Thermo-mechanical and micro-structural properties of xylanase containing whole wheat bread

G.GhoshlU.S.ShivhreU.C.Bnerjee

a Dr.S.S.Bhatnagar University Institute of Chemical Engineering and Technology,Panjab University,Chandigarh 160014,India

b Department of Pharmaceutical Technology,National Institute of Pharmaceutical Education and Research,SAS Nagar 160062,Punjab,India

Abstract Xylanase is a hemicellulase that can hydrolyses the complex polysaccharides.Hemicelluloses are main components of cell walls of cereal grains.Moreover, hemicelluloses are considered as potential sources of mono-and oligosaccharides.In this study, influence of xylanase on the physicochemical properties and sensory qualities of the whole wheat bread during storage was investigated.Studies of whole wheat bread on microstructure,texture,thermotics,Scanning Electron Microscopic(SEM),X-Ray Diffraction(XRD)were conducted at ambient temperature of 25 and 4°C respectively.During storage at different temperatures,bread containing xylanase exhibited less firmness but larger volume with whiter crumb color and longer shelf life as compared to control bread.Results of firmness,enthalpy,Fourier Transformation Infra Red(FTIR)and X-Ray Diffraction (XRD) studies suggested a lower staling rate of bread containing xylanase as compared to control one.Bread containing xylanase showed a smoother surface and more uniform pore size than the control.Significant differences in microstructure of control and bread containing xylanase were observed which might be attributed due to the change in water starch gluten interaction.These differences were also found to be interrelated to the textural properties of bread.Better sensory features were achieved in bread containing xylanase.

Keywords: Bread;Firmness;Enzyme;Texture;Microstructure

1.Introduction

Superior quality bread is defined in terms of sponginess and even texture, better aroma, brighter color, nutritious, with extended shelf life.Deliberately preventing mould growth and eliminating major reaction conditions, delaying staling and eventually longer shelf life bread can be made [1].In recent years, the baking industry is paying attention to the replacement of several chemical additives by microbial enzymes.Improved dough properties, better machinability, better quality, and enhanced shelf life of bread resulted by incorporating selected enzymes instead of chemical additives.Chemical free processed food products are growing fast.Biotechnology can play a crucial role to meet the demands for fiber enriched healthier,nutritious bread[2].

Epidemiological interpretation revealed that several diseases such as high blood serum cholesterol,cardiovascular,coronary heart, diabetes and colon cancer can be prevented by consuming fiber rich diet[3,4].Fiber rich bread is manufactured using whole wheat flour as a replacement for refined wheat flour.This replacement can lead to a reduction in concentration of gluten protein and undesired features in the final products[5,6].Presence of fiber in whole wheat flour perturbs the starch–gluten protein matrix in the dough and eventually in the gelatinization process during baking, and thereby leads to reduction in swelling power of starch granules.Consequently,small gas cells swell in a particular direction and adversely affect the viscoelastic behavior of dough,restrict gas holding power of dough and subsequently poor machinability.Ultimately, poor bread volume due to insufficient hydration of gluten, falling of gas retention power which produces hard bread of poor quality with unpleasant granular texture, taste and mouthfeel.Addition of external gluten, surfactant, surfactant/shortening blend and hemicellulases minimize these undesirable effects [7–9].Application of microbial hemicellulase is lacking in comparison to other synthetic texture modifying agent.Incorporation of cellulases and hemicellulases in dough produce larger sized and evenly distributed porous crumb structure [10].Hemicellulases/pentosanases generate free sugars such as pentoses and hexoses by hydrolyzing starch.Subsequently free sugars are consumed by the yeast and as a result theporous crumb develops.Among hemicellulase,xylanase plays an important role on bread quality by degrading arabinoxylan as well as increasing water absorption,interaction and cross linking with gluten.It improves manufacturing conditions making the dough softer,more elastic,less sticky,glutinous,machine friendly during sheeting,molding operation as well as xylanase acts as an anti-staling agent in bread manufacturing[11–13].

Staling(retrogradation)rate of bread can be enumerated by the change of physicochemical and thermal properties of bread during storage[14–16].Reduction of moisture content,drop of quantity of water soluble starch,change of enzyme digestibility,increase of firmness, alteration of glass transition temperature and change of enthalpy can be correlated with crystallization of starch during staling [11,17].However, systematic studies on quality of bread manufactured using freshly prepared and purified xylanases are lacking.

