Studies on Polyphenol Oxidase Activity of Heat Stabilized Whole Wheat Flour and its Chapatti Making Quality

Abstract

Studies were conducted to evaluate the effect of heat treatment on polyphenol oxidase (PPO) activity of whole-wheat flour (WWF) as well as its chapatti making quality. Whole wheat flour samples were heat treated through various means such as (i) dipping in boiling water (BW) for 20, 30, 40, 50, and 60 min; (ii) inpack heating under pressure (PH) at 0.352 kg/cm² for 4, 6, 8, 10, and 12 min; (iii) microwave heat treatment (MW) at 900 Watt, 2450 MHz for 20, 40, 60, 80, and 100 s. Studies showed that the heat treatment effectively reduced PPO level in whole wheat flour; although, it had adverse effect on the dough-making quality. Based on textural analysis of dough and chapatti as well as sensory scores of chapatti, the conditions for each of the treatments were optimized, i.e., (i) Dipping in boiling water (BW) for 30 min; (ii) inpack heating under pressure (PH) at 0.352 kg/cm² for 10 min; and (iii) microwave heating (MW) for 80 s. A maximum reduction (71.2%) in PPO activity of WWF using microwave treatment could be achieved followed by PH (56.9%) and BW (38.3%). The changes in colour of unbaked chapattis (flattened circular dough, diameter 150.0 mm and thickness 2.0 mm) and changes in quality of baked chapattis were measured to assess the effectiveness of the heat treatment. The L-value (lightness) decreased from 65.2 to 55.8, 65.7 to 58.3, 65.9 to 61.4, and 64.8 to 49.1 in case of BW, PH, MW treated, and control samples, respectively during the 72 h of storage under refrigeration temperature (5–6°C).

Keywords:

• heat treatment
• polyphenol oxidase (ppo)
• textural analysis
• mixograph
• dough
• chapatti
• brown discolouration

INTRODUCTION

Chapatti, a flat unleavened, hot plate baked product prepared from whole wheat flour with the application of fat during baking, is the staple food of more than 60% of the Indian population. It is similar to tortilla, which is prepared either from corn or wheat flour without application of fat during baking. On baking, chapatti is slightly puffed and gives a golden brown colour, with a few brown spots on the surface. On an average 90% of the wheat produced in India is consumed in the form of chapatti and only 10% is for the making of biscuits-bread-cakes and such other products.[1] Chapattis are generally prepared from whole-wheat flour by converting it into dough with water. Sometimes, oil, sugar, and salt are added to the dough to make chapatti soft and tasty. This dough is divided into small pieces of 30 to 40 g each and flattened into a round shape of about 150.0 mm in diameter with about 2.0 mm thickness. This circular dough is put on a hot plate (230–240°C) and baked with the application of fat. Chapatti preparation is itself a tedious process, starting from dough kneading to baking. In India, each time chapattis are prepared from fresh dough to get the good quality. Capattis prepared from stored dough (3–4 h either at ambient or refrigerated temperature) has a dull appearance because dough surface turns into brown colour due to the enzymatic browning. Polyphenol oxidase (PPO) has been implicated for enzymatic browning reactions in whole-wheat flour used for making chapattis.[2,3] PPO is present largely in the bran fraction of milled wheat and their level in flour rise with increasing extraction rate.[4–6] Since, in India whole-wheat flour (100% extraction rate) is used for making chapattis; therefore, the storage of dough is a big problem. Variations in PPO activity were correlated with quality characteristics,[7,8] cultivars and growing locations,[6,8,9] as well as milling fractions.[4,7,10] PPO is a heat labile enzyme and is inactivated readily by a short heat treatment at 70–90°C in fruits and vegetables tissues.[11] Hot air drying of wheat and the baking quality of the resulting flour had been studied by several authors.[12–14] Loaf volume, crumb grain, mixing time, and protein solubility were adversely affected at grain temperature above 71°C. Vadlamani and Seib[15] studied the effect of heat at different moisture levels (13–19%) in a rotating drum at 80 and 100°C for different durations (4–12 min) on PPO inactivation and reported that the noodles dough made with the flour from heat and moisture treated wheat had improved lightness. They also reported that autoclaving of the wheat flour at 121°C for 6 min fully inactivated the PPO activity but the flour completely lost its dough making quality. Although much work have been done to inactivate PPO in wheat flour but all work have been reported in lower extraction rate flour (60–70%), where PPO itself present in lower concentration and these flour mainly used for bread or noodles making purpose. PPO is present in higher concentration in whole-wheat flour (WWF) which is used for making chapattis in India. Therefore, this study was undertaken with the following objective: To study the effectiveness of different heat treatment methods including pressure heat treatment of whole wheat flour in inactivation of PPO and their impact on dough and chapattis quality.

