digestible starch, SDS slowly digestible starch, RS resistant starch, RAG readily available glucose. Page 120

38 Nutritionally important starch fractions of starch from two pearl millet varieties and corn (control) is presented in table Gelatinization is an important step in starch processing; hence all the starch samples were gelatinized prior to analysis (sample to water ratio of 1:4) and expressed on an as eaten basis. Starch is the main carbohydrate in human nutrition and is chiefly divided into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). RDS and SDS in pearl millet starches were higher than corn starch (control). However RS content was higher in corn (2.8) and K (2.2) while lower in MRB (1.4). The lower RS content of these gelatinized starches was apparently due to the elimination of structural obstruction to amylase hydrolysis during the process of starch isolation (Perera et al 2010). Also during the process of gelatinization which involves uptake of water and heat by starch granules lead to the disruption of the crystalline structure and consequently increased accessibility of glucose chains to amylolytic enzymes (Sadequr et al 2007). The pearl millet starches had similar total starch (TS) content which was higher than corn starch. A measure of the relative rate of starch digestion is given by starch digestion index (SDI). In the present investigation, SDI ranged from 50 (K and corn) to 57 (MRB). Similar values were reported for freshly cooked spaghetti (52), millet (55) and lentils (44). The rate of starch digestion was comparatively low in the starch isolated from pearl millet and corn (control) may be due to the fact that the physical form of these starches which are millet based possibly render starch partly inaccessible to the digestive enzymes (Englyst et al, 1992). The simple in vitro measurement of RAG and SAG is of physiologic relevance and could serve as a tool for investigating the importance of the amount, type, and form of dietary Page 121

39 carbohydrates for health (Klaus et al, 1999). RAG values are those which represent the amount of glucose that can be expected to be rapidly available for absorption after a meal. It is a better indicator of blood glucose and insulin response as it includes RDS and FG (Free glucose). RAG ranged from 11.3 (Corn) to 13.6 (MRB). Similar RAG values were reported for instant potatoes, millet and spaghetti (approx. 13%) (Englyst et al 1992). Low RAG values could be attributed to the fact that time for gelatinization was too short for complete gelatinization of starch molecules and due to the dense matrix which hindered enzymatic hydrolysis of starch. In conclusion, the yield of starch from pearl millet was lower (about 26.5%) than that reported (Wankhede et al 1997; Hoover et al 1996). Water and oil holding capacity of pearl millet starched were higher that corn starch. Swelling power of corn and pearl millet starches increased with increase in the temperature (55 to 95 o C). However solubility patterns did not follow the trend set by swelling power. As the swelling power increased with temperature, its solubility decreased. X Ray diffraction of corn and pearl millet starches exhibited semi crystalline structure. Relatively sharper peaks exhibited in the starch compared to respective whole flour suggested the interference of other components such as proteins, fat etc. An invitro method for measuring nutritionally important starch fraction provided a means for predicting the rate and extent of digestion in the human digestion. Although RDS and SDS were lower than corn starch, RS and TS were relatively higher. Page 122

40 CHAPTER 3 ANTIOXIDANT COMPONENTS AND ACTIVITY OF PEARL MILLET AS INFLUENCED BY PROCESSING The term phytochemicals or plant chemicals refers to every naturally occurring chemical substance present in plants, which also has a potential for antioxidant activity. Antioxidants play an important role in the body defense system against reactive oxygen species (ROS), which are the harmful byproducts generated during normal cell aerobic respiration (Boxin et al 2002). In foods, antioxidants prevent undesirable changes in flavor and nutritional quality of a product (Zielinski et al 2000). Several methods have been developed to measure Antioxidant activity. Commonly used assays are reducing power assay (RPA), ferric reducing antioxidant power (FRAP) and DPPH free radical scavenging activity. There are two basic categories of antioxidants, namely, natural and synthetic. Examples of nature antioxidants are phenolic compounds (tocopherols, flavonoids, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll derivatives, amino acids, and amines), carotenoids and ascorbic acid. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are examples of commonly used synthetic antioxidants that have been in use since the beginning of this century. Restrictions on the use of these compounds, however, are being imposed because of their carcinogenicity (Velioglu et al 1998; Hudson 1990). Thus, natural antioxidants have gained considerable interest in recent years. Cereals and millets are the most commonly consumed food items in India. They contain wide range of phenolics which are good sources of natural antioxidants. Studies report that methanolic extracts from red sorghum showed higher antioxidant Page 123

41 activity and contain higher polyphenolic levels compared to rice, foxtail millet, prosomillet and barley (Youngmin et al 2007). Bran, a byproduct of milling has antioxidant potential due to phenolic acids such as p- coumaric acid and vanillic acids that are concentrated in the bran portion of cereal kernels. Antioxidant activity of five bran extracts exhibited appreciable levels of total phenolics, flavonoids and DPPH radical scavenging activities (Shahid et al 2007). Processing such as soaking and roasting have been shown to influence total phenolic, flavonoid and antioxidant contents in selected dry beans. Raw kodo millet and finger millet have higher DPPH radical scavenging activities. However cooking of these millets by roasting or boiling reduced their antioxidant activity (Prashant et al 2005). Millets contain phytic acid, tannins, phenols which can contribute to antioxidant activity important in health, ageing and metabolic diseases. Pearl millet (Pennisetum typhoideum) is the most widely grown type of millet. Nutritionally, pearl millet is superior to major cereals with reference to energy value, high quality proteins, fat and minerals such as calcium, iron, zinc. Besides, it is also a rich source of dietary fiber and micro nutrients (Anu Sehgal et al 2006; Malik et al 2002). While, extensive information is available on proximate composition and mineral accessibility, information on antioxidant activity and its influence on processing in pearl millet are scanty. Research on the effect of processing on retention of bioactive components with potential antioxidant activity is very important. The objective of this investigation was to evaluate the effect of various processing methods (milling, heat treatments and germination respectively) on antioxidant components as well as antioxidant activities of pearl millet extracts. The methanolic extracts of raw and processed pearl millet were analyzed for DPPH free Page 124

42 radical scavenging activity; reducing power assay (RPA) and ferric reducing antioxidant power (FRAP) assays respectively. The samples were also evaluated for tannin, phytic acid and flavonoid content and were correlated with the antioxidant activity assayed using three methods. Table Yield of Methanolic Extracts from Pearl Millet as Influenced by Processing (g/100g) Processing Kalukombu MRB Whole flour (Raw) 5.8 cd ± bc ± 0.87 Semi refined flour 4.8 bc ± abc ± 0.26 Bran rich fraction 2.1 a ± ab ± 0.15 Boiling 2.4 ab ± ab ± 0.14 Pressure cooking 2.2 a ± a ± 0.04 Roasting 3.0 ab ± ab ± 0.04 Germination 7.7 d ± c ± 0.34 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB - Maharashtra Rabi Bajra. Page 125

43 Antioxidants are difficult to extract due to differences in the active compounds. A wide variation is seen in detecting antioxidant components due to differences in the polarity of the extracting solvents. Methanol is a relatively polar organic solvent and appears to be efficient in extracting compounds such as phenolics, flavonoids, and other polar material from millets (Florence et al 2010; Singh et al 2002). Methanol was therefore selected as an extracting solvent in the present investigation. Table 4.15 exhibits the yield of methanolic extracts obtained from the raw and processed two pearl millet varieties. The K variety (5.8g/100g) had higher yield than MRB (4.4g/100g). Wide variations in the yield as a result of processing were seen. Lower yields were seen in the millet subjected to heat treatments (boiling, pressure cooking, roasting) as well as in the bran rich fraction while, the processes of germination facilitated maximum extraction in both varieties; however this increase was not statistically significant. Page 126

44 Table Effect of Processing on Antioxidant Components of Pearl millet. Processing treatments Tannins (g/100g) Phytic acid (g/100g) Flavonoids (mg/g) K MRB K MRB K MRB WF (Raw) 0.23 c ± a ± c ± cd ± ab ± a ± 0.02 SRF 0.21 b ± a ± b ± ab ± a ± a ± 0.02 BRF 0.31 d ± b ± d ± d ± a ± a ± 0.00 Bo 0.18 a ± a ± b ± ab ± abc ± a ± 0.00 PC 0.18 a ± a ± b ± d ± bc ± a ± 0.01 Ro 0.20 b ± a ± a ± bc ± c ± a ± 0.07 G 0.36 e ± a ± a ± a ± a ± a ± g tannic acid equivalents/100 of dry flour - mg rutin equivalents/g of extract. Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, Bo boiling, PC Pressure cooking, Ro roasting, G germination. Page 127