Differential Scanning Calorimetry (DSC) can be used to measure the rate of staling quantitatively[18].Another way to quantify staling is X-Ray crystallography study,specifically the crystalline nature of the starch in the system.Starches appear as amorphous in a freshly baked bread but gradually restructure to crystalline state during storage.The re-crystallization is reflected in X-Ray Diffraction (XRD) patterns.Therefore, X-Ray crystallography can be used to determine molecular organization of starch in bread [19].X-Ray crystallography can be correlated with DSC for determining the increase in crystallinity during storage of bread.In conjunction with DSC,X-Ray crystallography can also be used in the analysis of the effect of xylanase on the whole wheat bread.

The present work was undertaken to find out the effect of xylanase on (i) whole wheat bread prepared using xylanase(produced in small scale laboratory fermenter usingPenicillium citrinumMTCC 9620);(ii)the evaluation of physicochemical,textural,thermal and micro-structural properties of fresh bread prepared using xylanase;(iii)the addition of xylanase on staling properties of bread during storage at ambient(25°C)and cold(4°C)temperature.

2.Materials and methods

2.1.Materials

Whole wheat flour, salt, sugar, yeast, shortening were purchased from the local market.Chemicals were procured from Hi-media(India)and are of analytical grade.Xylanase was manufactured in laboratory scale fermenter usingP.citrinumMTCC 9620 and purified[20–22].

2.2.Proximate analysis of whole wheat flour

In fresh as well as during storage moisture content of bread was estimated according to the American Association of Cereal Chemists (AACC) method [23].Protein, ash, crude fibre, dry gluten and fat content were measured according to the method described in American Organization of Analytical Chemists(AOAC)[24].

2.3.Preparation of bread dough

Dough was made by mixing whole wheat flour (100 g);instant dry yeast(3 g);sugar(6 g);salt(1.5 g);shortening(3 g);xylanase (20 μL, 299.5 U/mL) extracted and purified fromP.citrinumMTCC 9620 [21]; and, water according to requirement.Bread was prepared according to the process described by Ghoshal et al.[25].Control bread was prepared without the addition of xylanase keeping other ingredients unchanged.

2.4.Determination of physical properties of bread

Quality analysis of fresh bread samples was carried out by measuring the physical properties such as mass(m),volume(v),specific volume (m/v), height/width ratio of central slices and moisture content(%).Weight loss after baking and during storage was recorded.Volume was determined by seed displacement process[26].Moisture content was determined by keeping the bread samples at 100°C in hot air oven till constant weight and expressed in%(wet basis).

2.5.Color measurement

Crumb and crust color of fresh and stored bread was determined in terms ofL*,a*, andb* values using Hunter Lab Colorflex spectrophotometer (Hunter Associate Laboratory, Reston, VA, USA) according to the method described by Ghoshal et al.[25].Total color difference(ΔE)was calculated as:

2.6.Textural properties of bread

Bread texture was determined by penetration test (double)using Texture Analyzer(TA-XT plus,Stable Micro System,UK)using plunger type probe of 36 mm diameter(P/36R)according to the method described by Ghoshal et al.[25].Firmness (N),cohesiveness,springiness,resilience,gumminess(N)andchewiness(N)were calculated from the data analysis.Staling kinetic was determined by using Avrami equation(Eqs.(2)and(3))to determine rate constant K,and Avrami exponent n using change of firmness with time[27].

Upon linearization of Eq.(2)yields,

where θ represents the uncrystallized material at time t expressed in terms of firmness(dimensionless);F0is initial firmness at 0th day,Ftis firmness(N)at t(day);Fαis limiting value of firmness(N)at 20th day stored at refrigerator;k is rate constant(day?1);n is constant(dimensionless);t is storage time(days).

2.7.DSC study of stored bread

Glass transition temperature and enthalpy was determined using Differential Scanning Calorimeter (DSC-Phox, Netzsch,Germany).About 10 mg of bread sample was taken in the hermetically sealable aluminum pan and 10 μL distilled water was added.After sealing, bread samples were heated at 10°C/min rate from 20 to 200°C in the sealed pan.The parameters measured were the onset temperature (To), the peak temperature(Tp),and the conclusion temperature(Tc).Change of enthalpy(ΔH)was calculated using proteus analysis software provided with the instrument.Midpoint of onset and conclusion temperature was considered as glass transition temperature(Tg).