MATERIAL AND METHODS

Wheat and Flour Sample

In the present study, commercial, medium hard variety (Bansi, Triticum aestivum), grown in the state of Karnataka (2006) was procured from local market, manually cleaned and milled to 400–500 micron particle size in a disc mill (Model EGM- 467K, Dia: 45.0 cm, Ganesha & Company, Chennai, India) to obtain whole wheat flour (100% extraction rate), which is commonly used in household for making chapattis. All chemicals used in the investigation were of analytical grade and procured from M/S E-Merck, Mumbai, India.

Chemical Composition of Flour

The chemical parameters like protein, fat, ash, and gluten contents were determined according to AACC methods 46-11, 30-25, 08-11, and 38-11, respectively.[16] The moisture content of flour samples was recorded using Brabender Rapid-Moisture Meter (Duisburg, Type 890100, Germany).

Heat Treatment of Flour

600 g of whole wheat flour was filled in Retort pouches (size 200 × 230 mm, 12 μ PET/ 9 μ Al foil / 15 μ Nylon / 70 μ CPP) and sealed by heat sealer. The sealed pouches were placed in an autoclave set at 0.352 kg/cm² and heated for 4, 6, 8, 10, and 12 min. After removal from the autoclave, the pouches were opened, and the flour samples were immediately cooled at room temperature conditions by spreading on an aluminum tray. The treatment time was recorded when the pressure reached at 0.352 kg/cm². Similarly, for boiling water treatment, the filled and sealed retort pouches were placed in boiling water and heated for 10, 20, 30, 40, and 50 min and cooled as described above. For microwave treatment, the flour sample (600 g) was placed in a Borocil glass beaker (one liter capacity) and microwaved at full power, 900 Watt, 2450 MHz) for 20–100 s in a microwave oven (LG Electronics India Pvt Ltd, Model MG 607 APR, Delhi) and cooled as described above.

Quantitative PPO Assay

PPO activity in the samples was measured using spectrophotometeric method described by Bruiski and Dropkin[17]. Analytical accuracy of the procedure was assessed by multiple analysis of the standard mushroom PPO (Sigma). This sample had a known PPO activity level (84,000 units/mg solids) and was repeatedly analysed along with each sample set. The mean and coefficient of variation were 74,865 units/mg and 3.2%, respectively. According to specification of enzyme purity prescribed by sigma, the 89.1% mean recovery obtained in the study was within the acceptable level of at least 60.0% recovery. For PPO assay, samples (4 g flour) were ground in duplicate with 20.0 ml of sodium phosphate buffer solution (0.1M, pH 6.5) using a mortar and pestle followed by shaking using wrist shaker at reduced speed (100 rpm) for 1 h in a refrigerated room and filtered through muslin cloth followed by centrifugation at 6,250 × g for 15 min. An aliquot of supernatant that contained crude PPO extract was heated in a water bath for 3 min at 37°C and placed in crushed ice. A solution containing 1 ml of sodium phosphate buffer (0.5 M, pH 6.5), 1.5 ml distilled water, 0.25 ml catechol (0.5 M) was placed in a quartz cuvette. Crude enzyme extract (0.25 ml) was added and the contents were mixed immediately by inversion. Absorbance of the solution was recorded at 410 nm with a UV-visible spectrophotometer (SHIMADZU, UV 1601, Japan) for 3 min to obtain the change in absorbance per min. All the results were corrected for substrate auto-oxidation, using a blank with no PPO added and reported on 14 % moisture basis. PPO activity was expressed in terms of units/g sample/min. One unit of PPO activity was defined as the amount of the enzyme giving a change in absorbance of 0.001 unit/min as described above.

Colour Values

The colour values in terms of L, a and b of unbaked and baked chapattis were measured using a Hunter Colour Meter (Data lab, Silvasa, Gujarat, India) with illuminant D₆₅ and 10° observer. L-value expressed the whiteness of the sample with 100 as perfect white and 0 as black. A higher L value indicated a brighter or whiter sample. Values of a and b indicated the red-green and yellow-blue chromaticity, respectively.

Mixograph Analysis

The treated whole wheat flour samples as well as control (without heat treatment) were analysed for their mixing characteristics using 10 g moving mixograph (National Mfg, Division, TMCO, Lincoln, USA) according to AACC methods 54-40A.[16] Both midline and envelope (curves defining the centre top and bottom of the mixogram) analyses were produced. The peak time and height, ascending and descending slopes and work function were recorded from the midline analysis.

Instrumental Texture Profile Analysis

Textural analysis of dough and chapattis were performed on texture analyzer plus (LLOYD Instruments, Model 01/TALS/LXE/UK, Hampshire, UK) following the method of Szczesniak[18]. For dough extensibility test, dough strips (60 × 5 × 5 mm) were prepared and test was carried out with fixture (Part No 01/2759), volodke vitch bite test of chapatti was carried out using the part no 01/2663. The thickness and width of chapatti strips was 2.0 and 15.0 mm, respectively.