45 Millets contain phytates, phenols, tannins, trypsin inhibitors and dietary fiber which act as antinutrients by chelating minerals. Tannins are naturally occurring polyphenolic compounds linked to reduce protein digestibility by forming complexes with proteins and inhibiting enzymes (Aganga et al 2001). It is now established that phytates, phenols and tannins present in cereals are also good sources of natural antioxidants important in health, aging and metabolic diseases (Krings et al 2000). Phytic acid occurring in the grains acts as an antioxidant by the formation of chelates with prooxidant transition metals. Although, phytic acid is generally regarded as an antinutrient due to its mineral binding activity it is known to reduce the risk for colon and breast cancer in animals (Graf et al 1990). Table 4.16 exhibits tannin, phytic acid and flavonoid content of raw and processed pearl millet. The tannin content of K and MRB expressed as tannic acid equivalents was 23 and 21g/100g of dry flour. Semi refining reduced the level of tannins in the SRF thus increasing its levels in BRF suggesting its localization in the outer hulls of the grain. Millet subjected to various heat treatments lead to considerable reduction in tannin levels only in the K variety. On the other hand, the increase in the tannin content of the germinated flour extract in both varieties was pronounced, however, statistically not significant for MRB variety. The phytic acid content was highest in K (78g/100g) compared to MRB (57g/100g). Semi refining significantly reduced the corresponding values to 66 and 33g/100g for K and MRB respectively. They were mainly found concentrated in the bran rich fraction (99 and 61g/100g K and MRB respectively). Phytic acid content significantly (P 0.05) decreased due to heat treatments though maximum reduction was seen in the germinated millet. Page 128

46 The flavonoid content of raw and processed millet extract were expressed as mg rutin equivalents per gram of the extract (10mg/ml). The flavonoid content was high in K (27mg/g) than MRB (21mg/g). The milling fractions (SRF and BRF) irrespective of varietal differences, showed a reduction in the flavonoid content, nonetheless, the decrease was not statistically significant. Unlike tannins and phytic acid, the flavonoid levels considerably increased due to heat treatments (boiling, pressure cooking, roasting) while, germination did not alter the flavonoid content of pearl millet. Page 129

47 Table Effect of Processing on the Radical Scavenging Activity of Pearl Millet Extracts by DPPH Method Processing 200µg 400µg 600µg K MRB K MRB K MRB WF (Raw) 22 a12 ± ab1 ± b12 ± b1 ± b1 ± b1 ± 7.7 SRF 16 a1 ± bc1 ± b1 ± de1 ± cd1 ± e2 ± 3.6 BRF 19 a12 ± a1 ± c12 ± b1 ± d1 ± c1 ± 2.9 Boiling 26 a2 ± a1 ± b2 ± b1 ± c23 ± b1 ± 6.0 PC 24 a12 ± a1 ± b2 ± b1 ± c23 ± b1 ± 0.6 Roasting 23 a12 ± a1 ± b2 ± b1 ± c3 ± c2 ± 4.3 Germination 16 a12 ± a1 ± a1 ± b1 ± b1 ± b1 ± 3.8 Concentration of the extracts 10mg/ml, Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the row are significantly different (P 0.05), Values are mean ± SD (n = 4), Means with different superscripts (1, 2, 3) along the column are significantly different(p 0.05), K - Kalukombu, MRB - Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking. Page 130

48 Bars with different superscripts (a, b, c, d) are significantly different (P 0.05), WF whole flour, SRF semi refined flour, BRF Bran rich fraction, Bo boiling, PC pressure cooking, Ro roasting, G germination. Figure 4.7: Effect of Processing on the Radical Scavenging Activity of Pearl Millet Extracts by DPPH Method Table 4.17 and Figure 4.7 summarize data on DPPH radical scavenging activity of raw and processed pearl millet. In DPPH assay, the color stable DPPH radical is reduced in the presence of an antioxidant which donates hydrogen to non radical DPPH H (Sanna et al 2006). DPPH free radical scavenging activity was studied at three concentrations (200 µg, 400 µg and 600 µg). Radical scavenging activity varied with the processing methods used and was concentration dependent. Page 131

49 The greatest activity was obtained at a higher concentration of 600µg in the raw and processed flour extracts. For example, the antioxidant activity of K and MRB which was 22% and 31% at 200µg considerably increased to 42% and 40% respectively at 600µg. Heat treatments such as boiling; roasting and pressure cooking exhibited significantly higher antioxidant activity as compared to the raw flour. Similar findings were reported for little millet where roasting of the millet enhanced its radical scavenging activity (95.5%), compared to germinated (91.7%) and steamed (93.4%) millet (Pradeep et al 2011). In contrast, cooked peppers showed a marked reduction in the radical scavenging activity when cooked for 30 minutes in boiling water. This may be due to leaching of antioxidant compound from the pepper into cooking was during the prolonged exposure to water and heat (Ai Mey Chuah et al 2008). It was noteworthy that significantly high radical scavenging activity in heat treated millet had the lowest yield, whilst, germinated millet extracts which showed lowest activity had highest yield. Page 132

50 Table Effect of Processing on the Ferric Reducing Power of Pearl Millet Extracts at Various Concentrations Processing 200µg 400µg 600µg K MRB K MRB K MRB WF (Raw) 0.07 a12 ± a1 ± c23 ± b12 ± d1 ± d3 ± 0.03 SRF 0.06 a1 ± a1 ± b1 ± b1 ± c1 ± c123 ± 0.02 BRF 0.10 b2 ± a1 ± e3 ± c12 ± f2 ± d1 ± 0.05 Boiling 0.10 a2 ± a1 ± b23 ± b2 ± d2 ± c23 ± 0.03 PC 0.08 a12 ± a1 ± b2 ± b12 ± d1 ± c12 ± 0.03 Roasting 0.09 a2 ± a1 ± c23 ± b12 ± e12 ± d23 ± 0.01 G 0.07 a12 ± a1 ± b2 ± b2 ± c1 ± c23 ± 0.02 Concentration of the extracts 10mg/ml, Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d) along the row are significantly different (P 0.05), Means with different superscripts (1, 2, 3) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, WF Whole flour, SRF Semi refined flour, BRF Bran rich fraction, PC Pressure cooking, G germination. Page 133

51 Different superscripts (a, b, c) on each bars are significantly (P 0.05) different (n = 6), WF whole flour, SRF semi refined flour, BRF bran rich fraction, Bo boiling, PC pressure cooking, Ro roasting, G germination. Figure 4.8: Effect of Processing on the Ferric Reducing Power of Pearl Millet Extracts at Various Concentrations The ferric reducing power of raw and processed millet is presented in Table 4.18 and Fig 4.8. It was observed that the results followed a similar trend of DPPH free radical scavenging activity. The reducing power assay of methanolic extracts of the two pearl millet varieties differed between the processing treatments employed. Raw and processed millet extracts exhibited lower reducing power at 200 µg, nevertheless, it increased with the concentration (600µg). The reducing power of K and MRB flour extracts at 200µg concentration were A 700 = 0.07 and 0.06 respectively. The corresponding values increased to A 700 = 0.31 and 0.32 respectively at 600 µg. The Page 134

52 respective bran rich fraction, roasted and boiled millet extracts of K variety exhibited an increase in the absorbance (A 700 = 0.34, 0.39 and 0.47, respectively) compared to the raw millet extract (A 700 = 0.31 ) while, processing did not alter the antioxidant activity (reducing power) of MRB. Table Effect of Processing on FRAP of Two Pearl Millet Varieties Processing Kalukombu MRB Whole flour (Raw) 2.24 ab ± ab ± 0.21 Semi refined flour 2.89 b ± a ± 0.20 Bran rich fraction 2.08 ab ± ab ± 0.09 Boiling 2.18 ab ± ab ± 0.07 Pressure cooking 1.54 a ± ab ± 0.00 Roasting 2.58 b ± b ± 0.12 Germination 2.69 b ± a ± 0.23 Values are mean ± SD (n = 6), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB - Maharashtra Rabi Bajra. Page 135

53 Ferric reducing antioxidant power (FRAP) assay is a novel method for assessing antioxidant power where the ferric reducing ability of sample extract is tested. Ferric to ferrous ion reduction at low ph causes a colored ferroustripyridyltriazine complex to form. FRAP values are obtained by comparing the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions in known concentration (Iris et al 1996). In this study, FRAP was calculated using the equation Y = x, where x is the OD of the sample. The ferric reducing ability of the extracts was higher in K (2.24) followed by MRB (1.85) (Table 4.19). Pressure cooked K variety and germinated MRB exhibited lowest activity, although the decrease was not statistically significant. Overall, the processing methods employed in this study did not cause any significant changes in the activity (FRAP) of the millet. Table Correlations of Yield and Antioxidant Components with Antioxidant Activity of Two Pearl Millet Varieties Pearl millet Yield Phytic acid Tannins Flavonoids Kalukombu DPPH 0.629** ** 0.712** Reducing power * FRAP 0.478* MRB DPPH Reducing power FRAP * correlation is significant at the 0.05 level (2 tailed); ** correlation is significant at the 0.01 level (2 tailed), MRB Maharashtra rabi bajra, FRAP Ferric reducing antioxidant power. Page 136

54 The relationship of antioxidant activity with antioxidant components as well as the yield of methanolic extracts of raw and processed pearl millet is presented in Table There was variation in the antioxidant activity analyzed by different assays and antioxidant components in K and MRB varieties. The results indicated significant negative correlations between methanolic extract (r = 0.629, P 0.01) as well as tannin content (r = , P 0.01) while, a positive relationship between flavonoid content (r = 0.712, P 0.01) with DPPH free radical scavenging activity. There was a strong positive relationship between reducing power and flavonoid content (r = 0.456, P 0.05) and FRAP with methanolic extracts (r = 0.478, P 0.05). These results suggest that the DPPH radical scavenging activity and reducing power assay in K variety was largely due to the presence of flavonoids. However, in MRB, antioxidant components such as phytic acid, tannin and flavonoids were poorly correlated with antioxidant activity as determined by three methods suggesting that these components did not contribute to the antioxidant activity of MRB variety. In conclusion the antioxidant activity of pearl millet varied with the processing methods and between the two varieties (K and MRB) studied. K variety had higher content of antioxidant components reflecting its higher antioxidant capacity, compared to MRB. BRF showed high antioxidant activity in terms of RPA which was due to tannin, phytic acid and flavonoid levels. The millet subjected to various heat treatments exhibited higher antioxidant activity (DPPH scavenging activity and RPA) mainly due to flavonoid content. It was worthy of notice that significantly high radical scavenging activity in heat treated millet had the lowest yield, whilst germinated millet which showed lowest activity had highest yield. Page 137

