APP P E P ND N I D X 210

Transcription

1 APPENDIX 210

2 Research papers published / accepted/communicated for Publications so far based on the work reported in the Thesis 1. Hemalatha, M.S., Prasada Rao, U.J.S., Leelavathi, K. & Salimath, P.V. (2010). Influence of amylases and xylanase on chemical, sensory, amylograph properties and microstructure of chapati. LWT- Food Science and Technology, 43, Hemalatha, M.S., Bhagwat, S.G., Salimath, P.V. & Prasada Rao, U.J.S. (2012). Enhancement of soluble dietary fibre, polyphenols and antioxidant properties of chapatis prepared from whole wheat flour dough treated with amylases and xylanase. Journal of the Science of Food and Agriculture, 92, Manuscript under preparation: 1. Hemalatha, M.S., Prasada Rao, U.J.S., Leelavathi, K. & Salimath, P.V., Role of enzymes on microstructure, sensory and textural properties of chapati during storage 2. Hemalatha, M.S., SaiManohar, R., Prasada Rao, U.J.S. & Salimath, P.V., Effect of water soluble pentosans from different varieties on rheological characteristics of whole wheat flour dough and chapati making quality. 3. Hemalatha, M.S., Prasada Rao, U.J.S., Leelavathi, K. & Salimath, P.V., Effect of amylase and xylanase on amylograph characteristics of chapatis on storage. 4. Hemalatha, M.S., Salimath, P.V. & Prasada Rao, U.J.S., Effect of peroxidase on whole wheat flour dough characteristics, chapati quality and its influence on glycemic index of dough and chapati. 209

3 Posters presented at Academic Conferences: 1. Hemalatha, M.S., Salimath, P.V. & Prasada Rao, U.J.S., Studies on peroxidase-treated wheat dough: protein characteristics and digestibility of starch and protein. 20 th Indian convention of Food Scientists and Technologist Organised by AFST (I), CFTRI, DFRL, and NDRI, Bangalore, India. Nov 21-23, 2009, pp Hemalatha, M.S., Prasada Rao, U.J.S. & Salimath, P.V., Role of pentosans in relation to chapati making quality. 6 th International Food Convention, Organised by AFST (I), CFTRI and DFRL, Mysore, India. Dec 15-19, 2008, pp

4 LWT - Food Science and Technology 43 (2010) 1394e1402 Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: Influence of amylases and xylanase on chemical, sensory, amylograph properties and microstructure of chapati M.S. Hemalatha a, U.J.S. Prasada Rao a, *, K. Leelavathi b, P.V. Salimath a a Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore , India b Department of Flour Milling, Baking and Confectionary Technology, Central Food Technological Research Institute, Mysore, India article info abstract Article history: Received 21 July 2009 Received in revised form 22 April 2010 Accepted 23 April 2010 Keywords: Chapati Enzyme Amylograph Viscosity Microstructure Effect of enzymes on the quality of chapati e the flat unleavened bread made from different wheat varieties was studied. Doughs treated with fungal a-amylase, bacterial a-amylase, xylanase and combination of bacterial a-amylase and xylanase were examined for chapati making quality. The shear force of chapatis treated with bacterial a-amylase, xylanase, fungal a-amylase and combination of bacterial a-amylase and xylanase were in the range of 2.5e2.8 N, 2.3e2.5 N, 3.5e3.7 N and 2.2e2.5 N, respectively, and they were found to be lower and significantly different than that of their corresponding controls (4.7e6.5 N). Soluble starch and soluble amylose content of the chapatis prepared from dough treated with amylases were significantly different than that of control. Chapatis prepared from dough treated with bacterial a-amylase had shown higher amount of soluble amylose (around 0.5 g/100 g) compared to other treatments (0.2e0.25 g/100 g). In chapatis prepared from dough treated with bacterial a-amylase, and combination of bacterial a-amylase and xylanase, the amylograph paste viscosities were 38e66 BU, and were very much lower and significantly different from that of control (232e390 BU) may be due to the residual enzyme activities. The microstructures of chapatis prepared from dough treated with enzymes were uniform with distorted starch granules surrounded with protein matrix, while in control, the starch granules were spherical with protein matrix overlapping on one another to form aggregation. Chapatis prepared from dough treated with enzymes had softer texture, better pliability and higher overall quality scores compared to control. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Wheat flour contains about 82% starch, 12e14% protein, and 2e3% pentosans (Pomeranz, 1978). Starch is the major component of the wheat grain and its properties may affect the quality of flour and products derived from it. The starch consists of two types of glucose polymers, the highly branched amylopectin and the linear amylose. Pentosans originate in the endosperm cell walls of wheat grains and are mainly arabinoxylans. They play a key role in dough rheology and baking quality because of their functional properties. They exhibit high affinity for water absorption and viscosity of dough (Rouau & Moreau, 1993). Wheat flour contains various enzymes such as aeamylase and b-amylase, proteases, lipases, phosphatases and oxidases (Sahlstrom & Brathen, 1997). Amylase acts on starch resulting in increased proportion of low molecular weight fragments characterized by lower * Corresponding author. Tel.: þ ; fax: þ address: prasadarao_ummiti@yahoo.com (U.J.S. Prasada Rao). rates of retrogradation thus improving the baking quality (Palacios, Schwarz, & D Appolonia, 2004). Fungal a-amylase produces greater uniformity in dough properties and bread quality (Rouau, El Hayek, & Moreau, 1994). Pentosanases modify the functionality of pentosans during baking by reducing their water binding capacity thus inducing water redistribution in dough and lower the optimal amount of water needed for maximal gluten yield (Wang, Vliet,& Hamer, 2004). Rouau et al. (1994) reported that pentosanases incorporation at an optimal level improved the bread quality. Recently, Prabhasankar, Indrani, Jyotsna, and Venkateswara Rao (2004) reported that addition of enzymes modify the rheological properties of dough and overall quality of parotta, the flat unleavened bread. Chapati e the flat unleavened baked product prepared from the whole wheat flour is the main traditional staple food consumed by majority of the population in the Indian subcontinent. It is circular in shape with a diameter of 10e15 cm and a thickness of 3e4 mm, comprising mostly crust with little or no crumb. Desirable quality parameters for chapati are soft texture with chewy characteristics. Generally, it is consumed hot along with other adjuncts (Haridas Rao, 1993). In India, different wheat varieties are produced and /$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.lwt

