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FE.

Effect of physicochemical and empirical rheological wheat flour

properties on quality parameters of bread made from pre-fermented

frozen dough

Johannes Frauenlob a, Maria Eletta Moriano b, Ute Innerkofler a, Stefano D'Amico a,Mara Lucisano b, Regine Schoenlechner a, *

a BOKU - University of Natural Resources and Life Sciences, Department of Food Sciences and Technology, Institute of Food Technology, Muthgasse 18, 1190

Vienna, Austriab Universit�a degli Studi di Milano, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Mangiagalli 25, 20133 Milan, Italy

a r t i c l e i n f o

Article history:

Received 31 January 2017

Received in revised form

28 June 2017

Accepted 29 June 2017

Available online 30 June 2017

Keywords:

Frozen dough

Frozen storage

Flour quality

RVA

a b s t r a c t

The objective of this study was to examine the influence of flour quality on the properties of bread made

from pre-fermented frozen dough. The physicochemical parameters of 8 different wheat flours were

determined, especially the protein quality was analysed in detail by a RP-HPLC procedure. A standardized

baking experiment was performed with frozen storage periods from 1 to 168 days. Baked bread was

characterised for specific loaf volume, crumb firmness and crumb elasticity. The results were compared

to none frozen control breads. Duration of frozen storage significantly affected specific loaf volume and

crumb firmness. The reduction of specific loaf volume was different among the used flours and its

behaviour and intensity was highly influenced by flour properties. For control breads wet gluten,

flourgraph E7 maximum resistance and RVA peak viscosity were positively correlated with specific loaf

volume. However, after 1e28 days of frozen storage, wet gluten content was not significantly influencing

specific loaf volume, while other parameters were still significantly correlated with the final bread

properties. After 168 days of frozen storage all breads showed low volume and high crumb firmness, thus

no significant correlations between flour properties and bread quality were found. Findings suggest that

flours with strong gluten networks, which show high resistance to extension, are most suitable for frozen

dough production. Furthermore, starch pasting characteristics were also affecting bread quality in

pre-fermented frozen dough.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

Cereals and cereal products like bread are the largest energy

source for human nutrition (Goesaert et al., 2005). Bread making is

one of the oldest food production technologies, which underlies a

permanent fluctuation due to the changes in social habits and

consumer demands (Asghar et al., 2011; Rosell and G�omez, 2007).

One of the key advances in the last decades was the use of frozen

storage for preservation of bread and dough (Asghar et al., 2011).

Freezing technology can be applied at different processing steps of

bread production. Commonly, fully baked bread, partially baked

bread, pre-fermented dough or even unfermented dough are frozen

(Rosell, 2010). The use of pre-fermented frozen dough offers

an opportunity to meet both, product quality and economical

production of bread (Curic et al., 2008).

Since the first implementation, the quality of frozen dough has

increased markedly, yet, there is still a huge potential for process

improvement (Rosell and G�omez, 2007). Possible drawbacks asso-

ciated with this process to be solved are a decreased bread volume,

lack of texture, caused by a disintegrated crumb structure, and

dehydration of the crust, leading to flaking of crust parts (Rosell,

2010). In addition, Ribotta et al. (2001) reported faster staling for

breads prepared from frozen dough due to a higher degree of

amylopectin retrogradation. Factors that do have enormous influ-

ence on frozen dough quality are the dough preparation conditions,

freezing and thawing, use of additives, and of course the quality of

Abbreviations: dm, dry matter; RP-HPLC, reversed phase high-performance

liquid chromatography; RVA, rapid visco analyser; HE, haubelt units; GS, glutenin

subunits; HMW, high-molecular-weight; LMW, low-molecular-weight.

* Corresponding author.

E-mail address: [email protected] (R. Schoenlechner).

Contents lists available at ScienceDirect

Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

http://dx.doi.org/10.1016/j.jcs.2017.06.021

0733-5210/© 2017 Elsevier Ltd. All rights reserved.

Journal of Cereal Science 77 (2017) 58e65

Delta:1_given name

Delta:1_given name

Delta:1_given name

Delta:1_given name

Delta:1_given name

Delta:1_given name

mailto:[email protected]

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http://www.elsevier.com/locate/jcs




the raw materials (Rosell and G�omez, 2007). As the production

parameters (e.g. thawing time, baking program) in bake-off stations

cannot be adapted constantly, the possible impact of processing

conditions can be restricted to dough production in a centralized

plant. Additionally, food industry attempts to keep the use of

additives to a minimum, due to the steadily growing consumer

concerns (Smith et al., 2004). Therefore, a comprehensive knowl-

edge about the role of the rawmaterial, in particular flour quality, is

beneficial to further improve the quality of frozen dough.

Wolt and D'Appolonia (1984) studied the effect of flour quality

on frozen dough and indicated that the crude protein content is

not a reliable indicator for frozen dough quality. The findings of

Neyreneuf and Van der Plaat (1991) indicated that overly strong

wheat flours, with high values for Extensograph maximum resis-

tance can increase loaf volumes of bread from frozen dough.

However gluten network can also appear to be too strong, which is

reflected in poor loaf volume due to limited CO2 expansion (Lu and

Grant, 1999a). Flour reconstitution experiments conducted by Lu

and Grant (1999b) showed that the glutenin protein fraction had

the highest impact on frozen dough quality. A further aspect to be

mentioned is the role of starch in frozen dough. Lu and Grant

(1999a) indicated that repeated freeze-thaw cycles induce a

modification in the physicochemical properties of starch, which

consequently does have a substantial effect on the resulting dough.

A high amount of damaged starch is not desirable in frozen dough

production, as it shows adverse effects on loaf volume (Ma et al.,

2016). Besides protein and starch, alpha-amylase activity could

also have an influence on bread quality, because of their remaining

activity at low temperatures (Neyreneuf and Van der Plaat, 1991).

Currently, an elevated number of studies exist, describing

significant correlations between standardized flour analysis and

specific loaf volume of fresh bread, which was determined by

baking tests (Stojceska and Butler, 2012; Thanhaeuser et al., 2014).

However, only few researcher groups studied the influence on

quality of breadmade from frozen dough (Bhattacharya et al., 2003;

Kenny et al., 1999). These studies were often focused on specific

flour components or were conducted with a relatively small

number of different wheat flours, which limits the application of a

comprehensive correlation analysis.

The objective of this study was to define chemical, physical or

empirical rheological parameters that are possibly able to predict

the baking quality of flours for production of breads from pre-

fermented frozen dough. For this aim an extensive frozen dough

baking experiment was performed using 8 commercial wheat

flours. A detailed flour characterisation was conducted prior to the

baking experiment. Bread quality was characterised by determi-

nation of specific bread volume and texture (crumb firmness and

relative elasticity). Pre-fermented doughs were frozen over a stor-

age period of up to 24 weeks. By employing a thorough correlation

analysis, including all flour and bread parameters, the influence of

flour parameters on bread quality of pre-fermented frozen doughs,

was deeply investigated. Additionally, a further aim of this study

was to investigate, if the influence of certain parameters changes

during ongoing frozen storage.

2. Materials and methods

2.1. Materials

Eight different wheat flours (6 conventional, 2 organically pro-

duced) were provided from GoodMills Austria GmbH (Schwechat,

Austria) and Pfahnl Backmittel GmbH (Pregarten, Austria). It can be

assumed that none of them was produced from a single wheat

cultivar, since all of them were milled commercially. Flours were

stored at 4 �C in paper bags. Salt (iodised), dry yeast (saf-instant,

Lesaffre Austria AG, Wiener Neudorf, Austria) and sucrose were

obtained locally.

2.2. Methods

2.2.1. Flour quality

ICC Standard methods were used to determine flour moisture

(110/1), crude protein (105/2), a conversion factor of 5.7 was used,

ash (104/1) and fat (136). Wet gluten content (ICC 155) was

determined using the Glutomatic 2200 (Perten Instruments AB,

H€agersten, Sweden). Total Starch was determined enzymatically

(Megazyme International, Bray, Ireland) according to AACC

76e13.01. Empirical rheological properties of flours were analysed

by flourgraph E6 (Haubelt Laborger€ate GmbH, Berlin, Germany)

according to ICC standard method No. 179 and flourgraph E7

(Haubelt Laborger€ate GmbH, Berlin, Germany) according to ICC

standard method No. 180.

2.2.2. Pasting properties (RVA)

Pasting profiles of flours were determined using the RVA 4500

(Perten Instruments AB, H€agersten, Sweden). Flour (3.5 g, 14% dm)

was dispersed with 25.0 ± 0.1 ml of distilled water. The suspensions

were subjected to RVA General Pasting Method 1: holding time at

50 �C for 1 min, then heating to 95 �C over 3 min 42 s, holding

at 95 �C for 2 min 30 s, cooling to 50 �C over 3 min 48 s, holding at

50 �C for 2 min. Stirring speed was 160 rpm. The starch viscosity

parameters measured were peak viscosity, trough viscosity,

breakdown, setback and final viscosity. All measurements were

replicated three times; the results are presented as means of the

measurements.

2.2.3. Determination of glutenin subunits

Glutenin extracts were prepared according to Wieser et al.

(1998) and analysed as previously reported by Mansberger et al.

(2014), applying a gradient of 25e55% acetonitrile with 0.05% TFA

for 50 min. RP-HPLC was conducted on Shimadzu HPLC system

(Shimadzu Cooperation, Kyoto, Japan) equipped with DAD at

210 nm. Various glutenin-subunits (ub GS, HMW GS, LMW GS)

were quantified using LabSolutions Software (Shimadzu Coopera-

tion, Kyoto, Japan) as relative amounts of total chromatogram area.

The characteristic patterns shown byWieser et al. (1998) were used

to identify the subunits in the chromatograms. The ratio between

LMW GS and HMW GS was calculated, as it is a commonly used

quality index in other studies (Wieser and Kieffer, 2001).

2.2.4. Dough formulation and preparation of frozen doughs

The bread recipe was following ICC standard method 131 and

is summarised in Fig. 1. The amount of water used was deter-

mined by flourgraph E6. The baking formula was: 2500 g flour

(14% moisture basis), 2% sucrose, 1.8% salt, 1.8% dry yeast and

1500 g water (60% water absorption). First dry yeast was rehy-

drated with part of the water for 10 min (30 �C/85% RH). Then

flour, water, salt, sucrose and yeast solution were mixed with a

standard hook (Baer Varimixer RN10 VL-2, Wodschow & Co.,

Broendby, Denmark) for 1 min at 110 rpm and 5 min at 212 rpm.

Final dough temperature was 27 ± 1 �C. Pieces of 200 ± 1 g were

prepared and placed in a multiple baking pan (MULTISIZE Cake

Pan, Alan Silverwood LTD, Birmingham, UK) with 9 separate

compartments (10 � 10 cm), the central one was not used. After

a first fermentation for 30 min (30 �C/85% RH) dough pieces were

round by hand for 20 s. Fresh control breads were fermented for

further 30 min (30�C/85% RH) and then baked for 22 min (Model

60/3 W, MANZ Backtechnik GmbH, Creglingen, Germany). Frozen

doughs were fermented for 10 min and frozen in a blast freezer

(IF101L, Sagi S.p.a., Ascoli Piceno, Italy) to a core temperature of

J. Frauenlob et al. / Journal of Cereal Science 77 (2017) 58e65 59

�15 �C. Subsequently the dough pieces were packaged in air-

tight plastic bags, sealed and frozen according the defined

storage period at �18 �C. Selected measuring points (day 0, 1, 3,

7, 14, 21, 28 and 168) of bread quality were condensed in the first

period of storage, as it is known that the severest quality changes

occur during the first week of frozen storage. After frozen stor-

age, doughs were placed into baking pans and thawed in the

fermentation chamber for 45 min (30 �C/85% RH). Baking process

differed from fresh bread and lasted 28 min. Baking tests were

carried out in triplicates for all flour samples.

2.2.5. Bread quality evaluation

After baking, breads were cooled for 45 min at room tem-

perature and stored in a climate chamber (20�C/50% RH) for

135 min. Bread volume was measured twice for each loaf by

rapeseed displacement, specific loaf volume was expressed as

cm3/100 g bread. Relative volume reduction after 1, 28 and 168

days of storage was calculated according to (equ. (1)), where slvnis the specific loaf volume after n storage days and slvcontrol the

specific loaf volume of the fresh control bread, produced with the

same flour.