55 CHAPTER 4 STUDIES ON NUTRIENT DIGESTIBILITY (IN-VITRO) OF PEARL MILLET Pearl millet, a lesser known and underutilized crop can be grown at low maintenance cost, is relatively a cheaper source of nutrients and staple for population below poverty line for economic reasons. It has the distinct advantage of being a drought-resistant crop and hence acts as a principle source of energy, protein, fat and minerals for poor people living in these regions. However, it has some limitations, due to the presence of antinutritional factors such as phytate, tannins or dietary fiber. These compounds are known to interfere with mineral bioavailability, carbohydrate and protein digestibility (Fasasi, 2009; Anu Sehgal et al 2006; Malik et al 2002). The bioavailability of minerals from foods is defined as the proportion of the minerals that can be absorbed and utilized within the body (Lestienne et al 2005). Poor absorption of minerals leads to mineral deficiency resulting in conditions like anemia. The high prevalence of iron deficiencies in developing countries has several adverse effects on the population particularly on women and children (Zimmermann et al 2007). Carbohydrate digestibility in small intestine is influenced by the food form (physical form, particle size), type of preparation (cooking method and processing), type of starch (amylose or amylopectin) and presence of antinutrients, transit time, and amount of fiber, fat, and proteins (Julia et al 2007). For nutritional purposes, Englyst and Cummings (1993) classified the starch in foods as rapidly-digestible starch (RDS), slowly-digestible starch (SDS) and resistant starch (RS). RDS causes a rapid increase in blood glucose level after ingestion; SDS is digested slowly but Page 138

56 completely in the human small intestine and RS is a portion of starch that cannot be digested in the small intestine, but may be fermented in the large intestine (Englyst et al 1992). Protein digestibility is essentially a measure of the susceptibility of protein to proteolysis. A protein with high digestibility is potentially of better nutritional value than one with low digestibility because it provides more amino acids for absorption on proteolysis. Exogenous (interaction of proteins with non-protein components like polyphenols, non-starch polysaccharides, starch, tannins, dietary fiber, phytates and lipids.) and endogenous factors (changes within the proteins themselves) contribute to poor digestibility of proteins. During the process of milling and cooking, proteins interact with non-protein components and the proteins themselves thereby affecting their digestibility (Duodu et al 2003). Studies indicate that dietary fiber and tannins contribute to lower nutritional value of dietary proteins with soluble dietary fiber playing a major role in reducing its in vitro digestibility. In beans, soluble dietary fiber plays a more important role than insoluble dietary fiber in reducing protein digestibility (Joe Hughes, 1996). The purpose of the present study was to analyze mineral bioaccessibility, nutritionally important starch fractions and invitro protein digestibility of two pearl millet varieties as influenced by processing. Page 139

57 Table In-Vitro Bioaccessible Iron Content of Pearl Millet as Influenced by Processing Processing Kalukombu MRB Mg/100g % Mg/100g % Whole Flour 0.16 a ± c ± SRF 0.20 ab ± c ± BRF 0.16 a ± a ± Boiling 0.24 b ± b ± PC 0.33 c ± b ± Roasting 0.33 c ± b ± Germination 0.21 b ± c ± Values are mean ± SD (n = 6), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB Maharashtra Rabi Bajra, SRF semi refined flour, BRF bran rich fraction, PC pressure cooking. Table 4.21 represents data on bioaccessible iron content of pearl millet and its impact on processing. The bioaccessible iron content of K was 0.16 mg/100g while that of MRB was 0.44 mg/100g. Milling (SRF and BRF) did not alter the bioaccessible iron content of pearl millet, except for bran rich fraction (BRF) of MRB, where a two fold decrease was observed. On the other hand, heat treatments enhanced iron bioaccessibility which was evident only in K variety. The bioaccessible Page 140

58 iron content of K (0.16mg/100g) was increased to 0.24, 0.33 and 0.33 due to boiling, pressure cooking and roasting respectively, while germination caused a minimal increase of 0.21mg/100g. Although, bioaccessible iron content in MRB (0.44mg/100g) was the highest, it subsequently decreased as a result of processing with lowest decrease in the bran rich fraction followed by those subjected to heat treatments. Table In Vitro Bioaccessible Calcium Content of Processed Pearl Millet Processing Kalukombu MRB Mg/100g % Mg/100g % Whole Flour 30.1 b ± c ± SRF 22.4 a ± b ± BRF 20.8 a ± a ± Boiling 34.5 c ± c ± PC b ± c ± Roasting 35.9 c ± b ± Germination 35.7 c ± c ± Values are mean ± SD (n = 6), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), MRB Maharashtra Rabi Bajra, SRF semi refined flour, BRF bran rich fraction, PC pressure cooking Page 141

59 The bioaccessible calcium content of K and MRB was 30.1 and 34.5mg/100g respectively (Table 4.22). K variety subjected to heat treatments such as boiling or roasting increased the bioaccessible calcium content to 34.5 and 35.9 mg/100g respectively. Germination also brought about a similar increase in the calcium bioaccessibility (35.7mg/100g) of K variety. However, in case of MRB, neither of the processing treatments caused any improvement in the calcium bioaccessibility. Further, significant (P 0.05) decrease in the bioaccessible calcium of BRF and roasted millet was observed. Similar to iron bioaccessibility, processing did not enhance bioaccessible calcium content of MRB. Cooking is reported to modify seed composition and lowering of antinutritional factors in turn influencing dialysability of iron and calcium. (Sreeramaiah et al 2007). This was evident in case of calcium bioaccessibility studied in both varieties. Calcium in foods exists mainly as complexes with other factors (phytates, oxalates, fibre, lactate, fatty acids) from which the calcium must be released to be absorbed and heat treatments aided this process. Cooking effectively improved bioaccessible calcium while the same was less effective in improving bioaccessible iron content suggesting that calcium and iron combined in a meal may decrease iron bioaccessibility. Page 142

60 Table Effect of Processing on Bioaccessible Iron and Calcium as Influenced by Tap / De Mineralized Water (mg/100g) Processing Bioaccessible Iron Bioaccessible Calcium TW DMW TW DMW Kalukombu Boiling 0.25 a ± 0.01 (7) 0.24 a ± 0.02 (8) 35.5 a ± 2.61 (67) 35.9 a ± 2.99 (66) PC 0.35 a ± 0.02 (8) 0.33 a ± 0.03 (10) 32.7 a ± 2.41 (70) 30.0 a ± 1.61 (64) G 0.25 a ± 0.03 (3) 0.21 a ± 0.03 (3) 35.9 a ± (66) 35.7 a ± 2.51 (66) MRB Boiling 0.36 a ± 0.03 (6) 0.37 a ± 0.01 (5) 36.7 a ± 2.63 (69) 34.5 a ± 1.56 (65) PC 0.33 a ± 0.02 (4) 0.35 a ± 0.04 (5) 36.6 a ± 4.02 (70) 39.5 a ± 4.89 (75) G 0.46 a ± 0.05 (8) 0.47 a ± 0.01 (10) 40.8 a ± 4.93 (76) 38.0 a ± 3.37 (71) Values are mean ± SD (n = 4), means with different superscripts (a, b,) along the row are significantly (P 0.05) different, values in the parenthesis are % bioaccessible iron/calcium, MRB Maharashtra rabi bajra, TW tap water, DMW demineralized water, PC Pressure cooking, G Germination. Studies have report that contamination of iron originating from milling equipment (mills equipped with iron-containing grindstones) led to a considerable increase in bioaccessible iron, hence extrinsic iron supplied to food products by the milling equipment could play a role in iron intake in developing countries (Valerie et al 2011). Cooking water often contributes to mineral contamination influencing its bioaccessibility. In view of this, tap water (previously passed through a water purifier) Page 143

61 and de mineralized water was used in processing treatments like boiling, pressure cooking and germination respectively to see its effect on bioaccessible iron and calcium content (Table 4.23). Although iron and calcium bioaccessibility of millet processed using tap water was higher than de-mineralized water, the difference was not significant. Table Molar Ratios of Pearl Millet as Influenced by Processing Processing [Phytate]/[Calcium] 1 [Phytate]/[Iron] 2 [Calcium]/[Phytate] 3 [Oxalate]/[Calcium] 4 K MRB K MRB K MRB K MRB WF (Raw) SRF BRF Boiling PC Roasting Germination Recommended critical value of [Phytate]/[Calcium] is 0.24, (Morris et al 1985) 2 Recommended critical value for [Phytate]/[Iron] is 1, (Hallberg et al 1989) 3 Recommended critical value for [Calcium]/[Phytate] is 6.1, (Oladimeji et al 2000) 4 Recommended critical value for [Oxalate]/[Calcium] is 1.0, (Davis, 1979) K Kalukombu, MRB Maharashtra Rabi Bajra, WF Whole flour, SRF semi refined flour, BRF bran rich fraction, PC pressure cooking. Page 144