5 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e the quality of chapatis varies depending on the variety. The problem faced in preparation of chapati is availability of right variety of wheat flour suitable for chapati preparation. The use of unsuitable wheat flour for chapati preparation affects the quality of product resulting in a tough, non-pliable, fragile and undesirable chapati. To overcome these problems, the present study aims at determining the influence of enzymes on chemical and sensory quality characteristics as well as microstructure and amylograph characteristics of chapatis prepared from different Indian wheat varieties. 2. Materials and methods 2.1. Wheats Four Triticum aestivum wheat varieties namely GW-322, NI-5439, MACS-2496 and HD-2781 were procured from Agharkar Research Institute, Pune, India. GW-322 and NI-5439 are reported as good varieties for chapati making property, while MACS-2496 and HD-2781 are poor varieties for chapati making property (Hemalatha, Manu, Bhagwat, Leelavathi, & Prasada Rao, 2007). Wheat was ground in a commercial disc mill to obtain whole wheat flour Enzymes Bacterial a-amylase from Bacillus subtilis and fungal a-amylase from Aspergillus oryzae were procured in dry form from SigmaeAldrich, USA. The activities of these enzymes were determined by using the method described by Bernfield (1955) with minor modifications reported by Ajila, Bhat, and Prasada Rao (2007) and the activities were found to be 7771 U/g and 4163 U/g, respectively. Amylase activity was defined as release of 1mmole of maltose per min at ph 4.6 at 45 C. Xylanase from Hemicola insolins was procured in dry form from Novozyme, India, and was reported to have side activity of hemicellulase. The enzyme activity was found to be 1256 U/g. Xylanase activity was defined as release of 1 mmole of xylose per min at ph 4.8 at 50 C Determination of enzyme activity Residual amylase and xylanase activities in chapatis prepared from enzymes treated doughs were determined according to the methods as mentioned in Section Preparation of chapati Chapatis were prepared from whole wheat flour according to Haridas Rao, Leelavati, and Shurpalekar (1986) with slight modifications. Chapati dough was prepared by mixing 200 g flour and water in a Hobart mixer (Model N-50) at speed 1 (61 rpm) for 3 min. The quantities of water required for chapati dough preparation were determined using Research Water Absorption Meter (RWAM) as described by Haridas Rao, Leelavati, and Shurpalekar (1987). In case of chapatis prepared from enzymes treated doughs the enzymes (bacterial a-amylase; U, fungal a-amylase; U, xylanase; U) were dissolved in water and mixed with the whole wheat flour. Control chapati dough was prepared by adding water without enzymes. The dough was rested for 30 min at room temperature. The dough was divided into pieces of 25 g each and sheeted in a sheeter (Model SK0 64C Seewer Rondo AG, Burgdorf, Switzerland) to a thickness of 1.5 mm. The sheeted dough was cut into a circular shape of 12 cms diameter using a die. The sheeted and cut dough was baked on a hot plate maintained at 215 C for 70 s on side 1 and 85 s on side 2. The chapati was then transferred to a heated gas tandoor (370 C) in such a way that side 1 was placed on the grill and heated for 10 s. The puffed chapati was then cooled, packed in polypropelene pouches until further use Freeze drying of chapati Chapatis were cut into small pieces (approximately cms) and freeze dried in a Heto Dry Winner DW3 e Denmark. Drying was continued until no further loss in weight of the sample was noticed. Freeze dried samples were used for further studies Chemical analysis of chapati Moisture content (two stage method) was determined according to standard AACC methods (2000). Soluble proteins were estimated according to the method described by Lowry, Rosebrough, Farr, and Randall (1951). Soluble starch, total amylose and soluble amylose contents were determined according to the method described by Sowbhagya and Bhattacharya (1979) Texture (shear force) measurement of chapati Shear force of chapati was evaluated by using texture analyzer (Stable Micro Systems, Model TA-HDI, UK) using the Warner Bratzler blade (HDP/BSW). Chapatis, which were packed in polypropylene pouches, were removed and three strips measuring 4 cm 2 cm were cut from each chapati. One strip at a time was placed on the centre of the sample holder and the blade was allowed to cut the chapati strip. The force required to cut the chapati strip into two pieces was recorded. The speed was maintained at 1.70 mm/s. Measurement for four chapatis were recorded and average values of these are reported Amylograph characteristics of chapati The amylograph characteristics of freeze dried chapatis were determined using Micro Visco Amylograph (Brabender OHG, Duisburg, Germany). Freeze-dried chapati was powdered using mortar and pestle. Chapati powder (15 g on 14% moisture basis) was suspended in 100 ml of distilled water and heated in the viscoamylograph from 30 to 92 C at a rate of 5 C/min, held at 92 C for 5 min, cooled to 50 C and then held at 50 C for 1 min under constant stirring (250 rpm). Torque measuring range was 300 cmg. The viscosity was expressed in Brabender Units (BU) Scanning electron microscope studies of chapati The freeze-dried pieces of chapati were used to study the microstructure. A Leo Scanning Electron Microscope Model 435VP (UK) was used. Chapati pieces were mounted on sample holders with the help of double-sided scotch tape and sputter coated with gold (2 min, 200 Pa). The preparations were transferred to the microscope where it was observed at 15 kv and a vacuum of Pa. Scanning electron micrographs with appropriate magnifications were selected for presentation of results Sensory evaluation of chapati A panel of eight trained judges consisting of four males (40e55 yrs) and four females (35e55 yrs) evaluated the chapatis. The chapati was evaluated for color and appearance, tearing strength, pliability, taste and aroma, mouthfeel and overall quality. The panellists were asked to give the scores using a 9-point hedonic scale (9 e like extremely; 8 e like very much; 7 e like moderately; 6 e like slightly; 5 e neither like nor dislike; 4 e dislike slightly; 3 e dislike moderately; 2 e dislike very much; 1 e dislike extremely).