2.8.X-ray diffraction(XRD)study of stored bread

X-ray diffraction (XRD) profile of lyophilized powder of untreated and xylanase treated bread samples stored for different duration at room temperature and refrigeration temperature were studied in Philips X’Pert Pro X-ray diffractometer system.The radiation was used Cu Kα (λ=1.54060 ?) with 40 kV voltage and 40 mA intensity.Each time about 500 mg powdered samples were scanned from 5°to 60°2-θ.

2.9.Scanning electron microscopy(SEM)and Fourier Transform Infrared Spectroscopy(FTIR)of stored bread

Untreated and xylanase supplemented lyophilized bread samples were tested under scanning electron microscope (SEM)(JSM-6100, JEOL, Tokyo, Japan) at an accelerated voltage of 10 kV and magnification in the range of 100–2000×.The samples were visualized and the representative areas were photographed.

FTIR spectra of lyophilized powdered untreated and treated stored bread samples were recorded at room temperature(28±2°C) with a Tensor-27 spectrophotometer (Bruker,Germany)in the range of 600–4000 cm?1by accumulating 16 scans at 4 cm?1resolution.

2.10.Sensory evaluation of bread

Sensory evaluation was carried out using nine-point Hedonic scale (9=extremely good, 8=very good, 7=moderately good, 6=slightly good, 5=neither good nor bad, 4=slightly bad,3=moderately bad,2=very bad,1=extremely bad)[28].

Trained panel of 20 judges were chosen among the students and faculty members of Dr.S.S.Bhatnagar University Institute of Chemical Engineering and Technology, Panjab University,Chandigarh.Panel consisted of ten male and ten female (age group:20–40 years).

Each panelist was asked to assess the product and award the scores with respect to overall symmetry,smoothness,stickiness,uniformity of crumb cells,appearance,color,aroma and taste.

2.11.Statistical analysis

All the experiments were carried out in triplicate.Modeling and statistical parameters(coefficient of determination,standard error, analysis of variance, ANOVA) calculations were carried out using STATISTICA version 6.0(Statsoft Inc.,2002,Tulsa,USA).

3.Result and discussion

3.1.Proximate analysis of whole wheat flour

Composition of whole wheat flour was determined and expressed in wet basis.Flour contains 13.8%±0.56% moisture, 12.0%±0.48% protein, 10.5%±0.43% dry gluten,2.11±0.52%fat,1.98%±0.69%crude fiber,1.4±0.51%total ash,and 0.05%±0.07%acid insoluble ash.

3.2.Physicochemical properties

Sufficient hydration of gluten protein principally exhibits higher dough rising, while xylanase addition had resulted reduced absorption of water.Consequently, specific volume of bread containing xylanase (3.99±0.035 mL/g) was significantly higher(p<0.05)than that of control(3.52±0.003 mL/g)(Table 1a).Similar result has been reported by our group using another species ofP.citrinumMTCC 2553[25].Water absorption of dough containing xylanase was 64%, whereas it was 70% with control dough having similar dough consistency.Moisture content of bread is an important parameter related to specific volume, softness of bread crumb [29,30].Superiorquality of bread crumb contains 35%–40% moisture on wet basis.Retention of moisture in bread containing xylanase was higher (45.55%±0.46%, wet basis) as compared to control bread(41.73%±0.03%,wet basis)in fresh condition and during storage at room temperature(25±2°C).Xylanase is the most efficient hydrolase, it has the ability to degrade arabinoxylan,increases water absorption as well as interaction and cross linking with gluten protein.Similar observations are also reported in literatures[12,25,31].

Table 1a Physicochemical properties of control and bread containing xylanase at room temperature(25±2°C).

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Fig.1.(a)Variation of total color difference in control and xylanase-supplemented bread stored at room temperature.(b)Variation of total color difference in control and xylanase-supplemented bread stored at 4±1°C.

3.3.Color of bread

Homogeneous and glittering rusty brown crust and white spongy crumb color formation during baking is a hint of feature of good quality of bread.Crust and crumb of bread containing xylanase have significantly(p<0.05)higherL*values as compared to control during 6 days storage at room temperature and 20 days storage at refrigeration temperature(4±1)°C.Xylanase addition resulted significantly (p<0.05) higherb* values of crumb butb*value of crust did not affect significantly in bread containing xylanase neither at room temperature nor at refrigeration temperature.Significantly(p<0.05)lowera*values of crumb and crust of bread containing xylanase was observed as compared to control.Xylanase addition consequently,resulted brighter,yellowishandgrayish redcrumb ascompared to control(Tables 1b and 1c).Total color difference(ΔE)increased with duration of storage but the increase ofΔE was slightly lower in crumb of bread containing xylanase as compared to control and slightly higher in bread crust at both the temperatures of storage(Fig.1a and b).