62 The bioavailability of minerals depends on the amount of antinutrients and the ratio of antinutrients/minerals. These ratios are of significance when they are greater than the recommended critical values. In such cases antinutrients (phytates and oxalates) have a potential to complex with minerals (calcium/iron) thus impairing its absorption. The molar rations for [Phytate/Calcium], [Phytate/Iron], [Calcium]/[Phytate] and [Oxalate]/[Calcium] of raw and processed millet were calculated to study the effect of oxalate and phytate contents on the bioaccessibility of calcium and iron (Table 4.24). The phytate/calcium and phytate/iron molar ratios have to be lower than 0.24 and 1 respectively (Morris et al 1985; Hallberg et al 1989). Although, K and MRB exhibited molar ratios for phytate/calcium and phytate/iron above the reference value, it decreased with the processing. Furthermore, [Calcium]/[Phytate] and [Oxalate]/[Calcium] molar ratios of raw and processed millet varieties were well below the recommended critical values of 1 and 6.1 respectively (Davis 1979; Oladimeji et al 2000). Oxalate forms insoluble calcium salt with a 1:1 molar stoichiometry in the intestine thus, rendering calcium unavailable for absorption. In view of this, the importance of the oxalate content in limiting total dietary Calcium availability is of significance only when the calculated molar ratios are greater than the recommended critical values. Since under these circumstances the oxalate has the potential to complex not only the Calcium contained in the plant but also that derived from other food sources (Davies 1979). Page 145

63 Table Nutritionally Important Starch Fractions of Pearl Millet as Influenced by Processing Varieties Processing Treatments Dry Matter (%) Starch Fractions (g/100g as-eaten basis) RDS SDS RS TS K WF (Raw) 86.8 b ± a ± a ± b ± a ± 0.69 SRF 87.1 b ± b ± b ± c ± b ± 0.35 Boiling 83.9 b ± d ± d ± ab ± d ± 0.75 PC 84.7 b ± b ± c ± ab ± b ± 0.51 Roasting 86.6 b ± c ± c ± a ± b ± 0.55 Germination 71.8 a ± d ± c ± a ± c ± 1.01 MRB WF (Raw) 85.9 b ± a ± a ± b ± a ± 0.35 SRF 86.3 b ± b ± a ± c ± b ± 0.74 Boiling 85.2 b ± c ± c ± ab ± c ± 0.58 PC 85.4 b ± a ± b ± ab ± a ± 0.55 Roasting 85.3 b ± c ± d ± a ± c ± 0.38 Germination 74.4 a ± d ± e ± ab ± d ± 1.48 Values are means ± SD (n=4), Means with different superscripts (a, b, c, d) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra, SRF semi refine flour, PC pressure cooking, RDS rapidly digestible starch, SDS slowly digestible starch, RS resistant starch, TS total starch, Page 146

64 Starch is divided into 3 types considering the rate and extent of starch digestion which includes rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS). Food processing is known to render some portion of starch resistant to enzyme hydrolysis thereby influencing the starch fractions of food (Englyst et al 1992). Changes in the starch fractions of pearl millet as a result of processing are presented in Table The raw and processed pearl millet flours were gelatinized (flour to water ratio 1:4) prior to analysis and expressed on an as eaten basis. K and MRB contained approximately 22% TS, 11% RDS, 7% SDS and 5% RS content. In general, the processing methods employed in this investigation rendered the millet more digestible in comparison to the raw flour and also correspondingly increased RS content. Milling, an important step in millet processing has shown its influence on starch digestibility. The flour of pearl millet is coarse and contains antinutrients like phytic acid, tannins and oxalates that form complexes with nutrients leading to marked reductions in their digestibility (Arora et al 2003). Semi refining of K variety drastically increased RS content. Although, bran which is a physical barrier to enzymes and an important intrinsic factor limiting starch hydrolysis was partially reduced or eliminated as a result of semi refining, it was high in RS content. A combination of semi refining and gelatinization did not help in improving starch digestibility. The high RS content could be explained by the fact that the time taken for the process of gelatinization was perhaps too short to allow the starch to completely gelatinize. Starch molecules present were entrapped inside the granules which were not easily accessible to hydrolyzing enzymes (RS2) (Liljeberg, 2002). The seed coat/hull is an initial protective barrier for grains and plays an important role in starch digestibility. The removal of hull renders the starch more Page 147

65 accessible to hydrolytic enzymes leading to better digestibility. But in this study, the slow rate of starch digestion in semi refined flour might be due to the presence of the left out bran which led to the entrapment of starch in fibrous thick-walled cells (Richard et al 2009, Asp 1994). Millet subject to heat treatments (boiling, pressure cooking, roasting) reduced the TS content. While, boiling or pressure cooking of pearl millet did not alter the RS content a significant (P 0.05) drop in the millet subjected to roasting was noticed. The process of cooking destroys the semi crystalline structure of raw starch granules resulting in the loss of SDS and an increase of RDS. SDS content of food is important as it gets hydrolyzed to glucose slowly yet completely in the small intestine, thereby lowering the risk of chronic diseases such as cardiovascular diseases, obesity and diabetes (Sureeporn et al 2010; Cousin et al 1996; Richard et al 2009). In this study increase in the SDS and RDS content of heat treated millet in comparison to the raw counterpart was seen. Increased starch digestibility after boiling/pressure cooking has been reported in peas and beans (Richard et al 2009). The high RDS and SDS and low RS content could be attributed due to heat treatments/gelatinization and dispersion of the starch molecules rendering them more susceptible to attack by starch hydrolyzing enzymes. A combination of high temperature and moisture caused greater disruption of the cellular structure, increasing exposure of starch to amylolytic enzymes which lead to a decrease in RS in boiled or pressure cooked millet. Germination is a process which converts complex nutrients into simple ones thereby improving its digestibility. Germination led to the significant increase in RDS and SDS in both the varieties. A marked increase in the TS content as a result of germination was seen. Degradation of starch in grains during germination led to the Page 148

66 increase in dextrin and fermentable sugar. This change produces a special sweet flavor in germinated brown rice (Kayahara, 2004). In germinated cereal grains, hydrolytic enzymes are activated and decompose starch, non-starch polysaccharides, and amino acids. The decomposition of high molecular weight polymers during germination leads to the generation of bio-functional substances, and improvements in organoleptic qualities due to the softening of texture and increase of flavor in cereal grains (Kayahara 2004; Banchuen et al 2009). Table Starch Digestion Index & Rapidly Available Glucose Values of Processed Pearl Millet (g/100g) Processing Starch Digestion Index Rapidly Available Glucose K MRB K MRB Whole flour (raw) 49 bc ± b ± a ± a ± 0.7 Semi refined flour 43 a ± a ± b ± c ± 0.5 Boiling 47 abc ± b ± c ± e ± 0.3 Pressure cooking 46 ab ± a ± b ± b ± 0.3 Roasting 51 c ± b ± b ± d ± 0.6 Germination 52 c ± b ± d ± f ± 0.4 Values are mean ± SD (n = 4), Means with different superscripts (a, b, c, d, f) along the column are significantly different (P 0.05), K Kalukombu, MRB Maharashtra Rabi Bajra. Page 149

67 Changes in the SDI and RAG content of processed pearl millet is presented in Table Overall, processing treatments significantly (P 0.05) affected the SDI and RAG content of pearl millet. The changes in the SDI and RAG content of two pearl millet varieties, however, were different. Semi refining decreased SDI considerably. For example, raw flour of K and MRB varieties had SDI of 49 and 50 respectively; corresponding values were decrease to 43 and 41 after semi refining the whole flour. Heat treatments and germination did not cause any significant changes in the SDI of pearl millet. Rapidly Available Glucose (RAG) content is a major determinant of the magnitude of glycemic index. The % increase in RAG content for both the varieties varied with processing techniques used. The raw flour of K and MRB had RAG value of about 13%. The RAG content increased to about 27, 21, and 20% in germinated, boiled and roasted millet respectively. Medium increase was seen in semi refined flour and pressure cooked milled ranging from 14.6 to 17.5%. Page 150

68 Table Effect of Processing on % In Vitro Protein Digestibility of Pearl Millet Processing % In-Vitro Protein Digestibility Kalukombu MRB Whole Flour 45.5 ab ± a ± 7.9 Semi Refined Flour 54.8 abc ± a ± 7.9 Bran Rich Fraction 59.6 ac ± bd ± 8.9 Boiling 32.5 a ± ab ± 5.0 Pressure cooking 43.6 ab ± a ± 3.1 Roasting 65.8 c ± cd ± 1.4 Germination 88.2 d ± d ± 7.8 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra rabi bajra. Data in Table 4.27 indicated that protein digestibility of pearl millet was low (45.5% for K and 49.3% for MRB variety). The relatively low protein digestibility may be attributed to the influence of antinutrients such as enzyme inhibitors, lectins, phytates, tannins and dietary fiber which inhibits protein digestion and also due to presence of protein structures that resist digestion. Studies have demonstrated that high-tannin sorghum varieties formed indigestible protein tannin complexes which are a major limiting factor in protein utilization (Chibber et al 1980). Semi refining of the whole flour did not alter the protein digestibility, however, the BRF showed Page 151