Fig. 1. Pre-fermented frozen dough breadmaking procedure (WA ¼ water absorption; mb ¼ moisture basis).

J. Frauenlob et al. / Journal of Cereal Science 77 (2017) 58e6560

Vred:

¼ 100%�

slvnslvcontrol

(1)

Crumb firmness was measured by TA-XT2i texture analyser

(Stable Micro Systems™ Co., Godalming, UK) using the SMS P/100

probe and 5 kg load cell. Data were evaluated using the Texture

Expert Software (Stable Micro Systems™ Co., Godalming, UK). Two

crumb samples were cut out from every loaf of bread (3� 3� 3 cm)

with a tailor-made cutting device and analysed with following

conditions: pre-test speed 5.0 mm/s, test speed 0.5 mm/s, post-test

speed 10 mm/s and test distance 9 mm (corresponding to 30%

deformation, holding time 120 s). The resulting peak force of

compression was reported as maximum crumb firmness (Fmax).

Relative crumb elasticity (FREL, %) was calculated as ratio of Fmax to

F120 (force after 120 s test time) multiplied by 100.

2.2.6. Statistical analysis

One-way ANOVA was performed by using SPSS 21 for Windows

(SPSS Inc., Chicago, IL, USA) to analyse the significance of flour

type on standard quality parameters, pasting properties, glutenin

subunits and bread properties. To determine individual differences

between groups the Tukey test was used at p > 0.05. Relationships

within flour quality characteristics and between flour quality and

bread properties were estimated by Pearson correlation coefficients.

3. Results and discussion

3.1. Analytical and empirical rheological properties of flours

Significant differences in chemical and empirical rheological

properties within the eight flours were found. The results of the

basic flour characterisations are shown in Table 1. The ash contents

ranged from 0.54 to 1.43%. The lowest ash content was found in

flour 1 and the highest in flour 3, which was a flour with high

aleurone content that is used in some typical Austrian loaf breads.

Flour protein contents ranged between 10.89 and 15.00%. For wet

gluten content, values between 24.94 and 33.02% were obtained,

flour 3 was not analysed, because through its high aleurone content

an analysis with standard methodology was not possible. In flours

2, 4 and 6 a wet gluten content lower than 30% was found, which

was suggested as a minimum value for frozen dough production by

Olivera (2011). Gluten index ranged between 94 and 98, indicating

high gluten quality for all flours. Regarding the fat content, typical

values for wheat flour where found (1.03e2.16%) whichwere highly

significant correlated with ash content (r ¼ 0.861, p < 0.01). Total

starch content of these 8 flours varied from 72.04 to 80.95%.

Basic mixing and extensibility parameters such as dough

development time and the maximum resistance to extension are

also shown in Table 1. Great differences in Flourgraph E6 values

were found; for example water absorption at 500 HE varied from

57.3 to 67.9%. The increased value of flour 3 can be contributed to its

lower endosperm quantity as a result of the high ash content

(Goesaert et al., 2005). Maximum resistance measured by Flour-

graph E7 varied between 235 and 784 HE. The organic flours 2 and

3 showed the lowest values, this was the same for energy, an

explanation for that could be the influence of growing conditions

on protein composition (Pechanek et al., 1997). Also for Flourgraph

E7 ratio, a very broad spectrum of properties was found within the

eight wheat flours. These data must be interpreted with caution,

because flourgraph E7 values are correlated but not directly

comparable with the Brabender Extensograph; data of Iancu and

Ognean (2015) has shown that values for maximum resistance

and ratio are higher and extensibility is lower in the Flourgraph E7.

3.2. Wheat flour pasting properties

As presented in Table 1 RVA viscosities show high variation and

due to the low standard deviations, significant differences between

the flour have been detected for all parameters. A diagram with

average RVA pasting curves can be found as supplementary mate-

rial. RVA pasting parameters are influenced by amylose content,

a-amylase activity, proteins, lipids and also by particle size

Table 1

Flour characteristics of the eight used flours.

Quality Testsa 1 2 3 4 5 6 7 8

Ash,b % 0.54 ± 0.01a 0.76 ± 0.01b 1.43 ± 0.03e 0.64 ± 0.03c 0.68 ± 0.03c 0.72 ± 0.02bc 0.84 ± 0.05d 0.72 ± 0.01bc

Proteinb (N x 5.7), % 11.90 ± 0.20b 11.10 ± 0.50a 12.71 ± 0.37bc 12.12 ± 0.16b 13.14 ± 0.02c 10.89 ± 0.37a 14.56 ± 0.06d 15.00 ± 0.22d

Wet gluten (ICC 155),c % 31.89 ± 0.51cd 25.63 ± 0.77a e 24.94 ± 0.29a 31.46 ± 0.02c 27.88 ± 0.54b 31.02 ± 0.36c 33.02 ± 0.22d

Gluten Index (ICC 155) 96 ± 1ab 98 ± 1b e 94 ± 3a 97 ± 2ab 97 ± 1ab 96 ± 0ab 96 ± 2ab

Fat,b % 1.03 ± 0.09a 1.37 ± 0.08c 2.16 ± 0.01f 1.23 ± 0.01b 1.58 ± 0.00d 1.71 ± 0.10e 1.48 ± 0.03d 1.21 ± 0.03b

Starch,b % 80.95 ± 0.40d 78.75 ± 1.19cd 74.59 ± 0.63ab 75.12 ± 0.97ab 72.04 ± 0.65a 76.03 ± 1.17bc 73.48 ± 1.33ab 76.98 ± 2.41bc

Flourgraph E6 (ICC 179)

Water absorption 500HE, % 60.7 ± 0.3cd 58.1 ± 0.4ab 67.9 ± 0.3e 58.2 ± 0.3ab 58.5 ± 0.6b 57.3 ± 0.1a 61.2 ± 0.1d 59.9 ± 0.0c

Dough development time, min 9.4 ± 0.7d 6.6 ± 0.1bc 5.4 ± 0.4b 1.9 ± 0.3a 6.9 ± 0.6bc 6.8 ± 0.4bc 7.8 ± 1.0cd 7.0 ± 0.7cd

Stability, min 16.6 ± 1.7e 9.2 ± 0.7bc 7.1 ± 0.5ab 5.7 ± 0.2a 10.6 ± 0.5cd 10.4 ± 0.8cd 12.8 ± 0.5d 9.6 ± 1.6cd

Degree of softening, HE 41 ± 6a 51 ± 6ab 70 ± 12c 70 ± 3c 70 ± 5bc 52 ± 4abc 55 ± 8abc 56 ± 10abc

Quality number, HE 148 ± 25c 91 ± 15b 85 ± 8b 41 ± 21a 105 ± 9b 101 ± 15b 114 ± 6bc 108 ± 13b

Flourgraph E7 (ICC 180) - 90 min

Maximum resistance (R), HE 784 ± 31f 504 ± 50b 235 ± 6a 563 ± 47bc 747 ± 47def 649 ± 14cd 760 ± 6ef 675 ± 44de

Extensibility (E), mm 158 ± 8abc 138 ± 9ab 141 ± 10ab 141 ± 3ab 146 ± 18ab 132 ± 1a 163 ± 8bc 184 ± 8c

Energy, cm2 161 ± 9d 99 ± 5b 53 ± 5a 107 ± 6bc 141 ± 31cd 118 ± 1bc 159 ± 10d 165 ± 13d

Ratio (R/E) 5.0 ± 0.4cd 3.7 ± 0.6b 1.7 ± 0.1a 4.0 ± 0.4bc 5.2 ± 0.3d 4.9 ± 0.1cd 4.7 ± 0.3bcd 3.7 ± 0.3b

RVA (ICC 162, STD1 profile)

Peak viscosity, cP 2330 ± 45a 1686 ± 48b 1280 ± 23c 1885 ± 28de 1928 ± 8eg 1793 ± 30d 2081 ± 14f 2026 ± 14fg

Trough viscosity, cP 1263 ± 25a 819 ± 24b 607 ± 3c 1094 ± 18de 1078 ± 8de 1066 ± 22de 1123 ± 7e 1066 ± 3d

Breakdown, cP 1065 ± 27a 867 ± 25b 673 ± 23c 791 ± 12d 851 ± 0bd 726 ± 14c 958 ± 9e 960 ± 16e

Setback, cP 1296 ± 24a 1094 ± 20b 993 ± 3c 1179 ± 16d 1324 ± 5a 1172 ± 6d 1460 ± 18e 1602 ± 5f

Final viscosity, cP 2561 ± 43a 1913 ± 44b 1601 ± 3c 2273 ± 34d 2401 ± 11e 2238 ± 28d 2583 ± 23af 2667 ± 4f

Peak time, min 5.98 ± 0.08abc 5.85 ± 0.03ad 5.75 ± 0.03d 6.11 ± 0.03c 5.91 ± 0.08abd 5.93 ± 0.00ab 6.05 ± 0.03bc 5.98 ± 0.03abc

Within row, values with the same following letter do not differ significantly from each other (p > 0.05).a Mean and standard deviation of three replicates.b Water-free basis.c 14% moisture basis.


distribution aswell asmilling technology (Sahlstrøm et al., 2003). As

flour components are underlying some changes during flour stor-

age, pasting properties are also influenced by flour storage duration

(Brandolini et al., 2010). The RVA-analysis was conducted only a few

days prior to the baking experiment to eliminate this influencing

factor. Over all samples, peak viscosity ranged from 1280 to 2330 cP,

trough viscosity from 607 to 1263 cP and final viscosity from 1601 to

2667 cP. Flour 3 had the lowest viscosities, therefore its high ash

content could be responsible for. Hareland (2003) found a signifi-

cant negative correlation between ash content and RVA viscosities,

also in our study a significant correlation with peak viscosity was

found (r ¼ �0.831, p < 0.05) but none with trough viscosity or final

viscosity. From the three basic parameters, breakdown and setback

viscosities were also calculated. Breakdown was lowest for flour 3

(673 cP) and highest for flour 1 (1065 cP). For setback the highest

viscosities were found with flour 8 (1602 cP) and the lowest with

flour 3 (993 cP). Remarkable high values were found for flours 7 and

8, a possible explanation remains unclear. For peak time also

significant differences were found. Lowest peak time was found

with flour 3; this is in accord with Sun et al. (2010) who showed an

increase in peak time when fat content of flour was lowered.

3.3. Glutenin subunit composition of wheat flours

Table 2 provides the results obtained from the RP-HPLC of the

glutenin fraction. Glutenins contained ub GS in a range of

0.91e3.02%. Values for flours 3 and 4 were significantly lower and

for flours 7 and 8 significantly higher than for the others. Big

differences for this minor fraction were also reported in other

studies (Wieser, 2000), furthermore there is only very little infor-

mation about the functionality of ub GS. Relative amount of HMW

GS was significantly correlated (r ¼ 0.733, p < 0.05) with flour

protein content. This correlation is in agreement with findings of

Pechanek et al. (1997) who found a higher amount of HMWGS and

flour protein, due to increased fertilization levels. The LMW/HMW-

ratios are consistent with data from other authors (Pechanek et al.,

1997; Thanhaeuser et al., 2014), but rather high. The reason for that

might be that in our study ub GS were quantified separately.

3.4. Effects of freezing and storage

The results of the bread quality evaluation are summarised in

Table 3. A two-way ANOVA revealed that both, storage time and

flour type had significant influences on all bread parameters

(specific loaf volume, Fmax, FREL). With every flour, the highest

specific volume and lowest crumb firmness (Fmax) was obtained by

the fresh control bread. A direct comparison between frozen dough

and the fresh control bread should be made with caution, because

different baking procedures were applied. During increasing frozen

storage time (1e168 days), loaf volumewas decreasing significantly

for all flours, expect for flour 2 and 3. This volume decrease has

been shown in most research papers on frozen dough stability and

is mainly attributed to reducing substance as a consequence of

yeast damage and also to ice crystal growth during storage (Rosell

and G�omez, 2007). After 168 days of storage, very low bread vol-

ume occurred and bread quality was not satisfactorily, irrespective

of flour type.

