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Journal of Cereal Science 51 (2010) 205–212
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Journal of Cereal Science
journal homepage: www.elsevier .com/locate/ jcs
Storage-induced changes in einkorn (Triticum monococcum L.)and breadwheat (Triticum aestivum L. ssp. aestivum) flours
Andrea Brandolini a, Alyssa Hidalgo b,*, Luca Plizzari a
a CRA-Unita di Ricerca per la Selezione dei Cereali e la Valorizzazione delle varieta vegetali (SCV), Via Forlani 3, 26866 S. Angelo Lodigiano (LO), Italyb Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM), Universita degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
Article history:Received 18 March 2009Received in revised form21 November 2009Accepted 30 November 2009
Keywords:Alpha-amylase kineticsFalling numberRVASDS sedimentation
* Corresponding author. Tel.: þ39 0250319189; faxE-mail address: [email protected] (A. H
0733-5210/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.jcs.2009.11.013
a b s t r a c t
To assess the effect of ageing on alpha-amylase activity, falling number, pasting properties and SDSsedimentation volume, whole meal and white flours of einkorn (cv Monlis) and bread wheat (cv Serio)were stored in darkness at different temperatures and analysed several times up to 374 days.Pregerminated bread wheat flours (cv Blasco) were also evaluated.
Flour ageing deeply modified all the parameters examined. In general, alpha-amylase activitydecreased, while falling number and viscosity increased. SDS sedimentation values showed an increase,which in bread wheat flours was followed by a steep decline at high temperatures, and a moderatedecrease at low temperatures. Einkorn flours showed analogous trends for all the traits studied;however, SDS sedimentation had similar or higher values than the initial stand even after 374 days ofstorage at 38 �C, thus suggesting a more stable breadmaking performance during ageing.
In general, the changes were drastic at high temperatures (38 and 30 �C), but reduced or negligible atmedium and low temperatures (20, 5 and �20 �C). Storage improved the quality of pregerminated flours.
� 2009 Elsevier Ltd. All rights reserved.
1. Introduction technological and pasting properties (Salman and Copeland, 2007;
With 217.4 million hectares, wheat is the most widespreadcereal in the world (FAO, 2007) and, along with rice, contributesmost of the carbohydrates for human consumption. Wheat is har-vested during limited time periods, but consumed all around theyear. After harvesting, the kernels are stored in bins or elevators,where they are dried, fumigated to control pests, and possiblycooled with aeration (Bailey, 1992). Most of the wheat produced inthe world is then processed into white flour or semolina for thepreparation of bread, biscuits or pasta. The flour is stored in sacks ofdifferent material and preferably stockpiled in cool, dark rooms forvarying periods of time: white flour has a shelf life of 12 months(Catterall, 1998; Edwards, 2007) while whole meal flour has a shelflife of three months (Edwards, 2007).
During storage, seeds and flour undergo deep physical, chemicaland physiological modifications (Pomeranz, 1992), fostered bystorage temperature, moisture content, atmospheric oxygencontent, light and microbial activity (Tipples, 1995; Wang andFlores, 1999). Postmilling maturation influences colour, composi-tion (Hidalgo and Brandolini, 2008a; Hidalgo et al., 2009) and
: þ39 0250319199.idalgo).
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Wang and Flores, 1999) of the flours. Among the technologicalchanges observed are: increase in water binding capacity and batterviscosity (Shelke et al., 1992), falling number (Hruskova andMachova, 2002), starch gelatinisation temperature (Shelke et al.,1992) and viscosity (Salman and Copeland, 2007), gluten elasticity(Cenkowski et al., 2000) and bread loaf volume (Chen and Shofield,1996). Interestingly, improvement of some technological parame-ters in flour from presprouted wheat kernels has also been reported(Ariyama and Khan, 1990).
Einkorn (Triticum monococcum L. subsp. monococcum), a diploidhulled wheat closely allied to durum and bread wheat, is a potentialfood source with high nutritional properties because of its higherprotein (Borghi et al., 1996), carotenoid (Abdel Aal et al., 2002;Hidalgo et al., 2006) and tocol (Hidalgo et al., 2006) contents,coupled with some interesting technological properties (Borghiet al., 1996; Brandolini et al., 2008; Corbellini et al., 1999).
The objective of this research was therefore to assess theinfluence of flour ageing on several technological parameters ofeinkorn and bread wheat; additionally, it was of interest tounderstand if the quality of pregerminated wheat would improveduring storage. To reach this goal, alpha-amylase activity, fallingnumber, pasting properties and SDS sedimentation volume ofwhole meal and white flour from one einkorn cultivar, Monlis (nonpregerminated), and two bread wheat cultivars, Serio (non pre-germinated) and Blasco (lightly pregerminated), were repeatedly
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212206
measured up to 374 days during their storage at five differenttemperatures (�20, 5, 20, 30 and 38 �C).