69 higher %IVPD than whole flour and was comparable to that of wheat bran (69%) (Saunders et al 1972). Results on the effect of heat treatments like boiling, pressure cooking and roasting on IVPD significantly (P 0.05) varied between the two varieties. Wet heat treatments such as boiling or pressure cooking did not alter the protein digestibility of the millet which was in agreement with earlier reports on some cereals and legumes. Protein cross-linking mainly through disulphide bonding and reduced protein extractability in cooked samples appears to be the most important factor affecting protein digestibility in cooked cereals (Aisha et al 2004; Vijayakumari et al 2007; Singh et al 1981). In contrast, cooking improved %IVPD in foxtail, finger and common millet (Ravindran 1992). Nevertheless, roasting markedly improved IVPD of pearl millet from 45.5% to 65.8% in K variety and 49.3% to 75.4% in MRB suggesting that dry heat treatment is more effective in improving protein digestibility. The improvement as a result of dry heat treatment may be due to protein denaturation and/or decreasing resistance of protein to enzyme attack (Sathe et al 1982). Germination significantly (P 0.05) improved the protein digestibility in both varieties compared to the un-germinated millet (from 45.5% to 88.2% for K variety and 49.3% to 78.9% for MRB variety). These findings are in agreement with an earlier study on pearl millet (Anurag et al 1996). The increase in %IVPD can be attributed to an increase in soluble proteins, due to partial hydrolysis of storage proteins by endogenous proteases produced during the germination process. Such partially hydrolyzed storage proteins may be more easily available for pepsin digestion (Bhise et al 1988). Page 152

70 Table Association of % IVPD with Tannin and Dietary Fiber Content of Pearl Millet Dependent variable Independent variable Correlation coefficient Tannins ** % IVPD IDF SDF TDF ** Correlation is significant at 0.01 level (2 tailed), IDF Insoluble Dietary Fiber, SDF Soluble Dietary Fiber, TDF Total Dietary Fiber Tannins and dietary fiber are well known for their ability to bind and precipitate protein. A correlation study was carried out between tannins, TDF, IDF and SDF with %IVPD to ascertain whether protein digestibility of pearl millet was influenced by these factors (Table 4.28). A positive correlation, was found between % IVPD and tannin content (r = 0.605, P 0.01). For IDF, SDF and TDF the correlations were positive although not significant. In this study, protein digestibility of pearl millet showed a strong association with tannin levels. The low protein digestibility of pearl millet was not due to tannins or dietary fiber content. It could be due to other factors like interaction of proteins with non-protein components and the proteins themselves thereby affecting their digestibility (Duodu et al 2003; Abdalla et al 1997). Results from the present study shows that relatively bioaccessible iron and calcium content (in-vitro) was higher in MRB than K variety. Semi refining, heat Page 153

71 treatments or germination of pearl millet improved the bioaccessible iron and calcium in both varieties. Drastic improvement in bioaccessible iron content as a result of various processing methods was seen in MRB, while increase in bioaccessible calcium was similar in both varieties. Cooking effectively improved calcium accessibility while the same was less effective in improving bioaccessible iron suggesting that calcium and iron combined in a meal may decrease iron bioaccessibility. Use of tap/de mineralized water for boiling, pressure cooking or germination led to an increase in the bioaccessible iron and calcium content, nonetheless, this increase was not statistically significant. Changes in mineral and antinutrient content during processing led to significant variations in the antinutrient/mineral molar ratios which had a positive impact on the bioaccessible iron and calcium content. Milling fractions like WF, SRF and BRF exhibited higher RS values while a considerable decrease of the same due to heat treatment or germination was seen due to complete starch digestion. Lower in-vitro protein digestibility was seen in pearl millet. Milling, roasting and germination respectively were more effective in improving the protein digestibility of pearl millet. A positive correlation, was found between % IVPD and tannin content (r = 0.605, P 0.01). For IDF, SDF and TDF the correlations were positive but not significant. Page 154

72 CHAPTER 5 UTILIZATION OF PEARL MILLET IN FOOD PRODUCTS Pearl millet has a considerable potential to be used as foods and beverages. It can be used as cooked, whole, dehulled or ground flour or else as a grain like rice. The millet is mostly used as whole flour for traditional food preparation and hence confined to traditional consumers and to people of lower economic strata. The flour prepared out of pearl millet is coarse and has a grey to yellow color which imparts bitter taste (Olatungi et al 1982). Pearl millet is gluten-free and hence suitable for celiacs. Celiac disease is a permanent gluten intolerance elicited in the genetically susceptible subject after ingestion of gluten containing cereals (Laurin et al 2002). Celiac disease is a disorder of considerable and increasing importance in Western countries occurring in 1 of in the European population and in 1 of 111 of the US population (Green et al 2007; Joseph 1999). Celiac disease was recognized in northern India, primarily in children, since the 1960s (Walia et al 1966). Besides being rich in iron, calcium, zinc and high level of fat, it is nutritionally comparable and even superior to major cereals due to the energy and protein value (Fasasi 2009; Anu Sehgal et al 2006; Malik et al 2002). Owing to its superior nutritional quality, the aim of the present investigation was to explore the possibility of using pearl millet flour in food products. These products were analyzed for proximate composition, nutritionally important starch fraction, and mineral bioaccessibility. Sensory and shelf life studies were also carried out. Products like ladoo and burfi were stored in steel containers for 4 5 weeks while cookies were stored in PET PP and foil for 12 weeks. These products were analyzed for moisture Page 155

73 content, free fatty acids and peroxide value. Acceptability studies as affected by storage were also carried out. Table Proximate Composition of Breakfast Items Prepared from Pearl Millet (dry weight basis). Breakfast Moisture Protein Fat Ash Iron Calcium Phosphorus Items (g) (g) (g) (g) (mg) (mg) (mg) Dosa Rice (C) 56.9 b ± b ± a ± a ± a ± a ± a ± 2.6 K 51.6 a ± a ± b ± b ± b ± b ± c ± 28.0 MRB 51.6 a ± a ± ab ± b ± b ± b ± b ± 13.2 Roti Rice (C) 65.0 b ± b ± b ± a ± a ± a ± a ± 2.6 Ragi (C) 33.0 a ± b ± b ± b ± b ± c ± b ± 2.7 K 63.6 b ± a ± a ± c ± c ± b ± c ± 9.7 MRB 69.9 c ± b ± b ± c ± c ± b ± c ± 17.4 Puttu Rice (C) 61.5 b ± b ± b ± b ± a ± a ± a ± 4.1 Ragi (C) 28.6 a ± ab ± a ± a ± b ± c ± b ± 26.2 K 56.6 b ± a ± c ± c ± c ± b ± c ± 15.3 MRB 58.1 b ± a ± c ± a ± c ± b ± b ± 14.0 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), C Control, K Kalukombu, MRB Maharashtra Rabi bajra. Page 156

74 In order to evaluate the influence of pearl millet in product preparation, selected breakfast items typical of Indian cuisine such as dosa, roti and puttu were prepared and its nutritional profile was studied by comparing with traditionally prepared breakfast meals containing rice/ragi as major ingredients. The data indicated that incorporation of pearl millet in dosa significantly (P 0.05) lowered moisture and protein content, while, increased the fat content (Table 4.29). For instance, traditionally prepared rice dosa contained 56.9% and 7.8% of moisture and protein, the corresponding values decreased to about 51% and 5.7% respectively upon substitution with pearl millet, while the fat content increased from 5.0% to 6.5%. Addition of pearl millet in dosa greatly increased ash content and therefore rich in iron, calcium and phosphorus. Traditionally prepared rotis based of rice and ragi was used for comparison. The protein and fat content of MRB Roti was similar to that of rice and ragi roti (ranging from % and % respectively), although, a lower protein (6.6%) and fat (4.2%) content was seen in roti prepared using K variety. High ash and mineral content was seen in pearl millet rotis. However highest increase in calcium was seen in ragi roti. This is because ragi is a rich source of calcium than pearl millet and rice. Puttu prepared out of 100% pearl millet was compared with traditionally prepared rice and ragi puttu. The protein content of pearl millet puttu was significantly (P 0.05) lower than those of rice and ragi puttu. Significant (P 0.05) increase in the fat, ash and mineral content was seen in pearl millet puttu; however ragi puttu had the highest levels of calcium. Page 157

75 In general, the results indicated that breakfast items prepared by either partial or 100% substitution increased fat, ash and minerals like iron, calcium and phosphorus. The nutrient profile of breakfast items prepared out of K and MRB was similar. Table In-Vitro Bioaccessible Iron and Calcium Content of Breakfast Items Prepared from Pearl Millet (per 100g) Breakfast products Bioaccessible Iron Bioaccessible Calcium Mg/100g % Mg/100g % Dosa Rice (C) a ± a ± Kalukombu c ± c ± MRB b ± b ± Roti Rice (C) a ± a ± Ragi (C) c ± c ± Kalukombu b ± b ± MRB b ± b ± Puttu Rice (C) b ± a ± Ragi (C) a ± c ± Kalukombu bc ± b ± MRB bc ± b ± Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), C Control, MRB Maharashtra Rabi bajra. Page 158