Fig. 2 illustrates the different relative volume reductions

attributed to the freezing process itself (1 day), to normal storage

(28 days) and to prolonged storage (168 days) in comparison to the

fresh control breads. Roughly, four different behaviours of volume

changes during frozen storage can be categorized.

Flour 5 which had no significant volume decrease after one day

frozen storage had also the highest flourgraph E7 ratio. This flour

shows a great stability during the freezing process itself, neverthe-

less with ongoing storage the volume is decreasing markedly. Flour

with similar properties showed superior quality in an experiment by

Inoue and Bushuk (1992). A possible explanation for that could be

that this flour has too strong properties for conventional bread

making. Onlywith an additional freezing process the dough network

is weakened enough to be able to expand optimally. Since flour 1

showed similar behaviour in the extension test, but not for bread

volume, another influencing factor could be the high proportion of

HMW GS found in flour 5. For HMW GS a positive correlation with

loaf volume was described by Wieser and Kieffer (2001).

The volume loss of the two organic flours 2 and 3 remains

constant throughout the storage, only a slight reduction was ob-

tained for the longtime storage. This unusual volume stability is

contributed to the fact that very low loaf volumes of breads were

already measured after 1 day of frozen storage and a further

decreasing is only possible within a physical minimum.

With flour 8 also a constant reduction was found, but at a very

high percentage. The loaf volumewas dramatically reduced already

after one day; in this case the freezing process itself had a huge

impact on the loaf volume in comparison to the fresh bread, irre-

spective of storage duration. Interestingly, the highest volume for

fresh bread was observed with this flour, this data highlights that

flour requirements for fresh bread and for bread made from pre-

fermented frozen dough are different. The slight, non-significant

increase in specific loaf volume of flours 2, 3 and 8 after 168 days

of frozen storage can be attributed to the fact that specific loaf

volume is concealing the true volume in this case. Since during this

longtime storage also a loss of dough weight occurred (data not

shown).

For flours 1, 4, 6 and 7 a typical reduction of bread volume

with increasing frozen storage duration, as previously described

(Bhattacharya et al., 2003; Inoue and Bushuk, 1992), was found.

Relative volume reduction of these flours was quite similar at the

same storage duration, after one day (12.4 ± 1.4%), 28 days

(22.3 ± 4.5%) and after 168 days (38.3 ± 5.7%).

Regarding crumb firmness similar behaviour as for loaf volume

was found, as a result of its relation through bread density. A steady

increase occurred with all flours, expect for flour 2. In comparison

to the results of Bhattacharya et al. (2003), the increase of crumb

firmness was substantial, it should be noted that in their study the

final proofing time was variated to decrease the quality loss. For

flours 4, 5 and 6 it was not possible to cut a representative test cube

after 168 days storage, because of their very low loaf volume and

Table 2

Proportionsa (%) of glutenin proteins in wheat flours determined by RP-HPLC.

reversed-phase HPLCb 1 2 3 4 5 6 7 8

ub GS 1.62 ± 0.15a 1.46 ± 0.09a 1.02 ± 0.12b 0.91 ± 0.05b 1.47 ± 0.08a 1.72 ± 0.12a 3.02 ± 0.19c 2.87 ± 0.13c

HMW GS 23.84 ± 0.12a 21.82 ± 0.11b 25.10 ± 0.16c 25.11 ± 0.43c 26.61 ± 0.65d 25.32 ± 0.68c 21.17 ± 0.25b 21.28 ± 0.27b

LMW GS 74.51 ± 0.14a 76.72 ± 0.05b 73.88 ± 0.28ad 73.98 ± 0.49a 71.92 ± 0.59c 72.97 ± 0.64d 75.81 ± 0.21b 75.85 ± 0.26b

LMW/HMW Ratio 3.13 ± 0.02a 3.52 ± 0.02b 2.94 ± 0.03c 2.95 ± 0.07c 2.70 ± 0.09d 2.88 ± 0.10c 3.58 ± 0.05b 3.57 ± 0.06b

Within row, values with the same following letter do not differ significantly from each other (p > 0.05).a Calculated as percent (%) of total glutenins area.b Mean and standard deviation of three replicates.


uneven crumb structure. Therefore, Fmax and FREL were not ana-

lysed. Breads made from flour 1, 2 and especially 7, obtained

desirable soft crumb structures after frozen storage up to 28 days.

In Table 3 it is demonstrated that only slight changes in FRELwere

determined. The lowest values were found with flour 3. A tenden-

tious decrease with increasing storage time can be attributed to the

loss of moisture during frozen storage (Selomulyo and Zhou, 2007).

3.5. Correlation analysis

The results of the correlation analyses are summarised in

Table 4, only flour parameters with significant correlations were

listed, therefore different parameters were listed for specific loaf

volume and Fmax. Considering the data above, wheat flours with a

wide spectrum of properties were used in this study, also several

Table 3

Effect of frozen dough storage time and wheat flour type on bread characteristics.

Storage Time (Days) Flour

1 2 3 4 5 6 7 8

Specific loaf volume, cm3/100g

0 (control) 315 ± 52abA 283 ± 28aA 192 ± 21cA 314 ± 10abA 331 ± 23abA 322 ± 14abA 335 ± 7bA 342 ± 8bA

1 267 ± 5abB 220 ± 23cB 163 ± 5eB 281 ± 13bB 330 ± 18dA 279 ± 13bB 294 ± 16bB 241 ± 11caB

3 252 ± 9abB 207 ± 2cB 168 ± 10dAB 263 ± 10abeBC 284 ± 9fB 278 ± 10efBC 270 ± 4befCDE 248 ± 5aB

7 261 ± 6abB 215 ± 10cB 154 ± 3dB 246 ± 15aC 296 ± 22eB 258 ± 7abCD 275 ± 9beBCD 212 ± 7cCDE

14 276 ± 5aAB 205 ± 11bB 162 ± 5cB 242 ± 11dC 275 ± 7aBC 243 ± 15dDE 290 ± 11aBC 195 ± 9bDE

21 255 ± 4aB 204 ± 11bB 173 ± 3cAB 249 ± 14aC 247 ± 11aD 248 ± 12aDE 248 ± 5aE 191 ± 4bcE

28 256 ± 15abB 201 ± 19cB 163 ± 15dB 241 ± 21abeC 253 ± 9abCD 231 ± 3beE 267 ± 7aDE 215 ± 13ceCD

168 196 ± 16abC 226 ± 27bcB 176 ± 15aAB 212 ± 19abcD 202 ± 9abcE 201 ± 17abcF 180 ± 15aF 233 ± 15cBC

Fmax, N

0 (control) 1.3 ± 0.1aA 1.8 ± 0.4abA 4.5 ± 0.3cA 1.7 ± 0.0aA 1.4 ± 0.3aA 1.7 ± 0.1aA 1.6 ± 0.3aA 3.3 ± 0.6bA

1 2.2 ± 0.8aAB 3.2 ± 0.5abcB 7.2 ± 0.8dAB 4.6 ± 0.6bcB 3.3 ± 0.4abB 3.8 ± 0.6abAB 2.5 ± 0.4abAB 4.9 ± 1.3cC

3 2.9 ± 0.7abAB 3.9 ± 0.6abcB 7.8 ± 0.5eBC 5.0 ± 0.8cdBC 4.5 ± 1.1bcB 5.0 ± 1.2cdABC 2.5 ± 0.5aAB 5.3 ± 0.7dC

7 3.3 ± 0.4aAB 4.5 ± 0.4abB 7.5 ± 0.5cAB 5.9 ± 1.5bcBC 4.4 ± 1.3abB 5.5 ± 1.0bBCD 3.3 ± 0.5aB 4.9 ± 0.4abBC

14 4.3 ± 0.5abBC 4.4 ± 0.8abB 8.5 ± 1.5cdBC 7.0 ± 2.1bcdCD 6.0 ± 0.8bcC 9.2 ± 3.6dE 2.6 ± 0.2aAB 6.2 ± 0.8bcdCD

21 3.4 ± 0.4aAB 3.7 ± 0.4abB 8.2 ± 0.2dBC 6.8 ± 0.7cdBCD 8.3 ± 0.8dD 7.6 ± 1.8dCDE 3.1 ± 0.9aB 5.6 ± 1.3bcCD

28 3.7 ± 1.4abB 4.1 ± 1.2abB 10.0 ± 0.6cC 8.4 ± 0.8cD 7.7 ± 1.5cD 7.9 ± 1.7cDE 2.4 ± 1.2aAB 5.2 ± 1.5bC

168 6.5 ± 3.5aC 3.8 ± 0.3aB 8.0 ± 1.9aBC e e e 6.5 ± 2.1aC 7.6 ± 1.6aD

FREL, %0 (control) 71.3 ± 1.3aA 69.0 ± 0.5bcAB 67.3 ± 0.4cdA 70.2 ± 1.9abA 71.0 ± 0.7abA 70.2 ± 0.9abAB 69.3 ± 0.7bcAB 66.4 ± 0.6dA

1 72.0 ± 0.2aA 69.5 ± 0.6cdAB 65.6 ± 0.4eA 69.9 ± 1.3bcdA 71.3 ± 1.0abcA 70.2 ± 1.2abcA 72.4 ± 0.7abB 67.9 ± 1.4deABC

3 72.1 ± 0.8aA 69.5 ± 0.9cdAB 66.2 ± 0.4eA 69.7 ± 0.9bcdAB 71.5 ± 1.5abA 69.5 ± 1.4cdABC 71.3 ± 0.3abcB 68.3 ± 0.8dBC

7 71.4 ± 0.7abA 69.9 ± 0.4bcAB 64.5 ± 1.2eAB 69.1 ± 1.5cAB 72.1 ± 0.1aA 68.6 ± 0.9dABCD 69.5 ± 0.4cAB 67.5 ± 0.6dAB

14 69.6 ± 0.8abAB 68.7 ± 1.0abcB 66.4 ± 1.4bcA 68.5 ± 3.1abcAB 69.8 ± 1.3abA 64.7 ± 4.4cD 71.6 ± 3.3aB 66.9 ± 0.7bcAB

21 71.2 ± 0.6aA 71.1 ± 1.0aA 65.0 ± 2.0dAB 68.3 ± 1.3bcAB 66.4 ± 1.1cdB 66.0 ± 2.8cdBCD 69.4 ± 0.9abAB 67.6 ± 0.5bcdABC

28 70.3 ± 1.6aA 68.8 ± 1.0abcB 66.1 ± 0.6bcA 66.2 ± 2.1bcB 67.0 ± 1.8abcB 65.3 ± 3.8cCD 70.0 ± 2.1abAB 69.0 ± 0.9abcC

168 66.8 ± 4.1aB 65.6 ± 2.6aC 62.6 ± 2.1aB e e e 67.3 ± 1.1aA 67.3 ± 1.1aAB

a Mean and standard deviation of three replicates.

Values with same capital letter, in the same column and lower cases, in the same row are not significant different (p > 0.05).

Fig. 2. Relative bread volume reduction after 1, 28 and 168 days of frozen storage in comparison to fresh control breads.


different behaviours for bread volume loss during frozen storage

were found. Thus, some significant correlations were identified

between flour properties and bread quality parameters. Signifi-

cance of correlations was changing with storage time for some

parameters. After 168 days of frozen storage no significant corre-

lations were found. This indicates that pre-fermented frozen dough

is not suitable for such long storage times. Possible solutions for this

problem could be the use of additives, modified packaging or

freezing of pre-baked frozen bread or non-fermented dough.

A significant negative correlation of ash content was found with

bread volume at most storage times. This influence can be attrib-

uted to the decreasing effect of aleurone particles on bread volume

(Stojceska and Butler, 2012). Within the RVA pasting properties,

peak viscosity was positively correlated with specific loaf volume.

These results are likely to be related to alpha-amylase activity,

which is preferred to be low for frozen dough production according

to the theory of Neyreneuf and Van der Plaat (1991). There are,

however, other possible explanations, because RVA pasting profiles

are also affected by other flour constituents (Sahlstrøm et al., 2003).