3.4.Texture analysis of bread

Xylanase-addition resulted softer bread as compared to control bread during storage(Table 2).It may therefore be inferred that overall improvement in textural properties were observed when bread was manufactured with the addition of xylanase.In both the cases hardness increased with storage.Highest firmness values were obtained when control bread was stored at room temperature and lowest firmness value was obtained in bread containing xylanase stored at refrigeration temperature(4±1°C)(Fig.2).Xylanases cause transfer of water molecules from pentose phase to gluten phase eventually restructuring of the gluten-cross linking network during fermentation.Ulti-mately,increased specific volume in bread containing xylanase leading to softer bread crumb with better springiness value due to swelling of gluten-starch network.Increased specific volume can be correlated with the reduction in firmness value.It is well established that specific volume and moisture content are inversely proportional to the crumb firming rate[32].

Table 2 Effect of xylanase supplementation on bread texture(Avrami exponents and time constants)values stored at room temperature and refrigeration temperature.

Fig.2.Temporal variation of firmness of bread.

Eqs.(2) and (3) were used to describe temporal variation of firmness.The coefficients(n,k)of Eq.(3)were reported in Table 2.Variation of firmness with duration of storage is shown in Fig.2.Using Avrami coefficient calculated firmness values were determined and plotted(Fig.2).Eq.(3)signifies kinetics of alteration of a polymer from amorphous state to more organized crystalline state[33].Eq.(3)represents both starch retrogradation and bread staling quantification study.The coefficient,n is associated with crystal shape and is indicative of the growth of crystals.A 20%reduction of limiting firmness(Fα)values was observed in bread containing xylanase.Although in earlier report by Armero and Collar and Rojas et al.,n=1 was computed in Eq.(3)and good match of experimental values with predicted values were obtained.In the present study n and k were calculated from the regression parameter of Eq.(3)[34,35].Values of n for bread stored in refrigerated temperature were lower as compared to bread stored at room temperature(either control or xylanasesupplemented).The basic mechanism for starch crystallization is the instantaneous nucleation followed by linear growth of the crystals.Values of n were lower in bread containing xylanase as compared to control.Therefore, it may be concluded that xylanase addition caused reduction of crystallization rate and crystal enlargement in the bread.The rate constant(k)for bread containing xylanase was lower as compared to control bread at room temperature storage.It may therefore be inferred that rate of staling was reduced due to presence of xylanase.Similar results have been reported by other workers[34,35].However,k value was higher when bread containing xylanase was stored at 4±1°C.

3.5.Effect of xylanase on staling properties of the whole wheat bread

Bread staling is due to the starch retrogradation comprising of the following two processes:a time dependent rearrangement of amylose and time independent realignment of amylopectin[36].The formation of gel structure due to aging was linked to the development of crystallites,fringed miscelles.The amount of three dimensional network formations between the amorphous gluten and crystallites (starch) in bread increased with time during staling[37].

3.6.Thermal properties of whole wheat bread

Thermal properties of control and bread containing xylanase crumb were determined.The values of glass transition temperature Tg(To,Tp,TcTinf)and enthalpy change(ΔH)are reported in Table 3.Lesser enthalpy values were obtained in bread containingxylanase as compared to controlbread crumb.Larger size of crystal formation results with storage and eventually higher values of Tgwas obtained.An increase in the enthalpy value was also observed with storage.The degree of crystal formation was lower in bread containing xylanase which results in lowerTgandΔH values.Similar observations were also reported by other workers[38].

To investigate the variation of enthalpy with storage in bread containing xylanase Avrami analysis was carried out using the equations similar as 2&3 computing enthalpy values(ΔH)in place of firmness (F).The results showed that xylanase addition reduced the rate of crystallization and crystal growth in bread which is directly reflected in enthalpy values.Relationship between firmness and enthalpy values was determined to verify whether the values are mutually supporting each other or not.Linear correlation was established between change of firmness and enthalpy.Range of R2was above 0.9 in all the cases.