2. Experimental
2.1. Samples
Kernels of einkorn cv Monlis (recently released in Italy andparticularly suited for breadmaking) and bread wheat cv Serio andBlasco were harvested with a plot combine in 2007 at S. Angelo L.(Po plain, Italy) from 10 m2 plots with three replications, croppedfollowing standard cultural practices (Castagna et al., 1995).
2.2. Sample preparation
Approximately 3 kg of recently harvested seeds of the einkorncv Monlis were de-hulled with an Otake FC4S thresher (Satake,Japan); dehulling was not required for the free-threshing breadwheat cv Serio and Blasco.
Whole meal flours were produced using a Cyclotec 1093 lab mill(FOSS Tecator, Denmark); white flours were obtained using a Bona-GBR lab mill (Monza, Italy), that separates white flour from branand shorts. The white flour recovery rate was 60.9, 58.6 and 55.8%for Monlis, Serio and Blasco, respectively.
All flours were put in 500 mL glass bottles with screw caps andplaced under darkness in refrigerated cells (Igloo, Italy) for storageat �20�1.5 �C and 5�1.5 �C, and in thermostat cabinets (Heraeus,Germany) for storage at 20�1 �C, 30�1 �C and 38� 2 �C. Thestorage was maintained up to 374 days.
2.3. Analytical methods
The following determinations were performed on whole mealand white flours: dry matter content (method 44-15; AACC, 1994);SDS sedimentation volume, following the procedure described byPreston et al. (1982) with minor modifications; falling number(method 56-81B; AACC, 1994); alpha-amylase activity (method 22-02; AACC, 1994), using the Ceralpha assay kit (Megazyme Interna-tional Ireland Ltd., Bray, Ireland); amylose, determined with theMegazyme amylose assay kit (Megazyme International Ireland Inc.,Bray, Ireland). Alpha-amylase activity was measured in CeralphaUnits (CU): one CU is defined as the amount of enzyme, in thepresence of excess a-glucosidase and glucoamylase, required torelease one micromole of p-nitrophenol from BPNPG7 in oneminute under the defined assay conditions.
The pasting behaviour of starches during gelatinisation wasassessed with a Rapid Visco Analyzer (RVA, Newport Scientific Pty.Ltd., Warriewood, NSW, Australia). Briefly, whole meal flour (4.0 g,based on 14% moisture) or white flour (3.5 g, based on 14%
Table 1Pre-storage technological characteristics (mean values� s.d.) of flours from einkorn cv M
Monlis Ser
Whole meal White flour Wh
Moisture (g/100 g) 9.4� 0.05 10.4� 0.05 9Alpha-amylase (CU) 0.242� 0.008 0.184� 0.010 0.21Falling number (s) 346� 8.5 387� 5.0 34Amylose (g/100 g starch) 25.2� 0.73 25.1� 1.02 24Peak viscosity (cP) 2721� 11.3 2534� 8.5 151Through (cP) 1519� 24.7 1402� 3.5 92Breakdown (cP) 1203� 13.4 1133� 5.0 58Final viscosity (cP) 2629� 35.4 2468� 18.4 183Setback (cP) 1110� 10.6 1067� 14.8 91Peak time (min) 6.07� 0.000 6.10� 0.047 5.6Peak temperature (�C) 63.7� 0.04 62.4� 0.60 62SDS (ml) 29� 1.4 71� 0.7 4
moisture) was dispersed with 25 ml of distilled water in analuminium canister. With constant stirring, the flour-watersuspension was held at 50 �C for 1 min, heated to 95 �C over 3 min45 s, maintained at 95 �C for 2 min 30 s and progressively cooled to50 �C over 3 min 45 s. The starch viscosity parameters measuredwere peak viscosity, breakdown, final viscosity, setback, peak timeand pasting temperature.
All measurements were performed twice (amylose contentthree times); the results are presented as means of themeasurements.
2.4. Kinetics modelling
To determine the reaction order of alpha-amylase inactivation,zero- and first-order kinetics were hypothesised by applying thegeneral reaction rate expression –dC/dt¼ kCn, where C is theenzymatic activity, k is the reaction rate constant, t is the reactiontime and n is the order of the reaction (Atkins and De Paula, 2006).The order with the best correlation (r) and the best correspondenceamong the experimental values and the half-life of the compound(t1/2) [i.e. the time for the residual enzymatic activity to fall to halfits initial value, where t1/2¼ C0/2k for zero order and C0 is the initialactivity; t1/2¼ ln2/k for first order)] was selected.