6 1396 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e Statistical analysis and graphical representation The experimental data, including sensory scores of chapati, were treated statistically by Duncan s new multiple range test (DMRT) to determine the significance of results (Steel & Torrie, 1980). 3. Results and discussion 3.1. Influence of amylases and xylanase on textural and chemical properties of chapatis The moisture content of control chapatis in four different varieties, viz., GW-322, NI-5439, MACS-2496 and HD-2781 ranged from 28 to 31 g/100 g and the chapatis prepared from enzyme treated dough ranged from 27.4 to 31.2 g/100 g indicating that incorporation of amylases and xylanase did not show any significant difference on the moisture content of chapatis. However, incorporation of enzymes showed significant differences in shear force of chapatis (Fig. 1). In case of control chapatis (without enzyme treatment) prepared from different wheat varieties, the shear force varied from 4.7 to 6.5 N. HD-2781 had the highest value of 6.5 N while MACS had the lowest value of 4.7 N indicating that chapatis prepared from HD-2781 had the highest hardness compared to other three varieties. Interestingly, on incorporation of enzymes the variety HD-2781 yielded chapatis with lower shear force and these shear force values were comparable with other varieties. Incorporation of bacterial a-amylase, xylanase, fungal a-amylase and combination of bacterial a-amylase and xylanase had significantly decreased the hardness of chapatis prepared from all the varieties (Fig. 1). Chapatis prepared from the dough treated with bacterial a- amylase, xylanase, fungal a-amylase and combination of bacterial a-amylase and xylanase had shear force of 2.4e2.8 N, 2.3e2.8 N, 3.2e3.8 N and 2.2e2.5 N, respectively. Lower shear force denotes that the chapatis have softer texture hence easy to tear (Haridas Rao, 1993). Soft texture is a desirable character of chapati that makes it easy to chew. Amylase reduces the connectivity between the crystallites in the continuous starch phase and limits the size of the crystals by hydrolysing branched chains into smaller entities and thus contributing to soft texture (Boyle & Hebeda, 1990; Hug- Iten, Escher, & Conde Petit, 2003; Maninder & Jorgensen, 1983). Chapatis prepared from xylanase treated dough also had lower shear force compared to control. Similarly Shaikh, Ghodke, and Ananthanarayan (2008) have also reported that chapatis treated with xylanase had less shear force compared to control. Xylanase acts by cleaving the backbone of arabinoxylan thus modifying the functional properties of dough. Xylanase also removes arabinoxylan that interfere with the formation of gluten network by transforming the hydrated arabinoxylan carried by gluten into smaller oligomers (Martin, Wang, Lichtendonk, Plijter, & Hamer, 2005). This could be one of the reasons for improving texture of the product. The soluble starch content of chapatis prepared from amylase treated dough increased significantly in all the four varieties compared to control (Table 1). Soluble starch contents of control chapatis ranged from 2.11 to 2.24 g/100 g. The soluble starch content of chapatis prepared from commercial whole wheat flour is reported to be 2.11 g/100 g (Venkateshwara Rao, Leelavathi, Haridas Rao, & Shurpalekar, 1986). Soluble starch contents of chapatis prepared from bacterial a-amylase treated dough (3.06 g/100 g, 3.08 g/100 g, 3.11 g/100 g and 3.14 g/100 g) had no significant difference with the chapatis treated with fungal a-amylase (2.95 g/100 g, 2.95 g/100 g, 2.96 g/100 g and 2.98 g/100 g). Soluble starch contents of chapatis prepared from combination of bacterial a-amylase and xylanase treated dough were higher than the control (3.08e3.29 g/100 g). However, soluble starch contents of chapatis prepared from xylanase treated dough were not significantly different from control. Earlier, Jimenez and Martinez-Anaya (2001), Lent and Grant (2001) reported that the soluble starch content increased in bread and bagel, a bread product on treatment with a-amylase. During baking, starch gelatinises making it more susceptible to the action of amylases (Mathewson, 2000). The increase in soluble starch content in case of chapatis prepared from a e amylase treated dough compared to that of control may be due to the degradation of starch by amylase into low molecular weight fragments (Hug-Iten et al., 2003). The soluble amylose contents in control chapatis of all varieties were low and chapatis prepared from the dough treated with bacterial a-amylase were high (around 0.5 g/100 g) compared to other treatments. Soluble amylose contents of chapatis prepared from amylase treated dough increased significantly than control (Table 1). Maninder and Jorgensen (1983) have also reported that amylose content had increased in bread on treatment with amylases. Starch consists of amylose and amylopectin polymers. In the process of gelatinisation, intact starch granules absorb water, Fig. 1. Shear force of chapatis prepared from dough treated with enzymes of different wheat varieties. Data reported are expressed as means of four determinations. Means values followed by different letters in each variety are significantly different (p < 0.05).