Flattened Circular Dough Preparation

Dough from the treated and control wheat flours were prepared by mixing in Hobart dough kneader (HL 120 Hz 50/60, Troy, Ohio, USA) for 10 min, adding 68% water. The dough was allowed to rest for 15 min and divided into 40 g pieces. Each dough piece was formed into spherical shape, placed in a polyethylene pouch and pressed using a manual chapatti pressing machine (Sunrise Industry, Mysore, India) into a flattened circular dough sheet (Diameter 150.0 mm and thickness 2.0 mm). The polyethylene pouches were heat sealed and stored under refrigeration conditions (5–6°C).

Preparation of Chapattis

The flattened circular dough sheets were baked on a hot plate at 230 + 5°C for 1–2 min on either side with application of hydrogenated vegetable oil. The sensory characteristics of the chapattis were evaluated on a 9-point hedonic scale having a score of 9 for extreme liking and 1 for extreme disliking[19] by a ten member trained taste panel drawn from the scientific staff of the laboratory. The baked chapattis were evaluated for sensory parameters such as colour, texture, mouth feel, flavor, and overall acceptability.

Statistical Analysis

Experiments were performed in a 3 × 6 factorial design consisting of heat treatment methods (BW, PH, and MW) and time (0, 10, 20, 30, 40, 50 min; 0, 4, 6, 8, 10, 12 min, and 0, 20, 40, 60, 80, 100 s), respectively. All the experiments were performed in triplicate. Analysis of variance was carried out as per Snedecor and Cochran[20] using statistica software version-7 (State Soft Corporation, Tulsa, USA).

RESULTS AND DISCUSSION

The whole wheat flour (100% extraction) were analysed for its moisture, gluten, protein, fat, total carbohydrate and ash content, and the corresponding values were 10.6 ± 0.15%, 9.9 ± 0.02%, 13.4 ± 0.01%, 1.3 ± 0.03%, 73.2 ± 0.08% and 1.6 ± 0.06% respectively. The PPO activity in flour sample was 167.5 ± 3 units/g/min.

The PPO activity and its percent reduction in the heat treated flour samples by three methods for different durations have been presented in Table 1. It is clear from the table that all the treatments (a) as well as duration of heating; and (b) decreased PPO activity significantly (P ≤ 0.05) in flour samples. After 50 min treatment in BW, the PPO activity was reduced from 167.5 to 86.6 (48.3% loss), whereas PH treated sample had 73.1% less activity of PPO after 12 min of treatment. The percent reduction in PPO activity was more in PH samples as compared to BW treated samples due to high temperature under pressure and more penetration of heat encountered in PH samples. Vadlamani and Seib[15] reported complete destruction of PPO in autoclaved (1.05 kg/cm² for 6 min) flour samples, whereas only 73.1% reduction in PPO was obtained in present study during PH treatment after 12 min. Less reduction may be due to variation in heat transfer capacity of packaging material and due to lower pressure used during PH heat treatment. Vadlamani and Seib[15] also reported that such type of treated flour samples completely lost its dough making quality. An interesting result was observed during microwave heating of flour samples. Flour samples were treated in microwave for 20–100 s and 81.4% reduction in PPO activity was observed after 100 s of treatment. This shows that microwave treatment is more effective in inactivation of PPO in flour samples. This was mainly due to more penetration power of microwave as well as uniform heating of the samples.

Table 1 Polyphenol oxidase activity (PPO) of treated wheat flours (n = 3)

Treatment	Time	PPO activity,units/g/min	PPO reduction, %	Moisture content, %	Moisture loss, %
BW, min	0	167.5 ± 4^a	–	10.6 ± 0.06^a	–
	10	126.6 ± 3^b	24.4 ± 0.5^a	9.9 ± 0.05^b	5.9 ± 0.3^a
	20	111.6 ± 3^c	33.6 ± 0.6^b	9.7 ± 0.03^c	8.3 ± 0.2^b
	30	103.3 ± 4^d	38.3 ± 0.5^c	9.5 ± 0.05^c	9.8 ± 0.3^c
	40	91.6 ± 2^e	45.3 ± 0.4^d	8.8 ± 0.05^d	16.6 ± 0.2^d
	50	86.6 ± 3^f	48.3 ± 0.6^e	8.2 ± 0.04^e	22.3 ± 0.2^e
PH (at 0.352 kg/cm^2, min)	0	167.5 ± 4^a	–	10.6 ± 0.06^a	–
	4	109.9 ± 4^c	34.4 ± 0.8^b	9.8 ± 0.05^bc	6.8 ± 0.3^f
	6	92.4 ± 4^e	44.9 ± 0.8^d	9.7 ± 0.07^bc	8.1 ± 0.4^b
	8	80.5 ± 3^g	51.9 ± 0.7^f	9.7 ± 0.08^bc	8.5 ± 0.2^b
	10	72.6 ± 5^h	56.9 ± 0.7^g	9.5 ± 0.07^bc	9.9 ± 0.3^c
	12	45.3 ± 2^i	73.1 ± 0.6^h	9.2 ± 0.07^f	13.3 ± 0.2^g
MW, s	0	167.5 ± 4^a	–	10.6 ± 0.06^a	–
	20	106.6 ± 3^cd	36.4 ± 0.7^b	10.1 ± 0.06^b	3.9 ± 0.4^h
	40	95.2 ± 4^e	43.2 ± 0.5^d	9.9 ± 0.04^b	5.7 ± 0.3^a
	60	71.4 ± 4^h	57.4 ± 0.6^g	9.6 ± 0.04^c	8.6 ± 0.2^b
	80	48.3 ± 2^i	71.2 ± 0.5^h	9.5 ± 0.05^c	10.4 ± 0.3^c
	100	31.2 ± 3^j	81.4 ± 0.7^i	9.3 ± 0.05^cf	11.9 ± 0.4^i
BW: Dipping in boiling water; PH: Inpack heating under pressure; MW: Microwave heat treatment. Values expressed are mean ± standard deviation. Means in the same column with different superscripts were significantly (P ≤ 0.05) different.