3.7.Determination of staling properties by X-ray diffraction(XRD)

XRD has been used to study various starch crystal types,shape, the extent of starch melting and gelatinization and the extent of retrogradation[18,39,40].Different crystal types give their own specific difractogram and peak intensities.It has been demonstrated that fresh baked bread apparently shows only V-type structure, indicative of amylose complexation with fatty acids to form helical clusters [39].During storage, the B-type structure increases, while the V-type structure remains unaffected.In the present study, diffractogram of 6 days old/aged bread with and without xylanase addition at room temperature and 11 days at refrigerated temperature are presented in Fig.3a& b, respectively.Diffractograms of all samples in first day were similar and V-type pattern was observed.However,diffractogram on 6th day represented the highest peak value in control sample stored at room temperature (Fig.3a).From 2nd day onwards,the pattern changed due to staling and two prominent peaks were visible at 2θ18 and 20°and wheat bread recovered its A-type pattern without affecting V-type pattern.This incident was the evidence of staling.Further, no significant differences was observed in the diffractogram of bread samples containing xylanase stored at room temperature(Fig.3a).Two minor peaks at Brag’s angle (2θ) values could be observed super imposed on the diffuse background for starch and starch gluten.Comparison of control and samples containing xylanase was shown in Fig.3a.No difference was obtained in the diffractogram of control sample at 0th day and samples containing xylanase at 0th and 6th days samples, and all the three curves were overlapped.Control sample on 6th day showed separate peak.It may therefore be inferred that xylanase addition reduced staling in the bread.Fig.3b represents XRD of control samples stored at 4°C.Retrogradation was minimum when the control samples were stored at refrigerated temperature.In freshly baked bread, starches appear as amorphous, but gradually restructuring to crystalline state during storage.The re-crystallization is reflected in X-Ray diffraction(XRD)patterns.Therefore,X-ray crystallography can be used to determine molecular organization of starch in bread.

Table 3 Effect of xylanase supplementation on enthalpy(Avrami exponents and time constants)values stored at room temperature and refrigeration temperature.

3.8.Staling properties by scanning electron microscopy(SEM)

The microstructure of control and bread samples containing xylanase during storage at room temperature and refrigeration temperature was studied and the microphotographs are shown in Fig.4a&b.The microstructure of bread altered during storage due to staling.In fresh bread, small starch granules were visible at the surface.Gelatinization removed the defects and hilled small gas vacuoles and made the surface smoother.Starch granules undergone deformation and degradation during processing and appeared in the protein matrix but remain unchanged in structure during baking[18].Fig.4a shows the microstructure of control bread having more porous with non-uniform pore sizes.This may be due to the presence of coarse microfibrils of gluten.Fig.4a shows the microstructure of 6 days stored bread crumb at room temperature while Fig.4b represented the microstructure of 6 days stored bread crumb containing xylanase at room temperature.Clear differences were observed in control and crumb of bread samples containing xylanase stored at room temperature.In control bread crumb, size of starch crystal was bigger than that of in the crumb of bread containing xylanase.Crumb surface of bread containing xylanase was little smoother, pore sizes were uniform than control one.In control bread due to coalescence of gas cell,pore sizes are larger and non-uniform than that of xylanase containing bread.It may therefore,be concluded that xylanase could control staling.These results further supported the DSC and XRD results.Staling effect in bread crumb stored at refrigeration temperature exhibited similar trend as in bread crumbs stored at room temperature.

Fig.4.SEM of(a)control(b)xylanase supplemented bread crumb stored at room temperature for 6 days at×170 magnification.

3.9.Determination of staling properties of bread by FTIR study

The FTIR spectroscopy in combination with attenuated total reflectance (ATR-FTIR) has been used to study the various classes of organic compounds present in the bread.These substances generally contain saccharides, lipids, proteins besides other minor constituents.Thestretching frequency at 3000–3600 cm?1region indicated the presence of both intra and inter molecular hydrogen bonding abundantly.This may even include the formation of intra chain hydrogen bonding between two domains of protein resulting more compact crystalline structure.