7 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e Table 1 Chemical properties of chapatis prepared from dough treated with enzymes of different wheat varieties. Variations Soluble starch (g/100 g) Soluble amylose (g/100 g) Amylopectin (g/100 g) (by difference) GW322 NI5439 MACS2496 HD2781 GW322 NI5439 MACS2496 HD2781 GW322 NI5439 MACS2496 HD2781 Control 2.22 a a a a a a a a a a a a 0.03 Fungal a-amylase 2.95 b b b b b b b b b b b b 0.02 Bacterial a- amylase 3.14 c bc bc c c c c c bc b b b 0.03 Xylanase 2.22 a a a a a a a a a a a a 0.02 Combination* 3.08 c c c d b b b b c c c c 0.03 *Combination: Combination of bacterial a-amylase & xylanase Data reported are expressed as mean SD of three determinations. Means of the same column followed by different letters are significantly different (p < 0.05). swell and on cooling, amylose and amylopectin crosslinking takes place. The presence of exogenously added amylase may prevent the formation of these crosslinks. Biliaderis and Zawistowiska (1990) reported that incorporation of amylase prevents the formation of amylose and amylopectin crosslinks. Thus, the increase in soluble amylose and amylopectin in amylase treated chapatis may be due to the prevention of amylose and amylopectin crosslinking by amylases. The soluble protein contents of control chapatis and chapatis prepared from dough treated with bacterial a-amylase and combination of bacterial a-amylase and xylanase are significantly different in all the varieties whereas, no significant differences were observed in other treatments. The total amylose content in chapatis prepared from enzyme treatments were significantly different from that of control chapatis (Table 2) Influence of enzymes on amylograph studies of chapatis The amylograph characteristics were studied to determine the differences in starch gelatinisation in chapatis prepared from dough treated with enzymes. The amylograph characteristics of control chapatis were different from the chapatis prepared from enzymes treated dough. The paste viscosities of control chapatis were between 251 and 390 BU (Fig. 2). The paste viscosities of chapatis prepared from bacterial a-amylase treated dough were very low compared to other treatments (25 BU to 67 BU). The paste viscosities of chapatis prepared from fungal a-amylase treated dough were lower than the control (124e302 BU) but higher than the chapatis prepared from bacterial a-amylase treated dough. As can be seen in Table 3, chapatis prepared from both bacterial a- amylase and fungal a-amylase incorporated dough had shown the presence of amylase activities (residual activities) much higher than the control. In case of chapatis prepared from bacterial a- amylase treated dough it was five fold higher than that of control. This increase may be due to the difference in their thermal stabilities. Bacterial a-amylase was reported to be stable up to 95 C whereas fungal a-amylase was reported to be stable up to 65 C (Anderson, Adams, & Walter, 1983; Miller, Johnson, & Palmer, 1953). Harinder, Maninder, and Bains (1983) reported that dough incorporated with fungal a-amylase had low paste viscosity compared to control and more than dough treated with bacterial a-amylase. The paste viscosities of chapatis prepared from xylanase treated dough were also low when compared to control (220e336 BU) but higher than that of amylase treated. Xylanase cleaves the arabinoxylans into oligomers resulting in the decrease in peak viscosity (Redgwell et al., 2001). This may be due to the presence of residual xylanase activities in chapatis. As can be seen in Table 4, residual xylanase activities in chapatis prepared from xylanase treated dough were higher than that of control. In the chapatis prepared from combination of bacterial a- amylase and xylanase treated dough paste viscosities were low between 38 and 66 BU. The chapatis prepared from dough treated with combination of bacterial a-amylase and xylanase had showed no significant difference in amylograph characteristics to that of chapatis prepared from dough treated with bacterial a-amylase alone and therefore could be concluded was solely due to the action of bacterial a-amylase. Paste viscosities primarily depend on the degree of starch gelatinisation (Sidhu, Seibel, & Meyer, 1990), and treatment with enzymes has decreased the paste viscosity and has showed better retrogradation Influence of enzymes on microstructure of chapatis The microstructures of native wheat starch granules are spherical, circular to ellipsoidal with varying sizes and also pentagonal in shape (Anitha & Muralikrishna, 2008). In wheat dough, the small