76 The bioaccessible iron content of dosa, roti and puttu prepared from rice flour was 0.125, and mg/100g, respectively (Table 4.30). Whole or partial incorporation of pearl millet flour into these products brought about a significant (P 0.05) increase in the bioaccessible iron content, for example, the corresponding values increased to about 0.27, 0.33 and 0.61mg/100g respectively. However, these values were lower compared to ragi based roti and puttu. Ragi is a rich source of iron and calcium and hence it showed a positive influence on bioaccessible iron and calcium. Although bioaccessible iron content of pearl millet based products increased, its % bioaccessibility remained lower than rice products. The highest % bioaccessible iron content was registered for rice than pearl millet or ragi based products. The addition of pearl millet enhanced bioaccessible calcium content (mg/100 g) as well as % bioaccessibility in all the products. The % bioaccessibility of calcium was even higher than ragi based products. The highest increase in bioaccessible calcium was seen in roti followed by puttu prepared using pearl millet. Page 159

77 Table 4. 31: Nutritionally Important Starch Fractions (In-vitro) of Breakfast Items from Pearl Millet (g/100g) Breakfast Products Dry matter TS RDS SDS RS SDI RAG Dosa Rice (C) 43.2 a ± b ± c ± c ± a ± b ± c ± 0.09 Kalukombu 47.5 a ± b ± b ± b ± b ± a ± b ± 0.27 MRB 48.4 a ± a ± a ± a ± b ± a ± a ± 0.22 Roti Rice (C) 34.9 b ± a ± a ± a ± b ± a ± a ± 1.11 Ragi (C) 66.9 c ± b ± b ± b ± a ± c ± b ± 0.20 Kalukombu 36.4 b ± a ± a ± a ± b ± b ± a ± 0.20 MRB 30.3 a ± a ± a ± a ± b ± ab ± a ± 0.40 Puttu Rice (C) 38.5 a ± a ± a ± a ± b ± a ± a ± 0.63 Ragi (C) 73.9 b ± a ± b ± c ± a ± a ± b ± 0.23 Kalukombu 43.4 a ± c ± d ± b ± b ± a ± c ± 2.65 MRB 41.9 a ± b ± c ± a ± b ± a ± c ± 0.09 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra Rabi bajra, C Control, TS Total starch, RDS Rapidly digestible starch, SDS Slowly digestible starch, RS Resistant starch, SDI Starch digestion index, RAG Rapidly available glucose. Page 160

78 Nutritionally important starch fractions of breakfast products prepared from two pearl millet varieties (K and MRB) and rice/ragi (control) were analyzed in vitro and exhibited in Table Pearl millet was used in the preparation of roti, dosa and puttu. Dosa is a fermented breakfast item while puttu is a streamed product. Total starch content of dosa prepared out of rice and K variety (48%) were similar while MRB had low starch content (37.9%). Starch in rice dosa was rapidly available (25.8%) with low levels of SDS (19.6%) and RS (3.1%). Higher starch digestibility was found in rice dosa (control) as it was prepared with higher amounts of rice along with some minor ingredients. Replacement of rice with pearl millet reduced RDS and SDS to about 17.9% and 12% respectively, while, greatly increased RS to 14%. Significant reduction in RAG and SDI was also noticed. The rotis were prepared by adding warm water and a pinch of salt to the respective flour and mixed into dough, then flattened into round shaped roti and shallow fried ( o C) on a pan. Cereal and millet based rotis were used as controls. TS content of ragi roti (41.4%) was higher than rice (37.8%) and pearl millet roti (35%). Ragi roti had significantly (P 0.05) higher RDS (26.5%) and SDS (14.6%) content, where as rice and pearl millet rotis were low in RDS and SDS which of about 21% and 10.7% respectively. Ragi roti was found to contain minor amounts of RS (0.24%), whereas, higher RS was found in rice and pearl millet roti ranging from 3.6 to 4.5%. SDI and RAG values were higher for ragi roti and lower for rice and pearl millet roti respectively. Puttu is a steamed product served with desiccated coconut and sugar. Cereal (rice) and millet (ragi) based controls were used for comparison. Unlike dosa and roti, rice and ragi puttu were found to have low TS and RDS content. Compared to ragi Page 161

79 dosa or roti, puttu prepared out of ragi had considerable amount of RS (0.92%), yet, it was lower than rice and pearl millet based puttu. SDI was similar for all the products while RAG values ranged from 28.6% for rice to about 37% for pearl millet based products. Table Sensory Analysis of Breakfast Items Prepared from Pearl Millet Breakfast products Color Flavor Texture Taste Dosa Overall Acceptability Rice (C) 7.7 c ± b ± b ± b ± b ± 0.63 K 6.8 b ± a ± a ± a ± a ± 0.83 MRB 6.2 a ± a ± a ± a ± a ± 1.28 Puttu Rice (C) 7.6 b ± ab ± a ± b ± b ± 0.92 Ragi (C) 6.7 a ± b ± a ± b ± b ± 0.63 K 6.8 a ± ab ± a ± b ± ab ± 0.84 MRB 6.7 a ± a ± a ± a ± a ± 1.06 Roti Rice (C) 7.8 b ± b ± b ± ab ± b ± 0.83 Ragi (C) 6.8 a ± b ± b ± b ± ab ± 0.77 K 6.4 a ± ab ± ab ± ab ± ab ± 0.89 MRB 6.4 a ± a ± a ± a ± a ± 0.93 Means followed by different letters (a, b, c) in the same column differ significantly (P < 0.05), C Control, K Kalukombu, MRB Maharashtra Rabi bajra. Page 162

80 The sensory evaluation of breakfast products is presented in Table The sensory evaluation results for dosa prepared from rice and pearl millet respectively, revealed that the scores for parameters like color, flavor, texture and overall acceptability obtained for rice dosa were higher than that of pearl millet dosa. Based on the hedonic ratings, pearl millet dosa was less preferred by the panelists than rice dosa. The color score of pearl millet puttu was lower than rice puttu, while, no significant difference was found with the color scores of ragi puttu. Replacement with pearl millet did not affect the flavor, taste, texture and overall acceptability of puttu. Roti prepared from MRB had the lowest scores for all the parameters while; roti from K variety had scores similar to those prepared from rice or ragi. In a hedonic scale of 9 points, a score of 6 is considered a quality limit (Munoz et al 1992).The three different products prepared from rice, ragi and pearl millet respectively exhibited scores appreciably higher than the quality limit (6.2 to 7.9). Overall, pearl millet incorporation substantially affected the color of the products; however the scores were in the acceptable range. Page 163

81 Table Proximate Composition of Wheat flour (Control) and Pearl Millet Cakes Cake Moisture (g) Fat (g) Proteins (g) Ash (g) Iron (mg) Calcium (mg) Phosphorus (mg) Control 13.9 b ± a ± a ± a ± a ± b ± a ± 0.8 K a ± a ± b ± c ± c ± a ± b ± 0.6 MRB 13.3 b ± b ± c ± b ± b ± c ± c ± 2.0 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), K Kalukombu, MRB Maharashtra Rabi bajra. Cakes were baked by replacing refined wheat flour with 40% pearl millet and compared with cake prepared with 100% refined wheat flour. Data in Table 4.33 presents proximate composition of cake incorporated with 40% pearl millet (K and MRB). The fat content of the cake increased when fortified with 40% MRB. Since pearl millet is richer in protein content than refined wheat flour, incorporation of cakes with pearl millet enhanced protein content (increased from 5.9 (control) to 6.3 and 8.5 for K and MRB). The mineral content of cakes baked with 40% pearl millet was also higher. Page 164

82 Table Proximate Composition of Wheat flour (Control) and Pearl Millet Buns Bun Moisture (g) Fat (g) Proteins (g) Ash (g) Iron (mg) Calcium (mg) Phosphorus (mg) C 26.2 c ± b ± c ± a ± a ± a ± a ± 0.8 K 27.8 b ± a ± b ± b ± b ± b ± b ± 0.4 MRB 22.4 a ± c ± a ± b ± c ± c ± c ± 0.5 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), C Control, K Kalukombu, MRB Maharashtra Rabi bajra. Buns were prepared by incorporating 40% pearl millet (K and MRB) and compared with bun prepared using 100% refined wheat flour (Table 4.34). Buns fortified with Pearl millet contained fat and protein higher than control. Incorporation of pearl millet had higher levels of ash content thereby contributing to higher levels of mineral content. Minerals such as iron, calcium and phosphorus were considerably high in buns baked using 40% pearl millet than 100% refined wheat flour. Page 165

83 Table 4. 35a. Sensory Analysis of Wheat flour (Control) and Pearl Millet Cakes Cake Color Flavor Texture Taste Overall Acceptability Control 7.7 b ± b ± a ± b ± b ± 0.72 K (40%) 6.9 a ± b ± a ± ab ± b ± 0.82 MRB (40%) 6.7 a ± a ± a ± a ± a ± 0.84 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), Control 100% wheat flour, K Kalukombu (60:40 wheat flour: pearl millet flour), MRB Maharashtra Rabi bajra (60:40 Wheat flour: Pearl millet flour) Table 4. 35b: Sensory Analysis of Wheat flour (Control) and Pearl Millet Buns Bun Control K MRB Wt. of Bun (g) b ± a ± a ±0.95 Vol. of Bun (ml) 190 b ± a ± a ±2.9 Crust shape Normal Normal Normal Crumb grain Medium fine Medium fine Slightly soft Texture Soft Very soft Slightly soft Eating quality Typical Appealing wholesome taste Typical Means followed by different letters (a, b) in the same column differ significantly (P 0.05), K Kalukombu, MRB Maharashtra Rabi bajra. Page 166