In order to assess the effect of heat treatment of flour on dough and chapatti making quality, the dough samples were evaluated for extensibility and chapatti for bite test on Texture Analyzer Plus and results have been presented in Table 2. In all the samples, extensibility of dough decreased as duration of heat treatment increased. This was due to the alteration in functional properties of wheat gluten.[21] Hot air drying of wheat and the baking quality of the resulting flour has been studied by several authors,[12–14,21] and they reported that loaf volume, crumb grain, mixing time, and protein solubility were adversely affected at grain temperature above 71°C. Extensibility of dough decreased significantly (P ≤ 0.05) from 13.0 mm to 7.2 mm in BW treated samples after 50 min, however up to 30 min treated samples the dough have 10.2 mm extensibility, which was not significantly different from control (zero min). In PH treated samples, the extensibility (10.3 mm) of dough (from flour samples treated for 8 min) did not differ significantly from control. Extensibility of dough also decreased in MW treated samples, however reduction was less as compared to BW and PH treated samples due to very short duration of heating in microwave. In the experiment, the extensibility of dough was not affected significantly up to 60 s of heat treatment in microwave. Chapattis prepared from heat treated flour samples were also analysed for its bite test. The results showed that hardness, cohesiveness and chewiness increased significantly (P ≤ 0.05) in all the samples, whereas significant (P ≤ 0.05) reduction in springiness were observed during heat treatment. This was supposed to be caused due to the denaturation in protein resulting into weak formation of gluten network. The sensory data of chapattis prepared from heat treated flour samples have been presented in Table 3. A significant (P ≤ 0.05) negative correlation (r = − 0.82) was observed between hardness and overall acceptability scores of chapattis. A positive (r = +0.72) and negative (r = −0.78) correlation was observed between springiness and overall acceptability scores and between chewiness and overall acceptability scores, respectively. A decrease in all sensory parameters was observed in all heat treated flour samples. The sensory scores were more affected by heat treatment with respect to texture and lowered the overall acceptability scores. It can be observed from the table that up to 30 min treatment in BW did not have significant reduction in texture, however after that significant (P ≤ 0.05) reduction (8.6 to 7.5) was observed. Similarly, in PH treated samples, the texture did not differ significantly from control up to 10 min of heat treatment with respect to texture and overall acceptability scores. Whereas, microwave treated samples did not show significant (P ≤ 0.05) difference in sensory scores of texture and overall acceptability up to 80 s, although flour samples treated up to 100 s showed significant (P ≤ 0.05) difference. Based on textural analysis and sensory scores of chapattis prepared from heat treated wheat flour samples, the optimum heat treatment time of wheat flour was 30 min, 10 min and 80 s for BW, PH and MW, respectively.

Table 2 Texture profile of dough and chapattis prepared from treated flours (n = 10 measurements)