8 1398 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e1402 Table 2 Chemical properties of chapatis prepared from dough treated with enzymes of different wheat varieties. Variations Soluble protein (g/100 g) Total amylose (g/100 g) GW322 NI5439 MACS2496 HD2781 GW322 NI5439 MACS2496 HD2781 Control 4.1 a a a a c d e e 0.3 Fungal a-amylase 4.3 b,c c c a a c c d 0.2 Bacterial a- amylase 4.5 d b e d b b d c 0.3 Xylanase 4.1 a b b b a e b a 0.3 Combination* 4.4 c,d d d c a a a b 0.3 *Combination: Combination of bacterial a-amylase & xylanase. Data reported are expressed as mean SD of three determinations. Means of the same column followed by different letters are significantly different (p < 0.05). Fig. 2. Amylograph paste viscosity of chapatis prepared from dough treated with enzymes of different wheat varieties. Data reported and expressed as means of two determinations. Means values followed by different letters in each variety are significantly different (p < 0.05). starch granules string together by protein matrix (Sidhu et al., 1990). The microstructures of control chapatis of all the varieties were quite different from the chapatis prepared from dough treated with enzymes (Fig. 3). In case of control chapatis starch granules were distorted bound together in a mass found scattered around. Protein matrix was overlapping on one another forming aggregation. Uniformity was absent in control chapatis (Fig.3eA). Sidhu et al. (1990) also reported that the starch granules were greatly distorted and squeezed on the inner side of the chapati crumb. Compared to the microstructure of chapatis, microstructures of bread have open structure with interconnected cavities inside large gas cells (Rojas, Rosell, Benedito de Barber, Perez-Munuera, & Llunch, 2000) and the Arabic bread (Khaboos) have intact small and large starch granules with incomplete gelatinisation (Sidhu, Caceres, & Behbehani, 1997). Table 3 Amylase activity of chapatis prepared from doughs treated with different enzymes of different wheat varieties (Expressed in mmole/min/g). Treatment GW322 NI5439 MACS2496 HD2781 Control 1.11 a a a a 0.03 Fungal a- amylase 2.49 b b b b 0.04 treated Bacterial a-amylase 5.74 d d d d 0.04 treated Combination of Bacterial 4.52 c c c c 0.03 a-amylase and Xylanase treated Data reported are expressed as mean SD of three determinations. Means of the same column followed by different letters are significantly different (p < 0.05). The microstructure of chapatis prepared from bacterial a-amylase treated dough showed highly solubilized starch granules, which were prominently embedded, within the protein matrix. There was uniformity in the microstructure (Fig.3eB). The microstructure of chapatis prepared from fungal a-amylase treated dough showed slightly ruptured gelatinised starch granules and were scattered all around (Fig.3eC). A thin film of protein was surrounded on each granule. Unlike control, protein aggregation was not found. The micrographs of fungal a-amylase treated parottas, an unleavened bread, showed a few intact starch granules and the rest had lost their outline and shape (Prabhasankar et al., 2004). The micrographs of chapatis prepared from xylanase treated dough showed starch granules with slight distortion and rupturing with ellipsoidal shape and were embedded in the gluten protein (Fig.3eD). Xylanase solubilizes the insoluble pentosans, leading to a change in water distribution (Hilhorst et al., 2002; Martin & Anaya, 2003). Prabhasankar et al. (2004) reported that microstructure of parottas treated with xylanase showed slight distortion Table 4 Xylanase activity of chapatis prepared from doughs treated with different enzymes of different wheat varieties (Expressed in mmole/min/g). Treatment GW322 NI5439 MACS2496 HD2781 Control 0.98 a a a a 0.03 Xylanase treated 3.05 c c c c 0.04 Combination of Bacterial 2.48 b b b b 0.06 a-amylase and Xylanase treated Data reported are expressed as mean SD of three determinations. Means of the same column followed by different letters are significantly different (p < 0.05).

9 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e Fig. 3. The microstructure of chapatis prepared from dough treated with enzymes of a. GW-322 and NI-5439 wheat varieties; b. MACS-2496 and HD-2781 wheat varieties.