84 As shown in Table 4.35a, the color score was higher for control cake (7.7) than 40% pearl millet cake (6.9 for K and 6.7 for MRB). However, these sensory scores were within the acceptable range. Flavor scores were similar for control and 40% K variety cakes (7.6 each) than those prepared with 40% MRB flour (6.8). Texture, taste and overall acceptability scores for control, 40% K and 40% MRB flour fortified cakes were similar and were markedly higher than 6, which are considered as the quality limit of a product. All the sensory scores given by the panelists revealed that the cakes prepared by fortifying with 40% K or MRB flour were acceptable. The data presented in Table 4.35b showed that incorporation of pearl millet flour (40%) decreased the weight and volume of buns significantly (P 0.05) from 63g to about 48g in weight and from 190ml to 140 ml in volume. The crust shape of control and pearl millet buns was normal, while crumb grain which was medium fine in case of control and K bun, was slightly soft in case of MRB bun. On the whole, the addition of pearl millet particularly K variety flour improved the eating quality of the bun and resulted in a wholesome appealing taste. Page 167

85 Table Effect of Storage of Bun at Room Temperature on Mould Growth. Control Bun Mould growth appearance First evidence of mould growth on 4 th day of storage Kalukombu First appearance of mould growth on 5 th day of storage MRB First appearance of mould growth on 3 rd day of storage MRB Maharashtra Rabi bajra Table 4.37: Effect of Storage of Cake at Room Temperature on Mould Growth Control Cake Mould growth appearance First appearance of mould growth on 13 th day of storage Kalukombu First appearance of mould growth on 12 th day of storage MRB First appearance of mould growth on 11 nt day of storage MRB Maharashtra Rabi bajra Buns and cakes baked using pearl millet flour was packed in airtight polythene covers and were placed in an area sterilized with alcohol. They were observed for mould growth and compared with the standard (conventionally prepared). Appearance of mould growth in conventionally prepared bun and cake was observed on 4 th and 13 th day of storage respectively (Table 4.36 & 4.37). While those prepared by addition of MRB (40%) had mould growth on 3 rd and 11 th day of storage and K variety (40%) showed mould growth on the 5th and 12 th day respectively. The appearance of mould Page 168

86 growth in standard and pearl millet flour (40%) buns and cakes was almost same and was storable up to about 3 4 days for buns and days for cake. Table 4. 38: Proximate Composition of Traditional Sweets Prepared from Pearl Millet. Sweets Moisture (g) Fat (g) Proteins (g) Ash (g) Iron (mg) Calcium (mg) Phosphorus (mg) Ladoo C 0.11 a ± b ± c ± b ± a ± b ± c ± 5.5 K 0.22 c ± a ± a ± a ± b ± a ± b ± 1.3 MRB 0.18 b ± c ± b ± b ± c ± c ± a ± 1.1 Burfi C 0.12 b ± c ± b ± b ± a ± c ± a ± 2.1 K 0.13 c ± b ± a ± a ± b ± b ± b ± 0.6 MRB 0.11 a ± a ± b ± a ± c ± a ± a ± 3.1 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), C control, K Kalukombu, MRB Maharashtra rabi bajra. Page 169

87 Indian traditional sweets such as ladoo and burfi were prepared using pearl millet flours (K and MRB) and compared with traditionally prepared sweets for its nutrient composition (Table 4.38). The moisture content of ladoo and burfi prepared from K was higher than the control. Traditionally prepared sweets had higher protein content while those prepared using pearl millet were comparatively low in protein. However, significantly higher iron content was found in pearl millet sweets than those prepared using gram flour. The higher values of iron in pearl millet sweets can be attributed to the higher iron content in the millet. Table 4. 39: Sensory Analysis of Traditional Sweets Prepared from Pearl Millet. Traditional sweets Color Flavor Texture Taste Overall Acceptability Ladoo Control 7.2 b ± a ± a ± a ± a ± 0.90 Kalukombu 6.5 ab ± b ± a ± b ± b ± 0.71 MRB 6.0 a ± a ± a ± a ± a ± 1.07 Burfi Control 7.1 b ± a ± a ± ab ± a ± 1.01 Kalukombu 6. 4 a ± a ± a ± b ±x a ± 0.70 MRB 6.2 a ± a ± a ± a ± a ± 0.76 Means followed by different letters (a, b, c) in the same column differ significantly (P < 0.05), MRB Maharashtra rabi bajra. Page 170

88 Ladoo and burfi are the two traditional sweets prepared using gram flour as a base ingredient. In this study these traditional sweets were prepared with pearl millet and compared with the traditionally prepared. The control burfi and ladoo had a high color score of 7.2 and 7.1 respectively (Table 4.39). While those prepared out of pearl millet had low scores ranging between The flavor, taste, texture and overall acceptability scores for ladoo and burfi made from K variety were higher than control and MRB. The scores for all the sensory parameters of ladoo and burfi made from gram flour and pearl millet were higher than 6, suggesting that these products were acceptable. Page 171

89 Table 4.40: Effect of Storage on Sensory Attributes of Pearl Millet Ladoo LADOO Color Flavor Texture Taste Overall Acceptability Control Week ab ± a ± ab ± ab ± a ± 0.90 Week b ± a ± ab ± c ± b ± 1.12 Week ab ± a ± b ± c ± b ± 0.72 Week ab ± a ± ab ± bc ± ab ± 0.63 Week a ± a ± a ± a ± a ± 1.68 Kalukombu Week a ± a ± a ± a ± a ± 0.71 Week a ± a ± a ± a ± a ± 1.00 Week a ± a ± a ± a ± a ± 0.68 Week a ± a ± a ± a ± a ± 1.02 Week a ± a ± a ± a ± a ± 0.73 MRB Week ab ± a ± a ± a ± a ± 1.07 Week b ± a ± a ± a ± a ± 0.98 Week a ± a ± a ± a ± a ± 0.73 Week ab ± a ± a ± a ± a ± 1.06 Week b ± a ± a ± a ± a ± 0.91 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra Rabi bajra. Page 172

90 Initially, the overall acceptability scores for of control, K and MRB ladoo was 6.2, 7.4 and 6.6 respectively (Table 4.40). K ladoo was preferred over control or MRB ladoo. Color is an important sensory attribute of a food product. The color scores for control ladoo (7.2) were higher than K (6.5) and MRB (6.0) on day 0. This indicated that the yellowish color of the control ladoo was preferred by the panelists as compared with the grayish color of the pearl millet ladoo. However, the color scores for K and MRB ladoo increased to 6.8 and 6.4 respectively over the storage period, indicating that the panelist liked the color of pearl millet ladoo after they got adapted to it over the storage period of 4 weeks. The scores for all the sensory parameters of control ladoo decreased from 2 nd week onwards, while the scores of pearl millet ladoo (K and MRB) remained constant until the end of the study period. In particular, sensory scores for flavor, texture, taste and overall acceptability showed no significant differences throughout the study period. Page 173

91 Table 4.41: Effect of Storage on Sensory Attributes of Pearl Millet Burfi BURFI Color Flavor Texture Taste Overall Acceptability Control Week a ± a ± a ± b ± ab ± 1.01 Week a ± a ± a ± b ± b ± 0.81 Week a ± a ± a ± b ± ab ± 0.91 Week a ± a ± a ± ab ± ab ± 0.90 Week a ± a ± a ± a ± a ± 1.71 Kalukombu Week ab ± bc ± a ± b ± abc ± 0.70 Week ab ± a ± a ± a ± a ± 0.89 Week a ± c ± a ± b ± b ± 0.74 Week ab ± ab ± a ± a ± abc ± 1.17 Week b ± c ± a ± b ± c ± 1.40 MRB Week a ± a ± a ± ab ± ab ± 0.76 Week a ± a ± b ± b ± b ± 0.91 Week a ± a ± ab ± ab ± ab ± 0.94 Week a ± a ± ab ± a ± ab ± 1.20 Week a ± a ± ab ± ab ± a ± 1.52 Means followed by different letters (a, b, c) in the same column differ significantly (P 0.05), MRB Maharashtra rabi bajra Page 174

92 Sensory evaluation of burfi as affected by storage is presented in Table Similar to ladoo, the color score of control burfi (7.1) was higher than K (6.4) and MRB (6.2). However, the scores for color increased for burfi prepared from K over the storage period of 4 weeks. The color scores of control burfi remained highest throughout the storage period, while, values for taste and overall acceptability decreased after 3 weeks of storage. Significantly, burfi made from pearl millet retained acceptability throughout the storage period of 4 weeks. Therefore it may be concluded that prolonged storage of pearl millet burfi may have induced desirable changes in taste, texture and flavor. Burfi from K was more favored than MRB. It was also noticed that scores for color, flavor, taste and overall acceptability of burfi from K was highest at the end of 4 th week compared to MRB. Page 175