The physicochemical characteristics of starch are also influencing

RVA parameters and these have substantial effects on frozen dough

quality (Ma et al., 2016). However, in this study, peak viscosity had

the potential to predict the specific loaf volume of pre-fermented

frozen dough up to storage periods of 28 days.

Wet gluten content, which is a widely used quality indicator for

bread making quality in Europe, was significantly correlated only

with bread volume of fresh bread. Furthermore, the composition of

the gluten proteins is only poorly represented with wet gluten con-

tent, a better view can be obtained by extensibility tests which are

highly significant correlated with the gliadin/glutenin ratio (Horvat

et al., 2006). In this study the highest correlation coefficients were

found between specific loaf volume and Flourgraph E7 ratio. They

were correlated positively at a level of p < 0.01. Dough resistance

was also positively correlated with loaf volume, this is in agreement

with the correlation obtained by Kenny et al. (1999) who did a non-

fermented frozen dough experiment. Contrasting effects for fresh

bread were found by Thanhaeuser et al. (2014), where dough

resistance was negatively correlated with loaf volume of breads

produced by micro-Rapid-Mix-Test. Nevertheless, in the same study

it has been noted that a suitable baking test is essential for a repre-

sentative performance test of wheat flour, because with the applied

microbaking test different correlation coefficients were found.

Observing the correlationmatrix for Fmax (also shown in Table 4)

it must be noted that RSD for specific loaf volume (5.07%) wasmuch

lower than for Fmax (18.74%). Similar to loaf volume, significant

negative correlations were found with ash content, maximum

resistance, ratio and peak viscosity. Different to loaf volume other

significant correlations were found for Fmax. Flourgraph E6 stability

was negatively and degree of softening positively correlated to Fmax

at some storage durations. As stability was highly positively corre-

lated with dough development time (r ¼ 0.898, p < 0.01) a possible

explanation might be, that for some flours the applied kneading

time (which was set constant in this baking experiment) was too

short and were thus producing breads with increased crumb firm-

ness. Individual variation of kneading time in baking experiments is

not a common practice, but it could offer more information about

the breadmaking potential of wheat flours (Thanhaeuser et al.,

2014). Furthermore a positive correlation was found between

HMWGS content and Fmax after 21 and 28 days of frozen storage.

Taken together, these results suggest that RVA peak viscosity

and especially resistance to extension and ratio (maximum resis-

tance divided by extensibility) have great potential to predict bread

quality made from pre-fermented frozen dough. The intensity of

this correlation was not changing considerably during 28 days of

frozen storage. A note of caution is due here since a relatively

low number of 8 wheat flours were used for the calculation of

correlation coefficients.

4. Conclusions

The main aim of the current study was to identify flour quality

parameters, which can help to predict final quality of bread made

from frozen doughwithin a bakery orientated baking test setup. The

results after long-term storage highlighted that pre-fermented

frozen dough without any modifications is not suitable for storage

times up to 168 days, but after storage times of 28 days most breads

still showed acceptable quality, dependent on flour type. The most

significant influence on loaf volume was Flourgraph E7 maximum

resistance to extension and ratio and also RVA pasting parameters. It

can therefore be assumed that flours with high resistance to

extension and high pasting viscosities should be used in production

of pre-fermented frozen dough. The results of this study indicate

that loaf volume is decreasing with increasing storage duration, but

the intensity of this decrease is following different behaviours and

Table 4

Pearson correlation coefficients between flour characteristics and bread properties after different frozen storage durations (1e168 days).

Specific loaf volume, cm3/100g

Storage time (Days) fresh 1 3 7 14 21 28 168

Ash content �0.868** �0.735* �0.774* �0.754* �0.654 �0.717* �0.766* �0.542

Wet gluten 0.833* 0.288 0.435 0.212 0.262 0.056 0.471 �0.152

Maximum resistance - 90min 0.920** 0.847** 0.872** 0.882** 0.849** 0.752* 0.921** 0.178

Ratio - 90min 0.855** 0.914** 0.912** 0.962** 0.894** 0.882** 0.913** 0.132

RVA - Peak viscosity 0.830* 0.676 0.717* 0.724* 0.764* 0.682 0.860** 0.202

RVA - Setback 0.772* 0.484 0.569 0.433 0.393 0.196 0.558 0.301

Fmax, N

Storage time (Days) fresh 1 3 7 14 21 28 168

Ash content 0.834* 0.780* 0.728* 0.703 0.356 0.360 0.465 0.442

Maximum resistance - 90min �0.762* �0.842** �0.821* �0.874** �0.522 �0.406 �0.615 �0.141

Ratio - 90min �0.905** �0.876** �0.788* �0.776* �0.366 �0.251 �0.448 �0.357

Stability �0.477 �0.733* �0.715* �0.810* �0.571 �0.592 �0.693 �0.109

Degree of softening 0.379 0.625 0.628 0.640 0.436 0.748* 0.741* 0.507

RVA - Peak viscosity �0.677 �0.788* �0.818* �0.866** �0.596 �0.567 �0.681 �0.085

RVA - Breakdown �0.442 �0.701 �0.781* �0.884** �0.806* �0.782* �0.855** �0.195

HMW content �0.061 0.242 0.421 0.443 0.646 0.808* 0.793* 0.382

** Correlation is significant at the 0.01 level (2-tailed).

* Correlation is significant at the 0.05 level (2-tailed).


was highly dependent on flour properties. Another finding was that

wet gluten contentwas no reliable quality indicator for frozen dough

quality. In addition, glutenin subunit composition was not influ-

encing specific loaf volume significantly. This study suggests that

raw material quality has substantial impact on the quality of bread

made from pre-fermented frozen dough. Selecting flours according

to the described parameters can help to improve quality of frozen

dough-products. However, precise optimization of the whole pro-

duction process, from dough production to freezing and thawing,

plays also a key role tomaintain high-quality frozen dough products.

Acknowledgment

This work was financially supported by the Austrian Research

Promotion Agency (FFG Project No. 844234).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.jcs.2017.06.021.

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(Triticummonococcum L.) and breadwheat (Triticum aestivum L. ssp. aestivum)flours. J. Cereal Sci. 51, 205e212.

Curic, D., Novotni, D., Skevin, D., Rosell, C.M., Collar, C., Le Bail, A., Gabric, D., 2008.Design of a quality index for the objective evaluation of bread quality: appli-

cation to wheat breads using selected bake off technology for bread making.Food Res. Int. 41, 714e719.

Goesaert, H., Brijs, K., Veraverbeke, W., Courtin, C., Gebruers, K., Delcour, J., 2005.

Wheat flour constituents: how they impact bread quality, and how to impacttheir functionality. Trends Food Sci. Technol. 16, 12e30.

Hareland, G.A., 2003. Effects of pearling on falling number and a-amylase activity ofpreharvest sprouted spring wheat. Cereal Chem. 80, 232e237.

Horvat, D., Jurkovic, Z., Drezner, G., Simic, G., Novoselovic, D., Dvojkovic, K., 2006.

Influence of gluten proteins on technological properties of Croatian wheatcultivars. Cereal Res. Commun. 34, 1177e1184.

Iancu, M.L., Ognean, M., 2015. Use of flour-graphics technique in the compatibilityparameter extensograph brabender and flourgraph E7. J. Microbiol. Biotechnol.

Food Sci. 5, 277e281.Inoue, Y., Bushuk, W., 1992. Studies on frozen doughs. II. Flour quality requirements

for bread production from frozen dough. Cereal Chem. 69, 423e428.

Kenny, S., Wehrle, K., Dennehy, T., Arendt, E., 1999. Correlations between empirical

and fundamental rheology measurements and baking performance of frozenbread dough. Cereal Chem. 76, 421e425.

Lu, W., Grant, L., 1999a. Effects of prolonged storage at freezing temperatures onstarch and baking quality of frozen doughs. Cereal Chem. 76, 656e662.

Lu, W., Grant, L., 1999b. Role of flour fractions in breadmaking quality of frozen

dough. Cereal Chem. 76, 663e667.Ma, S., Li, L., Wang, X., Zheng, X., Bian, K., Bao, Q., 2016. Effect of mechanically

damaged starch from wheat flour on the quality of frozen dough and steamedbread. Food Chem. 202, 120e124.

Mansberger, A., D'Amico, S., Novalin, S., Schmidt, J., T€om€osk€ozi, S., Berghofer, E.,Schoenlechner, R., 2014. Pentosan extraction from rye bran on pilot scale for

application in gluten-free products. Food Hydrocoll. 35, 606e612.

Neyreneuf, O., Van der Plaat, J., 1991. Preparation of frozen French bread dough withimproved stability. Cereal Chem. 68, 60e66.

Olivera, S.D., 2011. The effect of basic raw materials in the process of wheat doughfreezing. Food Feed Res. 38, 9e19.

Pechanek, U., Karger, A., Gr€oger, S., Charvat, B., Sch€oggl, G., Lelley, T., 1997. Effect of

nitrogen fertilization on quantity of flour protein components, dough proper-ties, and breadmaking quality of wheat. Cereal Chem. 74, 800e805.

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Passos, M.L., Ribeiro, C.P. (Eds.), Innovation in Food Engineering: NewTechniques and Products. CRC Press, Boca Raton, pp. 59e79.

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Role of α-Amylase in the Pasting Behavior of WheatFlours Upon Storage

Johannes Frauenlob, Edwin Hetebrij, Stefano D’Amico,* and Regine Schoenlechner

Wheat flour pasting properties change during flour storage. Only little is

known about the contribution of α-amylase on this effect. This study aimed

to monitor the changes in rapid visco analyser (RVA) pasting viscosities of

four different wheat flours during 360 days of storage. To get an isolated

view on the influence of α-amylase, RVA curves were determined either with

water or with 2mM silver nitrate to inhibit α-amylase. After 360 days of

storage RVA final viscosity of the flours increased between 31 and 56%.

When α-amylase was inhibited it increased only by 12–28%. Remarkably,

trough viscosity did not change during a storage time of 180 days when

α-amylase was inhibited. To estimate the α-amylase activity of flours the ratio

between peak viscosities of flour with and without inhibition was calculated.

The estimation resulted in a decrease of α-amylase activity by 22–43% during

360 days of storage. These findings suggest that the decrease of α-amylase

activity is a major influencing factor in the increase of pasting viscosity

during flour storage. Additionally, differential curves from analyses with and

without α-amylase inhibition are a helpful tool to improve the understanding

of the enzymes mode of action.

1. IntroductionStarch as the major quantitative wheat flour component has asubstantial influence on its breadmaking properties.[1] There-fore, quality-determining methods, which focus on starch andstarch affecting enzymes, are used in the cereal industry tomonitor these flour characteristics. Frequently employedanalytical methods include Brabender Amylograph, RVA (RapidVisco Analyser) pasting tests, and the Falling Number

1

method.[2] It has been shown that RVA pasting viscosity andfalling number increase during ongoing storage of wheatflour[3,4] but also wheat kernels.[5,6] Pasting viscosities arerepresenting a wide range of interactions among flourcomponents during heating and cooling. They are influencedby amylose content,[7] α-amylase activity, proteins, lipids,[8] andalso by particle size distribution and milling technology.[9]

Better knowledge about the contributionof single flour components to theseviscosity changes would be beneficial touse suitable flours and/or additives forbakery products with precise raw materialrequirements. At least the influence of oneflour constituent, α-amylase, can be iso-lated, by inhibition with 2mM silvernitrate.[10] Determination of both curves,with and without α-amylase inhibition, andcalculation of their differences offers theoption to observe the impact of α-amylaseon pasting viscosities.[11] Too high, but alsotoo low α-amylase activities in wheat flourare resulting in insufficient baking qual-ity.[12] Therefore α-amylase activity of wheatflour is standardized frequently in the flourmill by the addition of fungal or maltamylases,[1] hence a fast and easy method isneeded to measure the actual status.