10 1400 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e1402 Fig. 3 (continued). in outline and shape of starch granules and some thinning of protein matrix. The micrographs of chapatis prepared from dough treated with combination of bacterial a-amylase and xylanase showed slightly distorted starch granules embedded with protein matrix similar to that of chapatis treated with bacterial a-amylase (Fig. 3a and b e E). Protein aggregation was absent unlike in control Influence of enzymes on sensory properties of chapatis Effect of enzymes on the sensory properties of chapatis is shown in Table 5. Results showed that there was a significant improvement in the color and appearance of chapatis prepared from enzyme treated dough especially those from GW-322 and NI-5439 varieties. Haridas Rao et al. (1986) reported that chapatis of good quality

11 M.S. Hemalatha et al. / LWT - Food Science and Technology 43 (2010) 1394e Table 5 Sensory properties of chapatis prepared from dough treated with enzymes of different wheat varieties. Variations GW322 NI5439 MACS2496 HD2781 Color and appearance Control 7.5 a a a a 0.3 Fungal a amylase 7.6 ab d bc abc 0.6 Bacterial a amylase 8.2 d bc abc abc 0.5 Xylanase 7.7 b ab bc bc 0.5 Combination 8.0 c de abc bc 0.4 Tearing strength Control 7.2 a a a a 0.4 Fungal a amylase 7.5 b b cd bc 0.6 Bacterial a amylase 8.1 d b b cd 0.4 Xylanase 7.5 b b bc bc 0.4 Combination 7.9 c c bc bc 0.6 Pliability Control 7.3 a a a a 0.5 Fungal a amylase 7.7 bc cd bc b 0.4 Bacterial a amylase 7.7 bc b bcd cd 0.3 Xylanase 7.6 b cd bc de 0.5 Combination 7.8 cd cd bc bc 0.6 Taste and aroma Control 7.3 a a a a 0.4 Fungal a amylase 7.5 b bc cd b 0.2 Bacterial a amylase 7.6 bc bc de cd 0.6 Xylanase 7.7 cd cd bc bc 0.5 Combination 7.7 cd cd b bc 0.2 Mouthfeel Control 7.4 a a a a 0.2 Fungal a amylase 7.7 bc b bc b 0.3 Bacterial a amylase 8.0 cd d cd de 0.2 Xylanase 7.6 b c bc c 0.6 Combination 8.0 cd c b cd 0.5 Overall quality Control 7.2 a a a a 0.3 Fungal a amylase 7.6 b c bc bc 0.5 Bacterial a amylase 8.0 de b bc de 0.4 Xylanase 7.8 c c bc cd 0.3 Combination 7.9 cd c bc de 0.2 *Combination: Combination of bacterial a-amylase & xylanase. Data reported are expressed as mean SD of eight determinations. Means of the same column followed by different letters are significantly different (p < 0.05). should possess an appealing color with light brown spots spread evenly over the surface. Chapatis made from enzyme treated dough had many light brown spots spread on the surface making the products look very appealing. Presence of the dark spots can be related to the action of these starch hydrolysing enzymes ultimately resulting in increase of glucose molecules taking part either in Maillard reaction or caramelization. Significant improvement in the tearing strengths of chapatis was also observed in all the four varieties and was not specific to any one of the enzymes used. Chapatis prepared from control dough were marginally tough requiring more strength to tear hence scored less, on the other hand, chapatis prepared from enzyme treated dough required less strength to tear therefore had significantly high score. This was also seen in the objective measurement of shear force as discussed earlier. Treating of chapati dough with starch hydrolysing enzymes has positive effect on the pliability of chapatis. Freshly prepared chapatis should be highly pliable resulting in softer textured products. Significant improvement was observed in all the four varieties prepared from enzyme treated doughs. Sensory scores for pliability ranged between 7.1 and 7.3 for control chapatis. These values ranged between 7.7 and 8.1 for those prepared from enzyme treated doughs. The addition of optimal level of enzyme improves dough properties, loaf volume and crumb softness in bread (Haros, Rosell, & Benedito, 2002; Rouau et al., 1994) Treating of chapati dough with amylases also showed a significant improvement in taste and aroma of chapatis. Chapatis prepared from enzyme treated dough had wholesome aroma and taste. These chapatis were sweetish after taste, which was found to be highly acceptable. These results can be attributed to the presence of residual sugars as a result of starch hydrolysis as well as due to the formation of melanin and caramels formed during baking of chapatis. Mouthfeel is a sense observed during chewing, biting and eating of chapatis. Chapatis having moderate tearing strength and more pliability result in a product requiring lesser effort during chewing of chapatis. Tougher chapatis require more effort in the mouth for biting, chewing and tearing action and such a mouthfeel is less desirable. Sensory score for mouthfeel for control chapatis varied from 7.1 to 7.4. But these values ranged between 7.5 and 8.0 for those prepared from enzyme treated doughs. Bacterial a-amylase treated doughs seemed to have impact on chapatis from all the four varieties. Possible explanation would be that since bacterial a- amylase continues to act throughout the baking process resulting in more intense breakdown of starch molecules. It is well known phenomenon that starch retrogradation begins as soon as the product starts cooling and retrogradation of starch molecules has been explained to cause firmness in bread crumb (Karim, Norziah, & Seow, 2000). On the other hand, presence of bacterial a-amylase in bread formulation is shown to lower the bread crumb firmness and keep it soft for a longer time (Martin & Hoseney, 1991). Similar reactions can be expected in chapati also. Overall quality of chapatis, which actually is influenced by all the above parameters, improved when their respective dough were treated with enzymes. The overall quality scores of control chapatis prepared from GW-322 and NI-5439 were 7.1 and 7.2, respectively, while the overall quality scores of chapatis prepared from enzyme treated doughs of these varieties increased significantly and their values ranged from 7.6 to 8.0. Similarly, the overall quality scores of control chapatis prepared from MACS-2496 and HD-2781 varieties were 6.8 and 6.9, respectively, while the overall quality scores of chapatis prepared from enzyme treated doughs of these varieties increased significantly and their values ranged from 7.8 to 8.0 (Table 5). From the above results it could be observed that treating of chapati dough with a-amylase had a significant improvement on the overall quality of chapatis. Xylanase treated chapati dough also showed improvement on chapati quality. This improvement could be attributed to its effect on pentosans present in the wheat flour. 4. Conclusion Chapatis prepared from dough treated with amylases and xylanases yielded soft textured chapati with improvement in overall quality. These improvements are likely due to alterations in starch structures by amylases during dough preparation, which is evident from increase in soluble starch, soluble amylose content and decrease in paste viscosity. Scanning electron microscope and amylograph studies also revealed changes in starch properties. In addition, improvement in chapati quality upon incorporation of xylanase may be due to changes in the pentosan structure. These studies suggest that incorporation of these enzymes may retard the staling of chapatis during storage. Acknowledgement The author MSH thanks DST, (SR/WOS-A/LS-156/2006), New Delhi for the award of fellowship.