The reaction rate to temperature relationship was quantified bythe Arrhenius equation ln k¼ ln k0� (Ea/RT), where Ea is the acti-vation energy of the reaction (kJ/mol), lnk0 is the pre-exponentialconstant, R is the gas constant (8.314 J/mol/K) and T is the meanabsolute temperature of the considered storage temperature range(K). From the slope of the Arrhenius line, the z value (z¼ 2.303 RT2/Ea) was computed: z represents the increase in temperature thatcauses a 10-fold rise in the reaction rate.
Kinetics data were analysed by regression analysis usingMicrosoft Excel 2000.
3. Results and discussion
3.1. Flour parameters
Table 1 reports moisture content, alpha-amylase, fallingnumber, amylose, RVA parameters and SDS sedimentation values infreshly milled whole meal and white flours of einkorn cv Monlis,bread wheat cv Serio and bread wheat cv Blasco.
The alpha-amylase activity was slightly higher in whole mealflours than in white flours; however, the difference was statisti-cally significant only for Monlis. Whole meal flours tend to presenthigher alpha-amylase activity because this enzyme is particularlyabundant in the external layers of the kernel (Rani et al., 2001);einkorn has smaller kernels and higher bran percentage thanbread wheat (Hidalgo and Brandolini, 2008b), thus further
onlis and bread wheat cvs Serio and Blasco.
io Blasco
ole meal White flour Whole meal White flour
.2� 0.01 11.0� 0.01 9.4� 0.12 11.0� 0.212� 0.013 0.202� 0.014 0.356� 0.021 0.344� 0.0189� 2.1 329� 9.2 216� 1.4 218� 5.0.8� 0.91 25.0� 1.21 25.3� 1.08 25.0� 0.650� 29.7 1420� 23.3 674� 4.2 803� 18.46� 2.1 762� 8.5 231� 5.0 248� 5.05� 31.8 658� 14.8 444� 9.2 556� 13.47� 44.5 1495� 33.2 548� 1.4 577� 14.81� 46.7 733� 24.7 318� 3.5 339� 9.92� 0.000 5.77� 0.042 4.90� 0.042 4.97� 0.049.9� 0.64 61.3� 0.00 61.7� 0.64 61.7� 0.646� 0.0 72� 2.8 43� 3.5 65� 0.0
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Fig. 1. Isothermal degradation kinetics of alpha-amylase activity during the storage of whole meal and white flour from einkorn cv Monlis, bread wheat cv Serio and bread wheat cvBlasco. The points represent experimental mean values; the lines follow the zero order kinetics equation (C¼ C0� kt).
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212 207
increasing enzymatic activity in whole meal with respect to whiteflour. On the other hand, Blasco showed signs of partial pre-germination, with moderately high alpha-amylase contents (0.356and 0.344 CU for whole meal and white flour, respectively) andsomewhat low falling number values (216 and 218 s). The status ofBlasco was confirmed by the results of the RVA analysis because,while Monlis and Serio presented normal pasting profiles, itsflours showed reduced peak and final viscosities, coupled withsteep breakdowns. While the trends were similar, no directcomparison between whole meal and white flour RVA results arepossible, because of the different substrate quantities (4.0 and3.5 g) used in the analyses.
No significant differences were observed among Monlis, Serioand Blasco or between flour type for amylose content, that wasalways around 25 g/100 g starch. On the other hand, the SDSsedimentation volume of the white flours (on average69.3� 2.19 ml) was significantly higher than that of the whole mealflours (39.3� 5.24 ml), because bran does not contribute to glutenswelling.
3.2. Alpha-amylase
The evolution of alpha-amylase activity during the storage ofwhole meal and white flours at different temperatures is presented
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Fig. 2. Variation of falling number during the storage of whole meal and white flour from einkorn cv Monlis, bread wheat cv Serio and bread wheat cv Blasco. The points representexperimental mean values; the best interpolation curve is shown.
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212208
in Fig. 1. In all samples, alpha-amylase activity decreased to variousextents during storage. The decline was absent or negligible at �20and 5 �C, but increasingly stronger at higher temperatures: forexample, after 374 days at 38 �C, alpha-amylase activity dropped,on average, 50.6, 35.6 and 28.5% for Monlis, Serio and Blasco,respectively; marginal differences were observed between wholemeal and white flours. Rehman and Shah (1999), in whole mealflour from wheat kernels stored at 10, 25 and 45 �C over six months,observed decreases of 9.2, 21.6 and 34.0%, respectively.