93 Bars with different letters (a, b, c) differ significantly (P 0.05), C Control, K Kalukombu, MRB Maharashtra Rabi bajra. Figure 4.9: Moisture, Peroxide value and Free Fatty Acid Content of Ladoo and Burfi Prepared from Two Pearl Millet Varieties as Affected by Storage Page 176

94 Moisture content is an important determinant of the shelf life of a product. Migration of moisture produces changes in the texture, water activity and flavor of the product. The moisture content of the ladoo and burfi prepared from pearl millet was about 0.24% initially which increased to about 0.48% at the end of the storage period (Fig. 4.9). The moisture content of pearl millet-burfi was higher while that of ladoo was lower than their traditional counterparts. This increase did not affect the texture of pearl millet sweets at the end of the 5 th week of storage; however those prepared traditionally, were soggy at the end of 5 th week. The peroxide value (PV) of an oil or fat is used as an indicator of rancidity reactions that have occurred during storage. Peroxide value is the most widely used method for determining autoxidation (oxidative rancidity), by measuring the amount of iodine formed by the reaction of peroxides (formed in oil or fat) with iodide ion. The peroxide value is the number that express, in milliequivalents of active oxygen, the quantity of peroxide contained in 100g of fat. When fatty acids react with oxygen they become peroxy radical. Hydroperoxides are unstable intermediates formed during oxidation process. They further disintegrated to form a wide range of products that are also unstable, in turn undergo oxidation to form stable products most of which are responsible for disagreeable flavors associated with rancid oils (Eskin et al 2001). There was a significant difference in the peroxide values between control and pearl millet burfi/lodoo (Fig. 4.9). The peroxide value for traditional and pearl millet burfi and ladoo was 0.00% which increased to about 1.41%. The differences between the PV of traditional and pearl millet products although significant, were not large. Free fatty acid (FFA) is a measure of hydrolytic rancidity in fats. A large majority of fats are triglycerides which is a combination of glycerol with 3 fatty acids Page 177

95 attached. Hydrolysis of triglycerides, in presence of water causes cleavage of ester bonds that attach fatty acids residue to glycerol thus setting it free from glycerol. The detached fatty acids from triglycerides are called free fatty acids. Essentially how soon the triglycerides deteriorate, determines the shelf life of a product. A fatty acid radical has an unpaired electron. Therefore they are short lived and are highly reactive as they seek a partner for their unpaired electron. Pearl millet triglycerides contains about 74% unsaturated fatty acids (oleic, linolic and linolenic acids) and the remaining fraction is made up of saturated fatty acid residues (palmitic and steric acids). Pearl millet germ is proportionally larger than other cereal grains and has major fraction (88%) of lipids stored in the germ. The relatively high proportions of polyunsaturated fatty acids that constitute the triglycerides negatively affect the shelf life of pearl millet flour (Lai et al 1980; Taylor 2004). Hydrolysis of triglycerides and subsequent oxidation of the released de esterified unsaturated fatty acids occur during the ambient storing conditions. This hydrolysis is catalyzed by lipase enzyme. It is these chemical changes that are manifested as undesirable tasted and odors in millet flours that has been stored (Taylor 2004). In this study, the free fatty acid value is expressed as palmitic acid equivalents/100g of ladoo/burfi. FFA content increased progressively during storage of ladoo and burfi (Fig. 4.9). The free fatty acid levels increased more rapidly in the sweets prepared from pearl millet compared to those traditionally prepared. The free fatty acid for stored control, K and MRB ladoo increased from 0.63, 0.69 and 0.74 to 0.89, 1.15 and 1.13% while FFA for stored control, K and MRB burfi increased from 0.46, 0.78 and 0.8 to 1.09, 1.15 and 1.10% respectively within 5 weeks of storage. Page 178

96 This was due to entry of moisture as indicated by a marked increase of moisture in the sweets. However this increase did not affect the sensory quality of the products. The burfi and ladoo of pearl millet remained sensorialy acceptable during the entire one month storage without any adverse effect on their acceptability by consumers, whereas those of the traditional sweets, the overall acceptability scores decline after 3 weeks of storage. Pearl millet flour is not stable during storage, as it develops disagreeable sensory flavors. Hence, the millet flour was roasted prior to its use in these products. Roasting stabilized lipids by inhibiting lipase activity in the pearl millet. Pearl millet can be successfully used in preparation of sweets that is preferred by consumers. Table Proximate Composition of Refined Wheat Flour (control) and Pearl Millet Cookies Cookies Moisture (g %) Fat (g %) Proteins (g %) Ash (g %) Iron (mg %) Calcium (mg %) Phosphorus (mg %) Control 2.57 b ± a ± a ± a ± a ± a ± a ± 5.4 K 0.58 a ± a ± b ± c ± b ± b ± b ±10.2 MRB 0.34 a ± a ± b ± b ± b ± c ± b ±6.2 Means sharing the same superscripts are not significantly different from each other (Tukey s B significant difference test, P 0.05), K Kalukombu, MRB Maharashtra rabi bajra. Page 179

97 Data in Table 4.42 presents proximate composition and mineral content of the cookies prepared out of refined wheat flour and pearl millet. The moisture content of control cookies (100% refined wheat flour) was significantly (P 0.05) higher than pearl millet cookies. This difference can be attributed to the use of roasted millet flour. Higher moisture content of cookies results in a soggy and soft texture which is a major cause for lower consumer acceptability. Fat content remained similar for control and millet cookies. Cookies fortified with pearl millet was had significantly (P 0.05) higher protein, ash and minerals like iron, calcium and phosphorus. Approximately 2.7 fold increase in the iron content was observed in pearl millet cookies. Other minerals such as calcium and phosphorus increased by 1.6 and 2.2 times respectively in the pearl millet cookies. Table Physical Characteristics of Refined Wheat flour (Control) and Pearl millet Cookies. Parameters Control Kalukombu MRB SEM ± Diameter (mm) 88.0 b 87.0 a 86.7 a 86.7 Thickness (mm) 10.6 a 10.9 a 11.1 b 0.20 Spread ratio (D/T) 8.30 b 7.98 a 7.81 a 0.10 Spread factor b a a 0.50 Breaking strength (g) 1600 a 1650 b 1680 b 10 SEM Standard error of means at 15 degrees of freedom, mean in the same row followed by different superscripts differ significantly (P 0.05), MRB Maharashtra Rabi Bajra. Page 180

98 Physical characteristics of cookies such as diameter, thickness, spread ratio and breaking strength were determined and are presented in Table The data showed that the diameter of control, K and MRB cookies did not vary between each other. Thickness of cookies prepared out of wheat flour (control) and K where similar while that of MRB increased. Spread ratio is based on the values obtained for thickness and diameter of the cookies. The spread ratio of the cookies decreased from 8.30 to 7.98 and 7.81 for K and MRB cookies respectively. Similarly, the breaking strength values for control, K and MRB cookies were 1600, 1650 and 1680g respectively indicating that the texture of the pearl millet cookies did not vary between varieties. Pearl millet cookies had light and crisp texture. OQ overall quality, C Control, K Kalukombu, MRB Maharashtra Rabi bajra Figure Sensory Profile of Wheat flour (Control) and Pearl Millet Cookies Page 181

99 Research findings from a study on the influence of flour mixes on the quality of gluten-free biscuits indicated that a mixture containing millet flakes was one among the three best mixtures selected based on sensory data (Tilman et al 2003). This shows that millets could exert beneficial influence on the quality of biscuits. However, in a comparative study on sorghum cookies and pearl millet cookies, latter were dark, less gritty and more fragile than the former (Badi et al 1975). A point to be noted here is that there was a wide variation in the composition and relative proportion of ingredients of the products studied, which, in all probability, resulted in the diverse quality of the products. In the present study, control cookies had off-white color and crisper texture. Perceptible vanilla like aroma, baked cereal aroma and sweet taste in control cookies were found to be low which consequently reduced its overall quality (8.1). On the other hand, pearl millet cookies (K&MRB varieties) had closely matching sensory profile (Fig. 4.10) which differed significantly from control in key attributes such as color, vanilla-like aroma, baked cereal aroma and sweet taste. Higher perceived intensities of these desirable sensory parameters significantly and positively impacted the overall quality of pearl millet cookies rated at 10.7 for K and 9.2 for MRB respectively as compared to control (8.1). These finding indicated that the pearl millet cookies were acceptable. Sensory panelists opined that pearl millet cookies had a combination of desirable and lasting vanilla like aroma coupled with typical baked millet aroma. In addition, crisp and crumby texture was perceived in these cookies which further enhanced their sensory appeal making them highly palatable. High acceptability for pearl millet cookies in a similar study was reported (Archana et al 2004) where depigmentation of pearl millet was carried out. Results showed that native or pigmented pearl millet cookies were rated slightly less for the Page 182

100 stated sensory attributes compared to depigmented cookies. However, in the present study, it was found that dark color of the pearl millet cookies did not adversely affect its acceptability, instead; it provided an interesting visual appeal. Page 183

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102 C Control, K Kalukombu, MRB Maharashtra rabi bajra, Figure 4.11: Sensory Profile of Wheat Flour (Control) and Pearl Millet Cookies as Affected by Storage Page 185