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13 Research Article Received: 29 April 2011 Revised: 27 July 2011 Accepted: 3 August 2011 Published online in Wiley Online Library: 26 September 2011 (wileyonlinelibrary.com) DOI /jsfa.4640 Enhancement of soluble dietary fibre, polyphenols and antioxidant properties of chapatis prepared from whole wheat flour dough treated with amylases and xylanase Mysore S Hemalatha, a Suresh G Bhagwat, b Paramahans V Salimath a and Ummiti JS Prasada Rao a Abstract BACKGROUND: Chapati preparation involves various processing steps such as mixing the flour into dough, sheeting and baking. During these processing steps, flour components are likely to undergo changes in their nutrient and polyphenol composition and their antioxidant properties due to phenol-mediated crosslinking of proteins and carbohydrates. Therefore, in the present study, changes in nutritional, nutraceutical and antioxidant properties of chapatis prepared from doughs treated with amylases and xylanase were determined. RESULTS: An increase in insoluble dietary fibre content and a decrease in soluble polyphenol content were observed during preparation of control chapatis from whole wheat flours. However, significant increases in soluble dietary fibre and soluble polyphenol contents were observed in chapatis prepared from amylase-treated doughs compared with control chapatis. Extracts of chapatis prepared from amylase- and xylanase-treated doughs showed better antioxidant properties than extracts of control chapatis. Among these enzyme treatments, chapatis prepared from amylase-treated doughs showed better antioxidant properties than chapatis prepared from xylanase-treated doughs. High-performance liquid chromatography analysis of extracts of chapatis prepared from doughs treated with amylases showed the presence of potential antioxidant phenolic acids such as caffeic, gentisic and syringic acids in addition to the phenolic acids present in control chapatis. CONCLUSION: Treatment of doughs with amylases increased the contents of soluble dietary fibre and soluble polyphenols as well as improving the antioxidant properties of chapatis. c 2011 Society of Chemical Industry Keywords: chapati; enzymes; antioxidant properties; dietary fibre 764 INTRODUCTION Diets rich in nutraceuticals are gaining importance because of their role in reducing the incidence of chronic diseases such as cardiovascular disease, diabetes and cancer. 1 Antioxidants are reported to contribute to health benefits through several possible mechanisms, such as quenching free radicals, chelating transition metals, reducing peroxides and stimulating the defence mechanism of antioxidant enzymes. 2,3 Dietary fibre is known to be beneficial against a variety of diseases, including colon cancer, diabetes, etc. 4 Whole wheat flour is a rich source of dietary fibre and antioxidants 5 7 and is used for the preparation of chapatis. Chapati, an unleavened flat baked product, is the main traditional staple food consumed by the majority of the population in the Indian subcontinent and is also widely consumed in the UK and other countries, particularly by the Asian ethnic community. 8 Processing of raw materials into food products has been found to decrease the content of soluble polyphenols. 9 Enzymes are increasingly being used as quality improvers in food products; however, in addition to quality improvement, recent emphasis has been on the improvement of nutraceutical content. Amylases and xylanase are known to improve the sensory quality of chapati and bread. 10,11 Previous studies have indicated that polyphenol content is increased by treatment with amylases. Zheng et al. 12 reported that the polyphenol content in raw apple pomace increased on treatment with amylases and pectinase, while Ahmed et al. 13 found that treatment with amylases increased the polyphenol content in sweet potato flour. In the present study the effect of processing during chapati preparation on the soluble phenolic acid contents and composition, antioxidant properties and nutritional composition of the final product was investigated. The enhancement of nutritional, nutraceutical and Correspondence to: Ummiti JS Prasada Rao, Department of Biochemistry and Nutrition, Central Food Technological Research Institute, CSIR Unit, Mysore , India. prasadarao ummiti@yahoo.com a Department of Biochemistry and Nutrition, Central Food Technological Research Institute, CSIR Unit, Mysore , India b Nuclear Agriculture and Biotechnology Division, BARC, Mumbai , India J Sci Food Agric 2012; 92: c 2011 Society of Chemical Industry

14 Improvement of chapati by treatment of dough with amylases and xylanase Table 1. Amounts of water (ml kg 1 ) added to whole wheat flours for preparation of chapati doughs Treatment GW-322 NI-5439 MACS-2496 HD-2781 Whole wheat flour (WWF) WWF + xylanase WWF + fungalα-amylase WWF + bacterialα-amylase antioxidant properties of chapatis prepared from doughs treated with amylases and xylanase was also studied. MATERIALS AND METHODS Wheat varieties Four wheat (Triticum aestivum) varieties, GW-322, NI-5439, MACS and HD-2781, were procured from Agharkar Research Institute (Pune, India). GW-322 and NI-5439 are reported as good varieties for chapati making, while MACS-2496 and HD-2781 are poor in chapati-making quality. 