In the unsprouted wheats, the alpha-amylase inactivationreaction followed a zero order kinetics (C¼ C0� kt) for both flourtypes in Monlis and Serio; high regression coefficients (0.93–0.99)
and good correspondence between experimental values andcalculated half-life time, especially at higher temperatures, wereobserved. The alpha-amylase activity of pregerminated wheatBlasco showed a linear decrease only in the first 100 days of storage,followed by a gradual reduction of the slope down to a plateau, thusleading to an asymptotic inactivation trend. The sometimesreduced r values at 5 e �20 �C were a consequence of randomexperimental errors on trends with minimal fluctuations.
During storage, alpha-amylase activity decreased as a functionof temperature and time; the reaction rate constant k increased asthe temperature augmented, indicating a quicker degradation ofthe compounds at higher temperatures. In the unsprouted wheats,
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Fig. 3. Changes of RVA final viscosity during the storage of whole meal and white flour from einkorn cv Monlis, bread wheat cv Serio and bread wheat cv Blasco. The pointsrepresent experimental mean values; the best interpolation curve is shown.
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212 209
rate constants were similar between flours, but different betweenspecies, as they were higher in Monlis (on average,k¼�0.292� 0.0078� 10�3 CU days�1 at 38 �C) than in Serio (k¼-0.193� 0.0062�10�3).
The Arrhenius model was used to determine the influence oftemperature on the reaction rate (k) for Monlis and Serio flours; theaverage activation energies in Monlis and Serio were 50.6�1.27and 47.5� 3.13 kJ/mol, respectively, with z values of 31.3� 0.78 and33.5� 2.20 �C and pre-exponential constants of 11.13� 0.37 and9.50�1.19. The overall variation of the Arrhenius parameters,considering the complex substrate analysed, was minimal. It is notpossible to compare these results with literature information
because, while several studies on inactivation kinetics of alpha-amylase in different systems are available (e.g. Apar and Ozbek,2004; Kumar et al., 2005; Riahi and Ramaswamy, 2004; Tanaka andHoshino, 2002), to the best of our knowledge no data on storedflours exist.
3.3. Falling number
The falling number variation during storage at differenttemperatures of whole meal and white flours is depicted in Fig. 2. Inall cases, falling number values increased during storage: theimprovement was minimal at low temperatures, but was relevant at
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Fig. 4. Evolution of SDS sedimentation volume during the storage of whole meal and white flour from einkorn cv Monlis, bread wheat cv Serio and bread wheat cv Blasco. Thepoints represent experimental mean values; the best interpolation curve is shown.
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212210
30 and, particularly, at 38 �C. At this temperature, the mean increaseranged from 56 to 70% in whole meal flours and from 89 to 176% inwhite flours; the least difference between flour types was observedin the pregerminated Blasco sample. These data are in good agree-ment with those reported by Meredith and Simmons (1975), whoexamined four bread wheats stored up to eight years and observedan increase of falling number throughout the ageing process.
Falling number and alpha-amylase activity are strongly corre-lated parameters: alpha-amylase hydrolyses a-1,4-glucosidic
linkages, thus degrading starch and reducing gel strength,measured by the falling number test (Lunn et al., 2001).
3.4. RVA parameters
RVA profiles of whole meal and white flours during storage at38 �C are presented as Supplementary Fig. 1: the upwards shift ofthe RVA curves clearly points to a change in pasting propertiesduring flour ageing. This result is evident also in Fig. 3, which
A. Brandolini et al. / Journal of Cereal Science 51 (2010) 205–212 211
depicts the variation of final viscosity at the different storagetemperatures. In all flour samples, with the exception of Monlisflour, the viscosity increased with a hyperbolic trend, reachinga plateau during the late storage stage. The variation was stronglytemperature-dependent. The increased rates were similar betweenflour types; however, they were different between accessions,because they were lower in Monlis (on average, 47.6�13.3% at38 �C) than in Serio (129.7� 2.5%) and in pregerminated Blasco(366.1�47.2%). Similar trends were observed for other relevantRVA parameters, i.e. peak viscosity, breakdown, setback and peaktime (not shown).
The effect of storage on pasting temperatures of wheat flour hasbeen sparingly explored in the past. Loney and Meredith (1974)described an increase in peak amylograph viscosity during natural(15–20 �C) and accelerated (50 �C) storage of commercial flours;controls kept at �25 �C, on the other hand, did not change. Forflours stored at 50 �C during one year, peak viscosity reacheda plateau after 20 weeks, then declined and after 40–50 weeks wassimilar to the original value. Salman and Copeland (2007) observedthat peak and final viscosity of whole meal wheat flours stored upto 12 months at 20 and 30 �C increased significantly with storagetime, compared with samples kept at 4 �C; the rise was correlatedpositively with a growth in fat acidity.