Several key parameters can be derivedfrom RVA curves, like peak viscosity (PV),trough viscosity (TV), final viscosity (FV) orpeak time (PT).[13] For all pasting viscosi-ties, an increase with storage time isreported, but not all values are influenced

to the same extent.[14] Fierens et al.[4] attribute this increase to therelease of free fatty acids. A slight decrease of α-amylase activityduring flour storage is reported by Brandolini et al.,[3] but itsinfluence on pasting behaviour was not considered in detail. Jiand Baik[5] showed that a decrease of α-amylase activity alsooccurs in stored wheat grains and is intensified by higher storagetemperatures. However, no controlled flour storage studies areavailable, where RVA curves of wheat flour with and without α-amylase inhibition were determined and considered together.

Detailed expertise on processes during flour storage wouldhelp to preserve product quality or even alter quality in a specificway through targeted storage techniques. Moreover, compre-hensive knowledge about this will be supportive to treat thewheat kernel as a biological unit[15] for further improvement andintroduction of new quality prediction tools.

To address these challenges, an RVA study was done on wheatflour stored up to 360 days under controlled atmosphere. RVAanalyses were performed with water or 2mM silver nitrate assolvents, in order to observe the isolated effect of α-amylase onpasting viscosity. Another aim of this study was to evaluate apossible influence of wheat flour quality and flour treatmentwith ascorbic acid and sunflower lecithin during storage.Obtained data of those pasting tests were evaluated by anextensive statistical analysis throughout the whole storage time.

J. Frauenlob, E. Hetebrij, Dr. S. D’Amico, Dr. R. SchoenlechnerDepartment of Food Sciences and TechnologyInstitute of Food TechnologyBOKU-University of Natural Resources and Life SciencesMuthgasse 18, 1190 Vienna, AustriaE-mail: [email protected]

The ORCID identification number(s) for the author(s) of this articlecan be found under https://doi.org/10.1002/star.201700123.

DOI: 10.1002/star.201700123

Keyword www.starch-journal.com

RESEARCH ARTICLE

Starch - Stärke 2017, 1700123 © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim1700123 (1 of 7)

https://doi.org/10.1002/star.201700123

http://www.starch-journal.com

2. Experimental Section

2.1. Materials

Four industrially milled wheat flours (W7c100) were providedfrom GoodMills Austria GmbH (Schwechat, Austria) andFrauenlob Muhle (Plainfeld, Austria). They were collected atthemills, directly after milling to guarantee maximum freshnessfor analysis. Flours 1 and 2 did not contain any additives. Flour 3was treated with 1.3 g/100 kg ascorbic acid and 30 g/100 kgsunflower lecithin, flour 4 with 2 g/100 kg ascorbic acid at themill. Flours with ascorbic acid and lecithin were chosen as theseadditives are frequently used in flour mills for flour standardi-zation. This means they are already in contact with flour duringstorage and possibly, interactions can occur. All flours werestored in paper bags (5 kg each) under controlled atmosphere(20 �C/50% RH). For each sampling, the contents were mixedthoroughly and 100 g flour were taken out as a representativesample.

2.2. Flour Quality Determination Prior to Storage

ICC standard methods were used to determine the (104/1), fat(136) and crude protein (105/2), using a factor of 5.7 forconverting nitrogen into protein content. Amylose content wasdetermined by enzymatic kit (cat. no. K-AMYL) obtained fromMegazyme International (Bray, Ireland). Gluten index method(ICC 155, Glutomatic 2200, Perten Instruments AB, Hagersten,Sweden) was used to determine the wet gluten content, the drygluten content and the gluten index. SDS-Sedimentation testwas performed as described by Axford et al.[16] with a final settle-time of 20min. Total titratable acidity (TTA) was determinedaccording to ICC standard method 145 with 67% ethanol assolvent and was expressed as ml of NaOH required to neutralizethe acids of 10 g flour. Rheological flour properties were analysedby flourgraph E6 (Haubelt Laborgerate GmbH, Berlin,Germany) according to ICC standardmethod 179 and flourgraphE7 (Haubelt Laborgerate GmbH, Berlin, Germany) according toICC standard method 180. All measurements were done intriplicate. Analyses were performed within a maximum storagetime of 3 days after milling.

2.3. RVA Pasting Properties

Pasting profiles of flours were determined using the RVA 4500(Perten Instruments, Hagersten, Sweden). Flour (3.5 g, 14% drymatter) was dispersed in 25.0� 0.1ml of distilled water or 2mMsilver nitrate solution. The suspensions were subjected to RVAgeneral pasting method 1 (ICC 162) with a duration of 13min.

The starch pasting parameters obtained by Thermoclinesoftware (Perten Instruments, Hagersten, Sweden) were peakviscosity (PV), trough viscosity (TV, minimum viscosity betweenPV, and FV), final viscosity (FV) and peak time (PT, time to reachPV). All measurements were replicated three times for eachsolvent; the results are presented as means of the measure-ments. Within the first 2 months of storage, pasting propertieswere measured four times (fresh, 14, 28, and 60 days) as is

common practice in the bakery industry. Additionally, long-termstorage (180 and 360 days) was also performed, in order to obtainmore information about the extent and mode of possiblechanges. To estimate α-amylase activity Eq. (1), defined byCollado and Corke,[11] was applied. To reveal the influence ofα-amylase during the whole pasting procedure, the formula wasapplied over the whole analysis time, as shown in Eq. (2).Average curves were used to calculate these coefficients.

αAAest: ¼PVAgNO3

� PVH2O

PVH2Oð1Þ

αAAest: tð Þ ¼vAgNO3

tð Þ � vH2O tð Þ

vH2O tð Þð2Þ

2.4. Statistical Analysis

One-way ANOVA was performed by using SPSS 21 forWindows (SPSS Inc., Chicago, IL, USA) to identify differencesamong the wheat flours and to analyse the significance ofstorage time on pasting viscosities and peak time. To determineindividual differences between groups the Tukey test was usedat p> 0.05.

3. Results and Discussion

3.1. Analytical and Rheological Properties of Flours Prior to

Storage

Table 1 provides an overview of the chemical and physicalproperties obtained from the freshly milled wheat flours. Ashcontent of flour 3 was significantly lower than for the otherflours, which showed no differences between them. Nosignificant differences were observed for protein content,which is a good predictor for breadmaking quality.[17] Proteincontent was higher than 13.5% for all flours, thus most likelyoutstanding breadmaking quality can be assumed for theseflours.[2] Fat content was significantly different between theflours. The lowest fat content was found for flour 3, which canbe described by its low ash content. No significant differenceswere found among amylose content. Wet gluten contentsbetween 30.5 and 33.1% were found with a constant high glutenquality, represented by a gluten index between 92 and 98. Anadditional indicator of high breadmaking quality for all fourflours was the high SDS-sedimentation volumes ranging from82 to 88ml. Significant differences were found for totaltitratable acidity, ranging between 2.88 for flour 3 and 3.30mlNaOH for flour 4. No significant differences in waterabsorption were observed by the Flourgraph E6 (a devicesimilar to the Brabender Farinograph) within the flours. Doughdevelopment time and stability were higher for flours treatedwith ascorbic acid. Flour 3, which was also substituted withsunflower lecithin showed the highest values. These increasedvalues are not necessarily connected to the flour treatment butcould also be caused by differences in wheat varieties or

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growing conditions. Nevertheless, an increase in doughdevelopment time and dough stability by emulsifier additionis expected.[18] The modifications by ascorbic acid treatmentsare clearly represented by the flourgraph E7 (a device similar tothe Brabender Extensograph) analysis. As expected, significantdifferences have been found for all flourgraph E7 parametersbetween flours with (3 and 4) and without ascorbic acid (1 and2). A considerable dough strengthening effect was observedfor flours treated with ascorbic acid. Maximum resistance,energy, and ratio increased, while extensibility was decreased byascorbic acid addition.

3.2. Storage-Induced Changes in Wheat Flour Pasting

Properties

RVA pasting viscosities and peak times are shown in Table 2for analyses with water and in Table 3 for analyses with 2mMsilver nitrate solution. The complete RVA curves can be foundin supplementary figure S1. For better visualization, the extentof change, as the percental increase since day 0, is reported.Comparing the viscosities obtained with water at day 0, PVranged from 1455 to 1867 cP, TV from 724 to 1046 cP, and FVfrom 1490 to 1957 cP. For all pasting viscosities, a continuousincrease with storage time was detected. The extent of increasewas different among the flours, however, after 180 days ofstorage, a significant increase has occurred in every flour foreach viscosity parameter. The highest percentual increase was

always found with FV, which is consistent with the dataobtained by Salman and Copeland[14] for whole meal flours.Contradicting results were reported by Fierens et al.[4] whofound the highest viscosity increase with TV, yet they applied aslightly different RVA profile and used a buffered solventduring analysis. The lowest increase for all viscosities wasfound with flour 2, followed by flour 3. The widest changeswere found for flour 1 and 4, a clear connection betweenthese changes and the flour quality parameters was notidentified. Other authors found similar differences amongwheat cultivars.[3] After 180 days of storage, peak time wassignificantly increased for all flours, except number 3, ifthis was the effect of the sunflower lecithin added remainsunclear.

With the addition of silver nitrate solution (Table 3), a similarbehaviour was identified, but changes were less pronouncedand some remarkable differences were found. Silver nitratestrongly inhibits the α-amylase activity during the RVA test, noeffect on other flour components are known today, but cannotbe excluded definitely.[10] As expected, for freshly milled flourhigher viscosities were found after AgNO3 addition comparedto the results with water as a solvent. Variations within thedifferent flours were lower, as PV varied from 2414 to 2533 cP,TV from 1550 to 1610 cP, and FV from 2604 to 2756 cP. Again,the highest percentual increase was found for FV followed byPV. Surprisingly, no significant viscosity increase was found forTV after 180 days of storage and after 360 days only in flour 1and 4, a significant increase was found. Trough viscosity can

Table 1. Quality characteristics of freshly milled wheat flour. Values are mean and standard deviation of three replicates.a)

Flour

Parameter 1 2 3 4

Ashb) (%) 0.68 � 0.01b 0.70 � 0.02b 0.53 � 0.01a 0.68 � 0.02b

Proteinb) (%) 13.63 � 0.40a 13.88 � 0.17a 13.57 � 0.11a 13.94 � 0.12a

Fatb) (%) 1.28 � 0.02b 1.42 � 0.07c 1.05 � 0.04a 1.39 � 0.03bc

Amylose (% of total starch) 25.3 � 1.5a 24.3 � 0.8a 25.1 � 1.5a 24.7 � 1.3a

Wet glutenc) (%) 30.5 � 0.6a 33.1 � 0.6c 31.1 � 0.5ab 31.8 � 0.6b

Dry gluten (%) 10.2 � 0.2a 11.1 � 0.2c 10.8 � 0.1b 10.6 � 0.1b

Gluten index (%) 96 � 2ab 92 � 3a 98 � 1b 94 � 2ab

SDS-Sedimentation (ml) 85 � 1b 88 � 0c 87 � 2bc 82 � 1a

Titratable acidity (ml NaOH) 3.05 � 0.03b 3.14 � 0.02c 2.88 � 0.03a 3.30 � 0.03d

Flourgraph E6 (ICC 179)

Water absorption (%) 61.4 � 0.2a 60.4 � 1.7a 59.8 � 0.2a 60.8 � 0.0a

Dough development time (min) 5.1 � 0.2a 5.4 � 0.3a 9.9 � 0.2c 6.6 � 0.4b

Stability (min) 6.5 � 0.5a 7.1 � 0.9ac 12.9 � 1.0c 9.0 � 0.3b

Flourgraph E7 (ICC 180) – 90min

Maximum resistance – R (HE) 427 � 18a 440 � 7a 876 � 75b 835 � 29b

Extensibility – E (mm) 208 � 1b 208 � 6b 155 � 6a 160 � 10a

Energy (cm2) 125 � 7a 129 � 2a 176 � 10b 173 � 8b

Ratio (R/E) 2.1 � 0.1a 2.1 � 0.0a 5.7 � 0.7b 5.3 � 0.5b

a)Within a row, values with the same letter do not differ significantly from each other (p> 0.05).b)water-free basis.c) 14% moisture basis.





possibly serve as a less storage dependent quality indicator forflour starch properties when α-amylase is inhibited. This resultmay be explained by the fact that during flour storage thecontent of free fatty acids increases[19] and as Tang andCopeland[20] concluded, the addition of fatty acids lowers TVof RVA profiles only marginally, but increases FV substantially.The percental increase of PV and FV was only half the sizewhen α-amylase was inhibited. Although Fierens et al.[4] andSalman and Copeland[14] contribute these viscosity increasemostly to the formation of free fatty acids, our results suggestthat the biggest influencing factor is the decrease of α-amylaseactivity during flour storage.