14 The wheat samples were ground in an EGM-467 K disc mill (Ganesha & Company, Chennai, India) to obtain whole wheat flours of sufficiently fine particle size to pass through a400 µm sieve. Enzymes Bacterial α-amylase from Bacillus subtilis and fungal α-amylase from Aspergillus oryzae were procured in dry form from Sigma- Aldrich (St. Louis, MO, USA). The activities of these enzymes were determined using the method described by Bernfield 15 with minor modifications reported by Ajila et al. 16 and were found to be 7771 and 4163 U g 1 respectively. Amylase activity was defined as the release of 1 µmol maltose min 1 at ph 4.6 and 45 C. Xylanase from Hemicola insolins was procured in dry form from Novozyme (Bangalore, India) and was reported to have additional activity of hemicellulase. The enzyme activity was found to be 1256 U g 1. Xylanase activity was defined as the release of 1 µmol xylose min 1 at ph 4.8 and 50 C. Preparation of chapatis Chapatis were prepared from whole wheat flours according to Haridas Rao et al. 8 with slight modifications. Chapati doughs were prepared by mixing 200 g of flour with water in a Hobart N-50 mixer (Hobart corporation, Troy, Ohio, USA) at speed 1 (61 rpm) for 3 min. The volume of water required for chapati dough preparation in each case was determined using a research water absorption meter so as to give an extrusion time of s. 17 The volumes of water added for preparation of doughs from different flours are given in Table 1. In the case of chapatis prepared from enzymetreated doughs, the enzymes (bacterial α-amylase, U; fungal α-amylase, U; xylanase, U) were dissolved in the water used for dough preparation. Control chapati doughs were prepared by adding water without enzymes. The doughs were rested for 30 min at room temperature, then divided into pieces of 25 g each and sheeted in an SK0 64C sheeter (Seewer Rondo AG, Burgdorf, Switzerland) to a thickness of 1.5 mm. The sheeted doughs were cut into circular discs of 12 cm diameter using a die. The sheeted and cut dough discs were baked on a hot plate maintained at 215 C for 70 s on side 1 and 85 s on side 2. The chapatis were then transferred to a heated gas tandoor (370 C) in such a way that side 1 was placed on the grill and heated for 10 s. The puffed chapatis were subsequently cooled, cut into small pieces (approximately 0.5 cm 0.5 cm) and dried in a Heto Dry Winner DW3 freeze-drier (Heto Holten A/S, Allerød, Denmark). Drying was continued until no further loss of sample weight was observed, and the dried samples were used for further studies. Proximate composition of chapatis Protein, fat and ash contents in freeze-dried chapatis were determined by AACC 18 methods 46-13, and respectively. Carbohydrate content was calculated by difference. The estimation of dietary fibre in the samples was done according to the enzymatic gravimetric method described by Asp et al. 19 Briefly, chapati powder (1 g) was suspended in 25 ml of 0.1 mol L 1 phosphate buffer (ph 6), then 0.1 ml of Termamyl (50 U) was added and the mixture was kept in a boiling water bath for 15 min to digest starches. The contents were cooled, 20 ml of water was added and the ph was adjusted to 1.5 with 4 mol L 1 HCl. Proteins were digested with 100 mg of pepsin at 40 C for 1 h. Then 20 ml of water was added and the ph was adjusted to 6.8 with 4 mol L 1 NaOH. Subsequently, 100 mg of pancreatin was added and the mixture was incubated at 40 C for 1 h. Finally, the contents were cooled, the ph was adjusted to 4.5 with 4 mol L 1 HClandthemixture was filtered through a dried and weighed crucible containing celite (0.5 g). To obtain insoluble dietary fibre, the residue retained in the crucible was washed with 950 g L 1 ethanol followed by acetone. The crucible was kept in an oven (105 C) until the weight became constant and its final weight was determined. The sample-containing crucible was then incinerated at 550 C for 5 h and its weight was determined. To obtain soluble dietary fibre, the volume of the filtrate was adjusted to 100 ml and the soluble fibre was precipitated by adding 4 volumes of warm (60 C) ethanol. The precipitate was filtered through a crucible containing celite, dried at 105 C and the weight of the crucible was determined. The sample-containing crucible was then incinerated at 550 C for 5 h and its weight was determined. Blanks were prepared as above but without the sample. Extraction of polyphenols in flours and chapati powders A 1 g sample was homogenised with 10 ml of 800 g L 1 acetone, stirred for 30 min using a magnetic stirrer and centrifuged at g for 15 min. The residue obtained was extracted twice more with 800 g L 1 acetone to achieve maximum extractability of polyphenols. The resulting supernatants were pooled and used for the estimation of polyphenol contents and composition and antioxidant properties. Total polyphenols of chapatis Total polyphenol content was estimated using the method of Swain and Hillis. 20 To 0.5 ml of extract, 4.5 ml of ethanol and 0.5 ml of phenol reagent (Folin Ciocalteu reagent diluted 1 : 2 (v/v) with water) were added and the mixture was incubated at room temperature for 3 min. Then 1 ml of saturated sodium carbonate solution was added and the reaction mixture was incubated at room temperature for 60 min. The absorbance was recorded at 675 nm. The total polyphenol content in the extract was expressed as gallic acid equivalent (GAE). Measurements of antioxidant activity Measurement of reducing power Generally, phenolics are good electron donors and can reduce Fe 3+ /ferricyanide complexes to ferrous form. 21,22 The reducing 765 J Sci Food Agric 2012; 92: c 2011 Society of Chemical Industry wileyonlinelibrary.com/jsfa