Ageing experiments carried out in rice, summarised in Zhouet al. (2002), indicate that rice paste viscosity increases consider-ably during storage; the effect is more pronounced at highertemperatures (29 �C) than at lower (2 �C). Some months after theinitial increase in peak and final viscosity, a plateau is sometimesobserved (Teo et al., 2000); over storage times lasting several years,a steady decrease during the latter part of the ageing period isdescribed (Sawbhagya and Bhattacharya, 2001).
The ageing effect on viscosity has been variously attributed tocell wall structure decomposition (Shibuya and Iwasaki, 1984),modification of the proteins (Teo et al., 2000), increases in solubleprotein content, particularly the high molecular weight gluteninfraction (Wilkes and Copeland, 2008), changes in free fatty acidsamount (Salman and Copeland, 2007) or in starch characteristics(Loney and Meredith, 1974).
One of the traits analysed in this research was amylose content,as percentage of total starch. However, amylose did not varysignificantly during storage, even at 38 �C (data not presented),therefore suggesting that it does not influence the variation ofpasting values during flour ageing. A stable content of total amylosein wheat grain stored at 10, 25 and 45 �C over six months wasobserved also by Rehman and Shah (1999).
3.5. SDS sedimentation
The variation of SDS sedimentation during the ageing of wholemeal and white flour is presented in Fig. 4. At high temperatures,a general trend for all samples was an initial increase in sedimen-tation values followed by a rapid decline, while at low tempera-tures, the improvement was slower but more pronounced andlonger lasting. The maximum sedimentation volume was reachedafter different times, depending on the storage temperature: forexample, in whole meal flours stored at 38 �C the peak, on average,was after 28 days, but it shifted gradually to 49 (30 �C), 72 (20 �C),128 (5 �C) and 203 days (�20 �C).
At the end of the ageing period the flours stored at 20, 5 and�20 �C maintained or improved their initial performance, while thebread wheat flours stored at higher temperatures had SDS sedi-mentation values largely lower than their initial value (on average,at 38 �C Serio sedimentation value decreased 26.8� 5.10 and Blasco35.8� 5.65%). A different trend was observed for Monlis: afterreaching a maximum, SDS values of whole meal and white flour
decreased but, even after being stored at 38 �C for 374 days, werehigher than the initial values by 37.9% and 7%, respectively. Theawareness of SDS sedimentation evolution during storage mayhave a relevant impact on flour utilisation pattern in breadmaking,suggesting the best employment periods for optimal results.
Ephrat and Sinmena (1976) reported a decrease of values inwheats with high initial sedimentation stored at 30, 20, 11 and 3 �C;however, they observed relevant genotypic differences in the sedi-mentation value dynamics and no decrease was observed in cultivarswith low initial values. High storage temperatures had a much morepronounced effect than lower temperatures. On the other handNishio et al. (2004), examining breadmaking quality improvementduring flour short-term ageing (eight weeks), described an increasein farinographic stability up to two weeks after milling, and inspecific loaf volume up to four weeks after milling.
The SDS sedimentation test evaluates the breadmaking qualityof the flours; breadmaking and gluten quality are mainly related tostorage protein content and composition (Taenzler et al., 2002).Wilkes and Copeland (2008), studying protein changes in wheatkernels stored at 4 �C and 30 �C for 270 days, observed a significantincrease in soluble protein content of the samples kept at highertemperature. Protein profiling and identification revealed that themost evident change was an increase in the content of highmolecular weight glutenin subunits in the soluble fraction.
Storing wheat flours at temperatures� 20 �C is recommendedto better preserve their breadmaking performance; moreover,conservation improves to some extent the quality of slightly pre-germinated flours.
Acknowledgements
This research was financially supported by project n. 1018‘‘MonICA-Monococco per l’innovazione agricola e colturale’’,sponsored by the Regione Lombardia, Italy.
Appendix. Supplementary information
Supplementary Fig. 1. RVA profiles of whole meal and whiteflour from einkorn cv Monlis, bread wheat cv Serio and breadwheat cv Blasco whole meal flours during storage at 38 �C. Thepasting profiles shift upwards with increasing storage times.
Note: Supplementary material associated with this article canbe found in the online version at doi:10.1016/j.jcs.2009.11.013.
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