Also, peak time was higher in comparison to the analyseswith water. Amylose/amylopectin ratio can influence peaktime.[7] However, flours in this study did not show significantdifferences for amylose/amylopectin ratio. Therefore it is mostlikely that changes in starch structure due to α-amylase activityoccur during the heating phase, which influences pastingproperties. No interrelation between the addition of ascorbicacid or sunflower lecithin and changes in pasting propertiesduring storage became obvious. The addition of ascorbic acid,for standardization of extensibility properties, did not influence

pasting properties of wheat flour during extended storagetimes.

3.3. Decrease of α-Amylase Upon Storage

Analysis of RVA data suggests that a major decrease in enzymeactivity occurs during storage. The estimated α-amylase activityand their changes during storage, according to the formula ofCollado and Corke,[11] are shown in Figure 1. Average PVs wereused to calculate this estimated value. If the different solventsdid not affect PVs, a value of 0 would result. If PV with α-amylase inhibition were twice as high as without inhibition, avalue of one would be obtained. The estimated enzyme activitywas highest for flour 4. A substantial decrease became obviousfor all flours after 180 days of storage. Estimated α-amylaseactivity decreased by 13.3–28.9% after 180 days and by 21.5–42.7% after 360 days of flour storage. Brandolini et al.[3] alsoshowed a continuous decrease of α-amylase activity with asimultaneous increase of pasting viscosities in einkorn andwheat flours. They observed a similar degree of loss in α-amylase activity analysed by an enzymatic test method (AACCI

Table 2. RVA pasting properties of four wheat flours after different storage times.

Flour Storage duration (Days) Peak viscosity (cP) þ% Trough viscosity (cP) þ% Final viscosity (cP) þ% Peak time (min)

1 0 1805 � 3a 1011 � 5ab 1918 � 2a 6.0 � 0.0a

14 1806 � 11a 0% 984 � 15a �3% 1941 � 19ab 1% 6.0 � 0.0a

28 1853 � 20ab 3% 1024 � 15ab 1% 1989 � 17b 4% 6.0 � 0.0a

60 1898 � 15b 5% 1047 � 18b 4% 2074 � 13c 8% 6.0 � 0.1a

180 2043 � 22c 13% 1127 � 9c 11% 2317 � 25d 21% 6.1 � 0.0ab

360 2504 � 30d 39% 1438 � 19d 42% 2990 � 15e 56% 6.2 � 0.1b

2 0 1867 � 11a 1046 � 4ab 1957 � 5a 6.1 � 0.0bc

14 1869 � 29a 0% 1034 � 7a �1% 1988 � 27a 2% 6.0 � 0.0ab

28 1887 � 23a 1% 1039 7� 7a �1% 2009 � 23a 3% 6.0 � 0.0ab

60 1923 � 5a 3% 1061 � 5b 1% 2068 � 4b 6% 6.0 � 0.0ab

180 2060 � 15b 10% 1148 � 9c 10% 2273 � 9c 16% 6.1 � 0.0abc

360 2169 � 7c 16% 1236 � 3d 18% 2556 � 7d 31% 6.1 � 0.0c

3 0 1716 � 34a 907 � 27a 1776 � 25a 6.0 � 0.1a

14 1792 � 16b 4% 929 � 8a 2% 1835 � 10b 3% 6.0 � 0.0a

28 1794 � 8b 5% 951 � 5ab 5% 1860 � 8b 5% 6.0 � 0.0a

60 1896 � 22c 11% 986 � 17b 9% 1979 � 21c 11% 6.0 � 0.1a

180 2018 � 20d 18% 1060 � 15c 17% 2207 � 13d 24% 6.0 � 0.0a

360 2134 � 20e 24% 1119 � 12d 23% 2491 � 8e 40% 6.0 � 0.0a

4 0 1455 � 16a 724 � 10a 1490 � 21a 5.7 � 0.0a

14 1517 � 10b 4% 753 � 8ab 4% 1554 � 10ab 4% 5.8 � 0.0ab

28 1534 � 6b 5% 773 � 10ab 7% 1589 � 4bc 7% 5.8 � 0.0ab

60 1589 � 12c 9% 797 � 15b 10% 1671 � 20c 12% 5.8 � 0.0ab

180 1706 � 4d 17% 906 � 34c 25% 1870 � 14d 25% 5.9 � 0.1bc

360 2029 � 22e 39% 1065 � 24d 47% 2284 � 50e 53% 6.0 � 0.0c

Within a column, values with the same letter do not differ significantly from each other (p> 0.05). This was calculated separately for each flour.





Method 22–02.01). The activity decrease is faster at highertemperatures and also significantly influenced by raw materi-als.[5] Although the obtained changes are in consistency withliterature, the results must be interpreted with caution, as theapplied formula was originally introduced for sweet potato

flour. However, the results of this study suggest that animplementation of this formula gives reasonable results andshould be evaluated in future studies for its comparability withstandard test kits for α-amylase activity determination.Continuous monitoring of α-amylase activity could help tostore wheat lots with problematic enzyme activities properlyand use them for blending with low activity wheat lots.Attention has to be paid to the recent work of Ral et al.,[21] whodemonstrated that α-amylases formed through pre-harvestsprouting or late maturity have different functionality inbreadmaking. These differences cannot be shown by solelymeasuring the falling number or PV because these keyparameters only react to the absolute amount of α-amylase, butnot its individual type and functionality. Therefore, it would bebeneficial for further understanding of functionality, to monitorthe contribution of α-amylases during the whole pastingprocess.

3.4. Contribution of α-Amylase on Pasting Curves

Differential curves, representing the role of α-amylase during thepasting process, shown in Figure 2, were plotted from minute 5

Table 3. RVA pasting properties of four wheat flours with α-amylase inhibition after different storage times.

Flour Storage duration (Days) Peak viscosity (cP) þ% Trough viscosity (cP) þ% Final viscosity (cP) þ% Peak time (min)

1 0 2475 � 31a 1585 � 19a 2731 � 27a 6.3 � 0.0ab

14 2484 � 31a 0% 1592 � 28a 0% 2705 � 33a 0% 6.2 � 0.1ab

28 2479 � 12a 0% 1605 � 37a 1% 2666 � 23a �1% 6.4 � 0.0b

60 2606 � 26b 5% 1622 � 19a 2% 2868 � 13b 6% 6.2 � 0.0ab

180 2591 � 8b 4% 1555 � 33a �2% 2925 � 8b 8% 6.2 � 0.1ab

360 3042 � 13c 22% 1828 � 41b 15% 3455 � 30c 28% 6.3 � 0.1ab

2 0 2525 � 6a 1607 � 33a 2722 � 5a 6.3 � 0.1a

14 2557 � 29ab 1% 1606 � 17a 0% 2702 � 14a �1% 6.2 � 0.0a

28 2575 � 16ab 2% 1621 � 19a 1% 2691 � 15a �1% 6.3 � 0.0a

60 2653 � 14c 5% 1623 � 22a 1% 2839 � 14b 4% 6.2 � 0.0a

180 2610 � 21bc 3% 1569 � 8a �2% 2866 � 30b 5% 6.2 � 0.0a

360 2715 � 14d 7% 1617 � 15a 1% 3055 � 13c 12% 6.2 � 0.0a

3 0 2533 � 1ab 1610 � 6a 2756 � 14ab 6.3 � 0.1bc

14 2504 � 45a �1% 1605 � 52a 0% 2660 � 54a �3% 6.4 � 0.1c

28 2588 � 52ab 2% 1623 � 22a 1% 2742 � 58ab �1% 6.3 � 0.0c

60 2643 � 20bc 4% 1607 � 35a 0% 2859 � 32bc 4% 6.2 � 0.1abc

180 2714 � 23cd 7% 1583 � 18a �2% 2991 � 26cd 9% 6.1 � 0.0a

360 2800 � 29d 11% 1618 � 21a 0% 3163 � 34e 15% 6.1 � 0.1ab

4 0 2414 � 11a 1550 � 24a 2604 � 4a 6.2 � 0.0a

14 2534 � 23bc 5% 1556 � 8a 0% 2714 � 18b 4% 6.2 � 0.1a

28 2467 � 28ab 2% 1543 � 37a 0% 2587 � 15a �1% 6.2 � 0.1a

60 2550 � 18c 6% 1578 � 26a 2% 2746 � 23bc 5% 6.2 � 0.0a

180 2520 � 12bc 4% 1549 � 12a 0% 2801 � 8c 8% 6.1 � 0.1a

360 2890 � 35d 20% 1776 � 22b 15% 3168 � 38d 22% 6.2 � 0.1a

Within a column, values with the same letter do not differ significantly from each other (p> 0.05). This was calculated separately for each flour.

Figure 1. Estimated α-amylase activity (αAAest.) according to Collado and

Corke[11] during 360 days storage (20 �C, 50% RH) of wheat flours.





of the RVA test when first a considerable viscosity increaseoccurred. For better visuality, curves for short time storage (<180days) were not plotted in the figure. Regarding the shapes ofthe curves, similar behaviour was found for all flours. Asubstantial increase occurred during 5 and 7min of test time.During this increase, the starch gelatinization takes place andPV is recorded. It can be assumed that during the holding timeat 95 �C all α-amylases were inactivated.[22] Between a test time of7 and 10min the calculated ratios (Figure 2) were reaching amaximum, during this time TV is recorded. Throughoutthe cooling phase, they decrease again until the final test timeof 13min is reached. This behaviour suggests an influence ofα-amylase reaction products on viscosity during cooling. Astudy by Leman at al.[23] has revealed that α-amylases in lowconcentrations have only little impact on FV, although theyare decreasing PV and TV substantially. This could be due tothe formation of oligosaccharides, which are influencing certainpasting parameters to a different extent.[24]

In the original formula (Eq. (1)) by Collado and Corke[11] PVwas used for estimation of α-amylase activity. Tables 2 and 3show differences in peak time when different solvents wereused, resulting in a comparison of viscosities at different testtimes (Figure 1). Overall, peak times varied between 5.7 and6.4min. This time shift was neglected when calculating thedifferential curves (Figure 2). The decrease of α-amylase activityafter 180 and 360 days of storage, described in Figure 1,becomes obvious also in the differential curves (Figure 2). Withflour 4 only slight changes in the differential curves wereobtained, when compared to the other flours, although a

continuous decrease of α-amylase activity wasmonitored by Eq. (1), as shown in Figure 1. Thisexample highlights again that during the occur-rence of PV, α-amylase is most likely still active.According to these data, we can infer that thecomplete influence until inactivation of α-amylaseon pasting properties is not expressed by PV alone.

4. Conclusions

The purpose of the present study was to thoroughlydetermine the changes in RVA pasting propertiesupon wheat flour storage. This study has identified decreasing α-amylases activity during flourstorage as the major impact factor on changes inpasting behaviour. As a second major finding TVwas identified as a storage-independent keyparameter when α-amylase was inhibited. Simulta-neous determination of RVA curves with andwithout active α-amylase and calculation ofdifferential curves leads to a better understandingof the enzymes impact on wheat flour pastingproperties. This analysis technique will be benefi-cial for future research on interactions between α-amylase and other constituents of complex foodmatrices. Overall, these findings enhance thecurrent knowledge of storage-induced changes inwheat flour quality and will help to preserve ormodify wheat flour quality in a defined and

targeted way. Further research might be useful to explore towhat extent these mechanisms occur already in wheat kernelsupon storage.

Abbreviations

FV, final viscosity; PV, peak viscosity; RH, relative humidity; RVA,Rapid Visco Analyser; TV, trough viscosity

Supporting Information

Supporting Information is available from the Wiley Online Library orfrom the author.

Acknowledgement

This work was financially supported by the Austrian Research PromotionAgency (FFG Project No. 844234).