		Dough		Chapattis
Treatment	Time	Extensibility, mm	Stiffness, N/m	Hardness, N	Cohesiveness	Springiness, mm	Chewiness, Nmm
BW, min	0	13.0 ± 0.05^a	97.8 ± 1^a	5.7 ± 0.1^a	0.23 ± 0.005^a	0.84 ± 0.02^a	1.12 ± 0.01^a
	10	12.9 ± 0.05^a	95.2 ± 1^a	5.8 ± 0.05^a	0.24 ± 0.006^a	0.84 ± 0.02^a	1.16 ± 0.02^ab
	20	11.5 ± 0.06^a	82.4 ± 2^b	6.0 ± 0.1^a	0.26 ± 0.006^ab	0.82 ± 0.02^ab	1.25 ± 0.01^b
	30	10.2 ± 0.05^ac	76.9 ± 2^c	6.3 ± 0.08^a	0.29 ± 0.007^ab	0.80 ± 0.02^b	1.37 ± 0.02^c
	40	9.2 ± 0.06^bc	50.8 ± 1^d	7.3 ± 0.06^b	0.31 ± 0.006^ab	0.76 ± 0.03^c	1.59 ± 0.01^d
	50	7.2 ± 0.07^d	42.4 ± 2^e	8.4 ± 0.1^b	0.35 ± 0.005^b	0.70 ± 0.02^d	2.38 ± 0.02^e
PH (at 0.352 kg/cm^2, min)	0	13.0 ± 0.05^a	97.8 ± 1^a	5.7 ± 0.1^a	0.23 ± 0.005^a	0.84 ± 0.02^a	1.12 ± 0.02^a
	4	12.3 ± 0.06^a	90.2 ± 2^f	5.8 ± 0.08^a	0.23 ± 0.006^a	0.82 ± 0.03^a	1.17 ± 0.02^ab
	6	11.6 ± 0.06^ac	82.7 ± 2^b	6.0 ± 0.06^a	0.25 ± 0.007^a	0.80 ± 0.02^a	1.32 ± 0.01^c
	8	10.3 ± 0.06^ac	75.3 ± 1^c	6.2 ± 0.1^a	0.26 ± 0.006^ab	0.75 ± 0.03^c	1.44 ± 0.02^c
	10	9.5 ± 0.05b^b	70.7 ± 2^g	6.8 ± 0.08^a	0.28 ± 0.007^ab	0.71 ± 0.02^d	1.55 ± 0.01^d
	12	8.2 ± 0.05^bd	60.2 ± 1^h	7.2 ± 0.08^b	0.32 ± 0.005^b	0.63 ± 0.03^e	2.47 ± 0.02^e
MW, s	0	13.0 ± 0.05^a	97.8 ± 1^a	5.7 ± 0.1^a	0.23 ± 0.006^a	0.84 ± 0.02^a	1.12 ± 0.01^a
	20	12.9 ± 0.04^a	95.2 ± 1^a	5.7 ± 0.07^a	0.23 ± 0.005^a	0.84 ± 0.03^a	1.12 ± 0.03^a
	40	12.8 ± 0.06^a	92.4 ± 1^a	5.8 ± 0.06^a	0.24 ± 0.005^a	0.84 ± 0.02^a	1.16 ± 0.03^a
	60	12.4 ± 0.05^a	89.7 ± 1^af	5.9 ± 0.08^a	0.24 ± 0.006^a	0.81 ± 0.03^ba	1.28 ± 0.02^b
	80	12.0 ± 0.04^a	84.7 ± 2^b	6.1 ± 0.1^a	0.26 ± 0.007^ab	0.80 ± 0.02^ba	1.51 ± 0.01^d
	100	10.3 ± 0.06^ac	77.4 ± 2^c	6.5 ± 0.08^a	0.27 ± 0.005^ab	0.76 ± 0.03^c	1.88 ± 0.02^f
BW: Dipping in boiling water; PH: Inpack heating under pressure; MW: Microwave heat treatment. Values expressed are mean ± standard deviation. Means in the same column with different superscripts were significantly (P ≤ 0.05) different.

Table 3 Sensory properties of chapattis prepared from treated flours (n = 10 panelists)

Treatment	Time	Colour	Texture	Flavour	Mouth feel	Overall acceptability
BW, min	0	8.2 ± 0.2^a	8.6 ± 0.2^a	8.5 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.2^a
	10	8.2 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.2^a	8.4 ± 0.1^a	8.5 ± 0.2^a
	20	8.2 ± 0.2^a	8.5 ± 0.3^a	8.5 ± 0.2^a	8.4 ± 0.1^a	8.5 ± 0.1^a
	30	8.1 ± 0.2^a	8.3 ± 0.3^a	8.4 ± 0.3^a	8.3 ± 0.2^a	8.3 ± 0.2^a
	40	8.1 ± 0.1^a	7.5 ± 0.3^b	8.2 ± 0.2^a	7.3 ± 0.2^b	7.2 ± 0.1^b
	50	8.0 ± 0.2^a	7.0 ± 0.3^c	8.0 ± 0.2^a	6.8 ± 0.2^c	6.6 ± 0.3^c
PH (at 0.352 kg/cm^2, min)	0	8.2 ± 0.2^a	8.6 ± 0.2^a	8.5 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.2^a
	4	8.2 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.1^a
	6	8.2 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.2^a
	8	8.2 ± 0.2^a	8.4 ± 0.1^a	8.3 ± 0.3^a	8.3 ± 0.1^a	8.4 ± 0.1^a
	10	8.1 ± 0.1^a	8.3 ± 0.1^a	8.3 ± 0.2^a	8.2 ± 0.1^a	8.3 ± 0.2^a
	12	8.1 ± 0.2^a	7.3 ± 0.2^b	8.3 ± 0.1^a	7.2 ± 0.3^b	7.2 ± 0.2^b
MW, s	0	8.2 ± 0.2^a	8.6 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.2^a	8.5 ± 0.2^a
	20	8.2 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.1^a	8.5 ± 0.1^a
	40	8.2 ± 0.2^a	8.5 ± 0.1^a	8.5 ± 0.2^a	8.5 ± 0.2^a	8.5 ± 0.2^a
	60	8.1 ± 0.2^a	8.4 ± 0.2^a	8.4 ± 0.1^a	8.4 ± 0.1^a	8.4 ± 0.1^a
	80	8.1 ± 0.2^a	8.4 ± 0.2^a	8.4 ± 0.1^a	8.4 ± 0.1^a	8.4 ± 0.1^a
	100	8.0 ± 0.2^a	7.2 ± 0.2^b	8.3 ± 0.1^a	7.0 ± 0.2^b	7.0 ± 0.3^b
BW: Dipping in boiling water; PH: Inpack heating under pressure; MW: Microwave heat treatment. Values expressed are mean ± standard deviation. Means in the same column with different superscripts were significantly (P ≤ 0.05) different.