Intensity of absorption band for H-bond region(3100–3500 cm?1) increased about 35% in samples of bread kept at room temperature for 6 days.The broadening of this peak was also noticed at 2850–2950 cm?1and 1743 cm?1bands for C-H stretching and ester stretching of triglycerides,respectively.An important band narrowing has been observed in the 1300–800 cm?1of starch region.These changes suggest that retrogradation process has taken place in 6 days.The peaks at 1020 and 1050 cm?1were of particular interest.The relative increase in the intensities of 1050 cm?1was the characteristic of crystalline region of starch.The bread samples after treatment with xylanase were submitted for I-R spectral analysis.The vibrational bands in 3600–3000 cm?1,2950–2850 cm?1, 1750–1700 cm?1, 1680–1500 cm?1and 1250–800 cm?1corresponding to H-bond, C-H stretching,stretching, C-N stretching,of amide in starch system,respectively were analyzed.These were found identical in samples of 0, 3, 4, 6 days.This indicated that the staling occurred in untreated samples of bread at room temperature,xylanase addition stopped staling (Fig.5a and b).The bread samples with and without xylanase treated under frozen(4°C)conditions were also studied by FTIR spectral analyses (5c and d).The vibrational bands for H-bonds in the region of 3600–3000 cm?1showed only 6% increase in intensity indicating less retrogradation of the untreated bread kept at 4°C than that of at room temperature.Similar trends have been observed in C-H stretching(2950–2850 cm?1)and starch system (1250–800 cm?1) also.The relative crystallinity in the bread samples under frozen condition (4°C) were indicated by relative peak intensities of bands at 1050 and 1022 cm?1.This suggests less reduction in amorphousness or an increase in organization of structure in comparison to control sample at room temperature.

Although the bread samples treated with xylanase and kept under frozen conditions showed no change from 0–4 days,some hardening was observed due to the broadening of Hbond region that is 3600–3000 cm?1.Similar changes were noticed in C-H stretching(2950–2850 cm?1)and starch region(1250–800 cm?1).This changes might be due to some dehydration at low temperature in these samples.This was also indicated by sharpening of starch bands at 1050 cm?1which was the characteristics of crystalline region of a starch system.

3.10.Sensory evaluation

Significant improvement (p<0.05) in sensorial properties were observed when xylanase was used in bread manufacturing(Table 4).Soft,non stick and even mouth feel,better flavor with better taste was observed in bread containing xylanase as compared to control.Specific volume, firmness value also can be correlated with the sensory evaluation results.The results of the texture analysis explained the softness of the xylanase containing breads which was also confirmed by the sensory evaluation findings.Cell uniformity (evenly distribution of similar shaped pore of similar size) was also better in bread containing xylanase as compared to control.Bread containing xylanase was whiter than control which confirms theL*values reported in Tables 1b and 1c.It may therefore be inferred that xylanase can be used as an additive to produce better quality spongy having bigger specific volume and excellent oven spring property with extended shelf life bread.

Fig.5.(a)FTIR of control bread stored at room temperature for 6 days.(b)FTIR of xylanase-supplemented bread stored at room temperature for 6 days.(c)FTIR of control bread stored at 4°C for 20 days.(d)FTIR of xylanase-supplemented bread stored at 4°C for 20 days.

Table 4 Sensory properties of bread samples.

4.Conclusion

The various bread qualities with respect to specific volume and moisture, textural properties, color, thermal and sensory properties etc.were improved by the addition of xylanase.Firmness and enthalpy values of stored bread samples were nicely fitted in Avrami equation.The calculated values are in agreement with experimental values.Xylanase addition resulted reduced rate of staling in bread.A 20% reduction of limiting firmness(Fα)was observed in bread containing xylanase.During storage,bread containing xylanase was softer as compared to control.From the analysis of various staling properties examined,it may be inferred that bread stales at both ambient (25°C) and cold(4°C)temperature,but the rate was lowest at 4°C in bread containing xylanase.By the addition of partially purified xylanase,the color,texture and sensory properties of whole wheat bread is improved.

Acknowledgements

G.Ghoshal and U.S.Shivhare are thankful to AICTE,New Delhi, India (Grant sanction no.8023/BOR/RPS-2/2006-07;dated 26/02/2007) for providing financial support to carryout this work.Authors are also thankful to Prof.Tejbir Singh from Department of Chemistry,Panjab University,Chandigarh for his generous help in interpreting FTIR results.

Appendix A.

L* Lightness

a* Green to red

b* Blue to yellow

ΔE Total color difference

θ Fraction of uncrystallized material at time t expressed in terms of firmness(dimensionless)

F0Firmness(N)of fresh bread,

FtFirmness(N)at time t;

FαLimiting value of firmness(N)at 20 days stored at 4°C;

k Rate constant(per day);

n Constant(dimensionless);

t Storage time(days).

ToOnset temperature

TpThe peak temperature

TcThe conclusion temperature

TαThe inflation temperature

TgGlass transition temperature

ΔH Enthalpy(J/g)