Conflict of Interest

The authors have declared no conflict of interest.

Keywords

α-amylase, rapid visco analyser, RVA

Figure 2. The contribution of α-amylase activity during RVA analysis of wheat flours

(1–4) after different storage times (0, 180, and 360 days). Differential curves were

obtained by calculation of ratio between RVA analysis with and without inhibition of

α-amylase (see Eq. (2))





Received: May 4, 2017

Revised: August 1, 2017

Published online:

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J. A. Delcour, Trends Food Sci. Technol. 2005, 16, 12.

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2015.

[3] A. Brandolini, A. Hidalgo, L. Plizzari, J. Cereal Sci. 2010, 51, 205.

[4] E. Fierens, L. Helsmoortel, I. J. Joye, C. M. Courtin, J. A. Delcour,

J. Cereal Sci. 2015, 65, 81.

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[7] M.Zeng,C.F.Morris, I. L.Batey,C.W.Wrigley,CerealChem.1997,74, 63.

[8] S. Sahlstrøm, A. B. Bævre, E. Bråthen, J. Cereal Sci. 2003, 37, 275.

[9] A. Becker, S. E. Hill, J. R. Mitchell, Cereal Chem. 2001, 78, 166.

[10] H. Olaerts, C. Roye, L. J. A. Derde, G. Sinnaeve, W. R. Meza,

B. Bodson, C. M. Courtin, J. Agric. Food Chem 2016, 64, 8324.

[11] L. S. Collado, H. Corke, J. Agric. Food Chem. 1999, 47, 832.

[12] E. S. Posner, A. N. Hibbs, Wheat Flour Milling, 2nd ed. American

Association of Cereal Chemists, St. Paul 2005.

[13] I. L. Batey, The RVA Handbook. in: G. B. Crosbie, A. S. Ross (Eds.),

American Association of Cereal Chemists, St. Paul 2007, pp. 19.

[14] H. Salman, L. Copeland, Cereal Chem. 2007, 84, 600.

[15] R. Lásztity, T. Abonyi, Food Rev. Int. 2009, 25, 126.

[16] D. W. E. Axford, E. E. McDermott, D. G. Redman, Cereal Chem. 1979,

56, 582.

[17] S. M. Thanhaeuser, H. Wieser, P. Koehler, Cereal Chem. 2014, 91, 333.

[18] A. Koocheki, S. A. Mortazavi, M. N. Mahalati, M. Karimi, J. Food

Process Eng. 2009, 32, 187.

[19] M. J. Warwick, W. H. H. Farrington, G. Shearer, J. Sci. Food Agric.

1979, 30, 1131.

[20] M. C. Tang, L. Copeland, Carbohydr. Polym. 2007, 67, 80.

[21] J.-P. Ral, A. Whan, O. Larroque, E. Leyne, J. Pritchard, A.-S. Dielen,

C. A. Howitt, M. K. Morell, M. Newberry, Plant Biotechnol. J. 2016,

14, 364.

[22] G. Muralikrishna, M. Nirmala, Carbohydr. Polym. 2005, 60, 163.

[23] P. Leman, H. Goesaert, G. E. Vandeputte, B. Lagrain, J. A. Delcour,

Carbohydr. Polym. 2005, 62, 205.

[24] L. B. Deffenbaugh, N. E. Lincoln, C. E. Walker, Starch/Stärke 1989,

41, 461.





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Die Bodenkultur: Journal of Land Management, Food and Environment 68 (1) 2017

A new micro-baking method for determination of crumb firmness properties in fresh bread

and bread made from frozen dough

Johannes Frauenlob1, Marta Nava2, Stefano D’Amico1, Heinrich Grausgruber³, Mara Lucisano2, Regine Schoenlechner1*

1 University of Natural Resources and Life Sciences Vienna (BOKU), Department of Food Sciences and Technology, Institute of Food Technology, Muthgasse 18, 1190 Vienna, Austria

2 University of Milan, Department of Food, Environmental and Nutritional Sciences (DeFENS), Via Mangiagalli 25, 20133 Milan, Italy3 University of Natural Resources and Life Sciences Vienna (BOKU), Department of Crop Sciences, Division of Plant Breeding, Konrad Lorenz

Str. 24, 3430 Tulln, Austria* Corresponding author: [email protected] Received: 23 January 2017, received in revised form: 9 March 2017, accepted: 10 March 2017

Summary

In general, micro-baking tests are used to determine the baking quality when only low amounts of test flour are available, for example, in grain breeding. Several micro-methods are described in literature, but none of them allows the determination of bread crumb texture parameters. Therefore, a micro-baking procedure that offers this option was developed, and it was also evaluated for bread made from pre-fermented frozen doughs. In this procedure, Rapid Visco Analyser (RVA) sample cans were used as baking pans. To examine the capability of this procedure, three wheat flours with different starch properties were chosen. The obtained breads were analyzed with respect to specific loaf volume, crust color and bread crumb firmness. Additionally, a storage test (0-5 days) was performed to determine the crumb firming parameters by kinetics of the Avrami equation. The obtained specific bread volumes revealed significant differences between the flours and the coefficients of variation ranged between 4.2 and 5.5%. Crumb firmness measurement was able to identify significant differences within the samples. The obtained data on firming kinetics reflected the expected properties of samples with dif-ferent starch properties. Overall, this work demonstrated the feasibility of crumb property measurement on breads on a micro-scale.

Keywords: bread staling, wheat quality, Avrami, bread crumb properties, Rapid Visco Analyser

Zusammenfassung

Mikrobackversuche werden zur Bestimmung der Backqualität bei Vorliegen von nur geringen Mehlmengen angewendet. In der Literatur sind dazu bereits mehrere Methoden beschrieben, jedoch bietet keine die Möglichkeit, Texturparameter der Brotkrume zu erfassen. Darum wurde in dieser Arbeit eine Methode entwickelt, die dies ermöglicht. Es wurde auch eine Methode für Brot aus vor-gegarten Tiefkühlteiglingen definiert. Als Backformen kamen Probenbehälter des Rapid Visco Analysers (RVA) zum Einsatz. Um die Differenzierungsfähigkeit dieser Prozedur zu bestimmen, wurden drei Weizenmehle mit verschiedenen Stärkeeigenschaften dem Test unterzogen. Von allen Broten wurde das spezifische Volumen, die Krustenfarbe und die Krumenfestigkeit bestimmt. Außerdem wurde ein Lagertest (0-5 Tage) durchgeführt, um basierend auf der Avrami-Gleichung die kinetischen Parameter des Altbackenwerdens zu er-mitteln. Die spezifischen Volumina der einzelnen Brote unterschieden sich signifikant voneinander, wobei die Variationskoeffizienten der einzelnen Versuche zwischen 4.2 und 5.5 % lagen. Auch bei der Messung der Krumenfestigkeit konnten signifikante Unterschiede zwischen den einzelnen Mehlen identifiziert werden. Die kinetischen Kennzahlen zur Beschreibung des Altbackenwerdens spiegelten wie erwartet die unterschiedlichen Stärkeeigenschaften der einzelnen Proben wieder. In der vorliegenden Arbeit konnte erfolgreich die Durchführbarkeit der Bestimmung von Krumeneigenschaften im Mikrobackversuch demonstriert werden.

Schlagworte: Altbackenwerden, Weizenqualität, Avrami, Brotkrume, Rapid Visco Analyser

Die Bodenkultur: Journal of Land Management, Food and EnvironmentVolume 68, Issue 1, 29–39, 2017. DOI: 10.1515/boku-2017-0003 ISSN: 0006-5471 online, © De Gruyter, www.degruyter.com/view/j/boku

Research Article

Entwicklung eines Mikrobackversuches zur Evaluierung der Krumeneigenschaften von frischen Broten und Broten

aus vorgegarten Tiefkühlteiglingen

Bereitgestellt von | Universitätsbibliothek Bodenkultur Wien

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Heruntergeladen am | 09.08.17 14:21

30 Johannes Frauenlob et al.


1. Introduction

Today, extensive numbers of analytical parameters are used

to describe wheat and wheat-flour quality, but accurate and

reliable predictions of end-use qualities for bakery-use are

still highly challenging (Békés, 2012a). Furthermore, the

sample sizes in early generation grain-breeding and testing

of newly tailored baking additives are limited. Therefore,

micro-methods are used to determine the quality charac-

teristics (Belitz et al., 1978; Kaur et al., 2004).

Several down-scaled versions of the common wheat-ana-

lyzing tools, such as Extensograph and Farinograph, have

been developed (Kieffer et al., 1998; Kaur et al., 2004).

Nevertheless, baking tests are still the best quality-predic-

tion tool to determine baking properties of wheat flour.

Therefore, various micro-baking tests have been estab-

lished (Sedlácek and Horćićka, 2011). Baking tests aim to

reflect the local bread-making practice; hence, many differ-

ent standardized procedures exist (Meppelink, 1981). For

instance miniaturized versions of the German Rapid-Mix-

Test (Pelshenke et al., 2007) are described by several au-

thors (Kieffer et al., 1993; Sedlácek and Horćićka, 2011)

and have been used extensively in recent studies (Schaf-

farczyk et al., 2014; Thanhaeuser et al., 2014). It was dem-

onstrated that the results of these micro-measurements are

highly associated with the conventional sized versions and

are immensely valuable in evaluating the dough and bread

characteristics (Békés, 2012b).

The available micro-baking methods are limited to de-

termination of bread volume, since analyzing the crumb

and crust properties is difficult to implement (Doekes and

Belderok, 1976). However, bread crumb and crust char-

acteristics are essential for end-use quality and consumer

acceptance of bread (Angioloni and Collar, 2009). Use of

wheat varieties with different starch pasting properties can

influence the crumb firmness significantly; also, different

surfactants can have substantial effects on the crumb prop-

erties (Goesaert et al., 2005). To screen the effects of these

factors on bread crumb properties at small scale, we de-

veloped a micro-baking test, which offers the opportunity

to measure the crumb properties and bread firming kinet-

ics. This micro-baking test used 15 g dough for one bread

and it was developed for fresh bread, but also for bread

made from pre-fermented frozen dough. To assess the dis-

crimination power and general feasibility of this test, three

wheat flours with differing starch pasting properties were

selected. These flours underwent a standardized dough

preparation and baking procedure. After a storage time of

up to 5 days, the bread volume, crumb firmness, crumb

elasticity and crust color were measured and evaluated.

2. Materials and Methods

2.1 Materials

Three different wheat cultivars have been used in this

study: bread making cv. Midas (A), waxy cvs. Waxydie (B)

and Waximum (C). The seed samples were provided by

Saatzucht Donau (Probstdorf, Austria), Dieckmann Seeds

(Rinteln, Germany) and BOKU Plant Breeding Division

(Tulln, Austria), respectively. Samples were milled on a labo-

ratory roller mill (E8, Haubelt Laborgeräte GmbH, Berlin,

Germany) and flour was sieved with 180 µm sieves. Flours

were stored for two weeks at 4°C in paper bags, before bak-

ing experiments were carried out. Salt (iodized) and dry yeast

(saf-instant, Lesaffre Group, France) were obtained locally.

2.2 Micro-Baking procedure

A schematic overview of the bread making process is shown

in Figure 1. For baking pans, unused Rapid Visco Analyzer

(RVA) sample canisters (h = 67.7 mm, d = 38 mm, wall

thickness 0.45 mm) were applied. Usually, these aluminum

canisters are exclusively used for viscosity measurements by

the RVA device (Perten Instruments, Hägersten, Sweden).