Mixing Properties

The heat treated flour samples by BW (30 min), PH (10 min) and microwave (80 s) and control samples contained 9.2 ± 0.05, 8.5 ± 0.05, 9.7 ± 0.05, and 9.9 ± 0.02 percent gluten, respectively. There was no significant reduction in gluten content of BW (30 min) and MW (80 s) treated samples, whereas it showed a significant (P ≤ 0.05) reduction in PH treated samples. However, the chapatti making quality of PH treated samples remained acceptable (8.3 OAA score) as shown by the sensory data (Table 3). Each optimally treated flour sample was analysed for its mixing characteristics using 10 g moving mixograph and the data has been presented in Table 4. The peak value or peak height decreased significantly (P ≤ 0.05) in BW and PH treated flour samples, whereas, no significant difference was observed in MW treated samples. It showed that there was degradation in gluten quality during heat treatment in BW and PH treated samples. Control wheat flour had a peak height of 60.4% torque, whereas BW treated had 46.6% torque, PH treated had 45.7% torque and MW sample had 58.5% torque. As indicated with peak value the gluten was significantly (P ≤ 0.05) affected in BW and PH treated samples. These heat treated flour may not be suitable for bread making where gluten play an important role but these are quite acceptable for making of chapattis as indicated with sensory data (Table 3). The peak time also significantly (P ≤ 0.05) increased in BW and PH treated samples, whereas the change was not significant for MW treated samples. The dough quality was also good in MW treated samples as compared to BW and PH treated samples as indicated by ascending and descending slope. It is also observed that excessive heat treated flour gave essentially a flat mixograph (Fig. 1B, C). Finney et al.[12] also reported that the peak height of flour dough always decreased when its gluten was heat damaged.

Table 4 Mixograph properties of treated flours (n = 3)

Sample	Peak time, min	Peak value, %torque	Ascending slope,%/min	Descending slope,%/min	Integral at peak,% torque ¥ min
Control	2.4 ± 0.05^a	60.4 ± 1.5^a	24.5 ± 0.5^a	−5.1 ± 0.3^a	90.4 ± 3.2^a
BW	4.3 ± 0.08^b	46.6 ± 1.3^b	2.4 ± 0.4^b	−2.6 ± 0.4^b	185.4 ± 2.3^b
PH	6.7 ± 0.05^c	45.4 ± 1.2^b	1.2 ± 0.5^c	−1.3 ± 0.3^c	251.5 ± 2.8^c
MW	2.5 ± 0.04^a	58.5 ± 1.5^a	13.3 ± 0.6^d	−4.5 ± 0.5^d	102.9 ± 3.5^d
BW: Dipping in boiling water for 30 min; PH: Inpack heating under pressure at 0.352 kg/cm^2 for 10 min; MW: Microwave heat treatment for 80 s. Values expressed are mean ± standard deviation. Means in the same column with different superscripts were significantly (P ≤ 0.05) different.

Figure 1 Mixograms of whole wheat flour treated by different heat treatment methods as well as control. A): Control (without heat treated); B): Dipped in boiling water for 30 min; C): Inpack heating under pressure at 0.352 kg/cm² for 10 min; and D): Microwave heat treatment for 80 s.

Changes in Colour Values of Stored Flattened Circular Dough

The changes in colour values of flattened circular dough stored at 5–6°C for 72 h has been shown in Table 5. To avoid differences in lightness caused by varying water absorption, all the wheat flour samples were made into dough at the same water absorption (68%). Visual differences in lightness are generally ascertained when L value differ by 0.5 units. Lightness (L) value decreased continuously with storage under refrigeration temperature (5-6°C) in all the samples. The decrease in lightness in treated samples was very slow as compared to the control one. By visual observation just after 24 h of storage the change in colour was found undesirable in control samples, whereas, in all the treated ones, the colour remained in acceptable range up to 72 h. Earlier we have discussed that inactivation of PPO was maximum in MW treated samples. This confirms that the change in lightness of flattened circular dough is related to the PPO activity in flour samples. Kruger et al.[22] and Baik et al.[23] also demonstrated that the rate of changes in lightness was correlated with level of PPO activity as well as various phenolic compounds in dough. The change in redness (a) value increased in all the samples during storage but the changes were not significant. Yellowness values (b) decreased in all the samples and rate of decrease was significantly (P ≤ 0.05) higher in control than the treated ones. This indicated that heat treatment of flour effectively controlled the change in colour due to enzymatic browning during storage of flattened circular dough. The effect of heat treatment on colour of baked chapattis prepared from stored flattened circular dough has been also given in Table 5. A similar trend was also found in baked chapattis. Baked chapattis prepared from the control flour dough was least acceptable after 24 h of storage in terms of its colour (6.8), while the chapattis prepared from heat treated flour dough retained good colour even after 72 h of storage and was found acceptable (Table 6). The maximum sensory score for colour was found in MW treated samples followed by PH and BW treated samples. A significant (P ≤ 0.05) negative correlation (r = −0.82) was found between PPO activity of flour and colour scores of the chapattis made from stored dough samples. The colour and overall acceptability scores were significantly (P ≤ 0.05) affected by the storage period in case of control samples and it showed a significant (P ≤ 0.05) reduction from 8.2 to 6.8 after 24 h of storage, limiting its acceptability. A sensory score more than 7.0 was considered acceptable. Although after 72 h of storage the change in sensory scores of all treated, as well as control samples, was significant (P ≤ 0.05); but all treated samples scored more than 7.0 for colour and overall acceptability. The sensory score for colour of MW treated sample was maximum (7.5), followed by PH (7.3) and BW (7.1) after 72 h of storage of flattened circular dough. Similar trend was observed for overall acceptability scores of chapattis. This indicated that the PPO activity of wheat flour was responsible for the colour changes during storage of dough as well as the colour and overall acceptability of chapattis prepared from the stored dough.

Table 5 Changes in colour value of flattened circular dough during refrigerated st°rage (5-6°C) (n = 3)

Colour parameter	Time, h	Flattened circular dough					Baked chapattis
		C	BW	PH	MW	C	BW	PH	MW
L	0	64.8 ± 0.4^a	65.2 ± 0.3^a	65.6 ± 0.4^a	65.9 ± 0.3^a	67.7 ± 0.3^a	68.2 ± 0.3^a	67.2 ± 0.4^a	67.4 ± 0.3^a
	24	55.3 ± 0.3^b	61.4 ± 0.3^e	62.5 ± 0.3^e	64.3 ± 0.2^a	58.1 ± 0.3^b	64.2 ± 0.2^e	65.3 ± 0.3^e	66.3 ± 0.2^a
	48	52.4 ± 0.4^c	57.3 ± 0.2^f	59.6 ± 0.3^g	62.1 ± 0.3^e	55.3 ± 0.2^c	60.1 ± 0.3^f	63.6 ± 0.4^e	65.1 ± 0.2^e
	72	49.1 ± 0.2^d	55.8 ± 0.3^b	58.3 ± 0.4^gf	61.4 ± 0.3^e	53.9 ± 0.2^d	58.3 ± 0.2^b	61.3 ± 0.3^f	64.4 ± 0.3^e
a	0	3.7 ± 0.02^a	3.7 ± 0.03^df	3.6 ± 0.02^e	3.7 ± 0.02^df	3.3 ± 0.03^a	3.2 ± 0.02^c	3.2 ± 0.03^ce	3.2 ± 0.02^e
	24	3.8 ± 0.02^a	3.7 ± 0.03^a	3.6 ± 0.01^ef	3.7 ± 0.03^df	3.4 ± 0.03^ab	3.2 ± 0.03^cd	3.2 ± 0.02^c	3.2 ± 0.03^ecd
	48	3.8 ± 0.03^b	3.7 ± 0.02^a	3.6 ± 0.02^ef	3.7 ± 0.03^df	3.4 ± 0.02^b	3.2 ± 0.02^cd	3.2 ± 0.03^c	3.2 ± 0.03^cd
	72	3.9 ± 0.03^c	3.8 ± 0.02^a	3.7 ± 0.03^df	3.7 ± 0.01^df	3.4 ± 0.02^b	3.3 ± 0.03^d	3.2 ± 0.03^cd	3.3 ± 0.02^cd
b	0	15.8 ± 0.1^a	15.1 ± 0.1^e	14.9 ± 0.2^fe	15.3 ± 0.1^a	13.6 ± 0.2^a	12.9 ± 0.1^dbe	12.5 ± 0.2^d	13.1 ± 0.1^e
	24	13.9 ± 0.2^b	14.2 ± 0.2^f	14.7 ± 0.2^f	14.9 ± 0.2^fe	11.9 ± 0.1^b	12.1 ± 0.2^db	12.3 ± 0.2^db	13.0 ± 0.2^edb
	48	13.1 ± 0.1^c	13.8 ± 0.1^b	14.1 ± 0.1^f	14.6 ± 0.1^f	11.2 ± 0.2^c	12.0 ± 0.1^b	12.0 ± 0.1^b	12.8 ± 0.1^edb
	72	12.6 ± 0.2^d	13.8 ± 0.2^b	14.1 ± 0.1^f	14.5 ± 0.2^f	10.9 ± 0.1^c	11.9 ± 0.1^b	11.9 ± 0.2^b	12.8 ± 0.2^edb
C: Control; BW: Dipping in boiling water for 30 min; PH: Inpack heating under pressure at 0.352 kg/cm^2 for 10 min; MW: Microwave heat treatment for 80 s. Values expressed are mean ± standard deviation. Means in the same column and rows for their respective colour parameter (L, a, b) and product (flattened circular dough and chapattis) with different superscripts were significantly (P ≤ 0.05) different.

Table 6 Changes in sensory characteristics of chapattis prepared from flattened circular dough stored under refrigeration temperature (5–6°C) (n = 10 panelists)

Sensory parameters	Sample	0	24	48	72
Colour	Control	8.2 ± 0.2^a	6.8 ± 0.2^b	6.2 ± 0.3^c	5.8 ± 0.3^d
	BW	8.1 ± 0.2^a	7.6 ± 0.1^hi	7.2 ± 0.2^f	7.1 ± 0.2^f
	PH	8.1 ± 0.1^a	7.8 ± 0.3^he	7.5 ± 0.1^g	7.3 ± 0.3^gf
	MW	8.1 ± 0.2^a	8.0 ± 0.2^ae	7.8 ± 0.2^eh	7.5 ± 0.3^gi
Texture	Control	8.6 ± 0.2^a	8.5 ± 0.2^ab	8.3 ± 0.2^bc	8.1 ± 0.2^c
	BW	8.3 ± 0.1^db	8.3 ± 0.2^bcd	8.1 ± 0.2^cd	8.0 ± 0.1^c
	PH	8.3 ± 0.2^d	8.3 ± 0.1^bcd	8.0 ± 0.1^c	8.0 ± 0.2^c
	MW	8.5 ± 0.1^ad	8.4 ± 0.1^ad	8.2 ± 0.2^c	8.1 ± 0.1^c
Flavour	Control	8.5 ± 0.2^a	8.5 ± 0.2^a	8.4 ± 0.1^ab	8.2 ± 0.2^b
	BW	8.4 ± 0.1^a	8.4 ± 0.2^a	8.3 ± 0.2^ab	8.3 ± 0.2^ab
	PH	8.3 ± 0.2^a	8.3 ± 0.1^a	8.2 ± 0.1^ab	8.2 ± 0.1^ab
	MW	8.4 ± 0.1^a	8.4 ± 0.2^a	8.3 ± 0.2^ab	8.3 ± 0.2^ab
Mouth feel	Control	8.5 ± 0.2^a	8.4 ± 0.2^a	8.3 ± 0.1^a	8.3 ± 0.1^a
	BW	8.3 ± 0.1^a	8.2 ± 0.2^a	8.2 ± 0.2^a	8.2 ± 0.2^a
	PH	8.2 ± 0.1^a	8.1 ± 0.1^a	8.2 ± 0.1^a	8.1 ± 0.1^a
	MW	8.4 ± 0.2^a	8.3 ± 0.2^a	8.3 ± 0.2^a	8.2 ± 0.1^a
Overall acceptability	Control	8.5 ± 0.2^a	6.8 ± 0.2^b	6.5 ± 0.3^c	6.0 ± 0.2^d
	BW	8.3 ± 0.1^a	7.8 ± 0.2^e	7.4 ± 0.2^f	7.2 ± 0.2^f
	PH	8.3 ± 0.1^a	8.0 ± 0.2^ae	7.7 ± 0.2^e	7.4 ± 0.2^f
	MW	8.4 ± 0.2^a	8.0 ± 0.2^e	8.0 ± 0.2^e	7.7 ± 0.2^e
BW: Dipping in boiling water for 30 min; PH: Inpack heating under pressure at 0.352 kg/cm^2 for 10 min; MW: Microwave heat treatment for 80 s. Values expressed are mean ± standard deviation. Means in the same column and rows for their respective sensory parameter with different superscripts were significantly (P ≤ 0.05) different.

CONCLUSION

It was concluded that the PPO activity level of wheat flour is responsible for the colour changes of dough during storage and can be significantly controlled by heat treatment of flour. However, the effect of heat treatment on PPO activity was significantly correlated with the duration and method of treatment. A 71.2% reduction in PPO activity could be achieved in the whole wheat flour by microwave heating for 80 s without affecting the quality of gluten. Dough prepared from the microwave treated flour could be stored under refrigeration conditions for 72 h, with minimum changes in colour values as well as sensory quality of chapattis. The acceptability of chapattis was comparable in terms of sensory attributes to a chapatti prepared from fresh dough.

ACKNOWLEDGMENTS

Authors express their thanks to the Head, fruits, and vegetable section for the help extended in the use of Hunter Colour Meter. Thanks are also due to Dr. M.C. Pandey and his colleagues for their help in analyzing the textural profile of dough as well as chapattis.