On the bottom of each pan, two holes (d = 3 mm) were

drilled for easier depanning of breads after baking (shown in

Figure 2). The baking formula included 100 g flour (14%

moisture basis), 2% salt, 2% dry yeast and 63 ml water. All

dry ingredients were equilibrated at room temperature be-

fore use. The water amount was kept constant for all flours

in this experiment, as changes would have had substantial

effects on crumb firmness of bread (Yi et al., 2009). All in-

gredients were mixed in a Flourgraph E6 (Haubelt Labor-

geräte GmbH, Berlin, Germany) for 5 minutes. Water was

pre-warmed to 30°C and added at the beginning of the

mixing. The mixing bowl of Flourgraph E6 was tempered

to 30°C, whereby a final dough temperature between 29

and 30°C was reached (and the dough temperature was

monitored). The dough was removed by hand, rounded

and placed into a fermentation cabinet (G66W, Manz

Backtechnik GmbH, Creglingen, Germany). After proof-

ing for 30 min at 30°C and 85% RH (relative humidity),

the dough was divided into 15±0.5 g portions. Each piece

was rounded by hand for 10 s and placed into an RVA

canister, which was greased carefully with baking spray


Angemeldet


31A new micro-baking method for determination of crumb firmness

properties in fresh bread and bread made from frozen dough


(Boeson-Trennwachs, CSM Deutschland GmbH, Bingen,

Germany) prior to use.

One part of such prepared dough pieces underwent a sec-

ond proofing step of 45 min (30°C/85% RH) for fresh

bread production, followed by the baking process. For the

production of frozen dough pieces, another part of the

dough pieces was submitted to a reduced second proofing

step of 30 min (30°C/85% RH) followed by freezing in

a blast freezer (IF101L, Sagi S.p.a., Ascoli Piceno, Italy)

at an ambient temperature of –36°C. The freezing dura-

tion of 20 min was experimentally determined before, as a

core temperature of –15°C was reached by these settings.

Then, the dough pieces were taken out of the cans, packed

in airtight plastic bags, sealed and stored for 1 week at

–18°C. After that, the frozen storage doughs were placed

into baking pans again, and thawed in the fermentation

chamber for 30 min (30°C/85% RH). Fresh and frozen

dough were baked under same conditions. First the bak-

Figure 1. Micro-baking procedure flow diagramAbbildung 1. Fließdiagramm des Mikrobackversuchs


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ing oven (60/3 W, Manz Backtechnik GmbH, Creglingen,

Germany) was pre-heated to 230°C, top and bottom heat.

Five bread pans were placed in the oven, evenly distrib-

uted inside the oven. Subsequently, the top temperature

was reduced to 200°C and bottom temperature to 180°C;

the breads were baked for 20 min. After baking, the breads

were cooled at room temperature within the pans for

10 min; then, the breads were removed from the pans and

were cooled for further 50 min in a controlled atmosphere

(20°C/50% RH). The baking trials were done in dupli-

Figure 2. Modified RVA cans used for micro-baking (right) and crumb texture determination (left)Abbildung 2. Bearbeitete RVA Probenbehälter zur Verwendung als Backform (rechts) und als Halterung für die Texturmessung (links)


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cate, resulting in 10 fresh breads and 10 breads from frozen

dough for each flour. Specific loaf volume and crust color

was measured for all breads. Bread crumb properties were

determined from 6 breads on the day of baking. The other

four breads were used for determination of firming kinet-

ics. For the storage study, breads were packed into plastic

bags and stored at 20°C. After 24, 48, 72 and 96 hours of

storage, one bread per sample was analyzed on each day.

2.3 Determination of bread properties

2.3.1 Specific loaf volume and crust color

Bread volume was measured twice for each loaf by rape-

seed displacement, as described by AACC method 10-05.

After weight measurements, the specific loaf volume was

expressed as cm³/100 g bread.

The determination of bread crust color was performed

with the DigiEye system (VeriVide Limited, Leicester,

GB). From top view, the bread images at controlled illu-

mination were taken, the crust color was measured with

DigiPix Software (VeriVide Limited, Leicester, GB) and

expressed according to CIELAB color space.

2.3.2 Bread crumb firmness and relative elasticity (REL)

Crumb firmness was measured by TA-XT2i texture analyz-

er (Stable Micro Systems™ Co., Godalming, GB) using the

SMS P/0-5 probe and 5 kg load cell. Data were evaluated

using the Texture Expert Software (Stable Micro Systems™

Co., Godalming, UK). Breads were cut with a sharp saw

at 2 cm height from the bottom. The lower parts were put

into a tailor-made RVA can (Figure 2, left) with 3 cm height

and 2 holes (d = 5 mm) on the bottom, for easier handling.

The RVA can with the bread was placed in the middle of

the texture analyzer instrument platform and a uni-axial

compression test was applied with the following test condi-

tions: pre-test speed 5.0 mm/s, test speed 0.5 mm/s, hold-

ing time 120 s and test distance 7 mm (corresponding to

35% deformation,). The resulting peak force of compres-

sion was reported as crumb firmness (Fmax

). Relative crumb

elasticity (FREL

, %) was calculated as a percentage ratio of

Fmax

to F120

(force after 120 s test time).

2.4 Statistical Analysis

Mean values, standard deviations and coefficients of vari-

ation were calculated using Microsoft Office Excel 2016

(Microsoft, Redmond, USA). One-way ANOVA was per-

formed by using SPSS 21 for Windows (SPSS Inc., Chi-

cago, IL, USA) to analyze the significance of flour type on

bread properties. To determine the individual differences

between groups, the Tukey test was performed at p < 0.05.

Bread staling parameters (k and n) were fitted to the Avra-

mi equation by using the website www.mycurvefit.com

(accessed 16th December 2016):

(𝐹𝐹∞ − 𝐹𝐹𝑡𝑡)(𝐹𝐹∞ − 𝐹𝐹0) = 𝑒𝑒−𝑘𝑘∗𝑡𝑡𝑛𝑛

where F∞ and F0 were the measured crumb firmness at the

beginning and final stage of bread staling and Ft corre-

sponds to firmness at time t (Cornford et al., 1964).

3. Results & Discussion

The physical properties of breads produced by the micro-

baking procedure are summarized in Table 1. For each

flour and procedure, the photos of two exemplary breads

are presented in Figure 3.

3.1 Bread Volume

Specific loaf volumes obtained by the micro-baking pro-

cedure varied between 330 and 425 cm³/100 g for fresh

bread and from 236 to 331 cm³/100 g for the frozen

dough procedure. The highest volume for fresh bread was

achieved by flour C, while for frozen dough bread, flour

B reached the highest volume. Specific volumes of waxy

wheat flours (B and C) were not significantly different or

even higher as compared to the volume of the standard

bread wheat (A). These results are likely to be related to a

higher loaf expansion of waxy wheat breads during baking

(Blake et al., 2015).

Guenzel (1981) argued that the baking process in micro-

baking is hardly comparable with standard baking tests,

because a very low oven rise occurs, and additionally,

the dough can shore up more at the walls of the baking

pans than in standard sizes. This could be critical in this

baking procedure, as the geometry of the RVA canisters

supports this disadvantage. For instance, flours B and C

show surprisingly high loaf volume for waxy wheat. Fig-

ure 3 demonstrates that breads obtained from these flours

have a rather flat top in comparison to the standard bread

wheat (A). This occurred due to the weak structure of the


Angemeldet




waxy wheat doughs, which allows a high oven rise, but

during baking, the dough partly collapses again. This ef-

fect would be higher when the bread is baked without pans

or in standardized pans with relatively larger base area and

lower height, as they are used in the ICC standard method

131, for example. However, within the used micro-pans,

the shearing of the dough at the walls is higher, thus the

collapse of the dough was probably diminished. Therefore,

the specific loaf volume could be higher in micro-pans

than in standard sized baking experiments.

The coefficients of variation were similar for each flour

and bread making procedure. Values were comparable to

data shown by Kieffer et al. (1998) for the micro-Rapid-

Mix-Test. Other studies (Sedlácek and Horćićka, 2011;

Thanhaeuser et al., 2014) reported extremely low (<0.5%)

coefficients of variation for micro-baking tests. However,

in this study, significant differences among wheat flours

could be detected.

In Figure 3, it is visualized that height measurements of mi-

cro-baking breads would only roughly describe the volume,

Figure 3. Exemplary front view images of breads obtained by micro baking procedure out of wheat flours A, B and C.Abbildung 3. Beispielhafte Fotos von Broten hergestellt mittels Mikrobackversuch aus Mehlen A, B und C


Angemeldet





as the bread volume increase does not occur evenly. For

example, a comparison between fresh breads produced by

flour C to frozen dough breads by flour A reveals on one

hand a “rectangular” shape, flat top and flat bottom (flour

A), and on the other hand a rather “circular” shape, round

top (higher rise in the middle part of the bread) and some-

times also round bottom (flour C).

3.2 Bread Firmness

The texture properties of the bread crumb were measured

by a compression test which describes the viscous and the

elastic deformation (represented by FREL

). Coefficients of

variation for maximum firmness (Fmax

) ranged between 7.5

and 21.3% and were higher for the frozen dough proce-

dure. For FREL

all coefficients of variation were lower than

7%. Significant differences between flours and bread mak-

ing procedure were found for both parameters. Maximum

firmness (Fmax

) of breads made from waxy wheat flours (B,

C) were significantly lower than from breads made with

standard bread wheat (A), which is a typical quality char-

acteristic of waxy wheat flours (Bhattacharya et al., 2002).

Moreover, there was no difference between the firmness

of fresh and frozen dough bread made by waxy wheats.

Standard bread wheat (A) showed substantial increase in

firmness with the frozen dough procedure, following the

results of numerous frozen dough studies (Rosell and

Gómez, 2007).

Results of relative elasticity (FREL

) showed diverse effects,

flour A and C showed an increase after the freezing pro-

Parameter Fresh bread (n = 10) Frozen dough bread (n = 10)

A B C A B C

Specific loaf volume, cm³/100g

Mean 359a 330ab 425c 236d 331ab 317b

Standard deviation 15 18 19 11 18 14

Coefficient of variation 4.2 5.5 4.5 4.7 5.4 4.3

Fmax

a, N

Mean 2.4a 0.9b 1.0b 5.9c 0.8b 1.0b

Standard deviation 0.2 0.1 0.1 1.3 0.1 0.2


FREL a, %

Mean 49.7a 37.6b 30.0c 59.3d 39.9b 40.5b



Color L*

Mean 80.8a 72.7b 63.1d 65.3cd 67.3c 57.5e



Color a*

Mean 11.1ab 10.6a 15.8c 15.1c 12.8b 16.7c



Color b*

Mean 37.5a 34.3bcd 35.3bc 35.8b 34.1cd 33.3d



a n = 6

Within row, values with the same following letter do not differ significantly from each other (p > 0.05)

Table 1. Specific volume, crumb firmness, crumb elasticity and crust color of breads made by micro-baking procedureTabelle 1. Spezifisches Volumen, Krumenfestigkeit, Krumenelastizität und Krustenfarbe der im Mikrobackversuch hergestellten Brote


Angemeldet




cedure, whereas flour B FREL

did not significantly change.

Crumb elasticity does not only affect the mouthfeel, but

also the cutting ability and therefore, it is an important

quality parameter in industrial bread production (Wasser-

mann, 1973).

3.3 Bread Color

To observe the repeatability of the baking procedure, color

is also a noteworthy parameter. For the measurements ex-

pressed as CIELAB parameters, only the top of the bread,

which was not in direct contact with the baking pan sur-

face, was used. The color of the sidewalls can be seen on

exemplary breads in Figure 3. For the parameter lightness

(L*), the coefficient of variation was 1.4 and 3.8%, for red-

ness (a*) 4.0 to 8.8% and for yellowness (b*) 0.5 to 3.4%.

Bread made from frozen dough had a significantly lower

L*-value than fresh bread with the same flour, this resulted

in a darker color, as shown in Figure 3. For all color pa-

rameters, significant differences between flours and bread

making procedure were identified.

3.4 Bread firming during storage

The increase in Fmax

of bread crumb, due to retrogradation

during 5 days of controlled storage is shown in Figure 4.

For both procedures, irrespective of the storage duration,

highest firmness was achieved with the standard bread

flour (A). Waxy wheat breads (B, C) showed similar be-

havior and only very little increase of firmness in the first

48 hours.

The kinetics of bread crumb firming can be described by

measuring crumb firmness after different storage periods;

this mechanism is following the model of Avrami equation

(Armero and Collar, 1998). The Avrami parameters deter-

mined by curve fitting are presented in Table 2. The pa-

rameter k is the firming rate and defines the initial stage of

firming. The Avrami exponent n indicates the nucleation

type and describes the behavioral approach to reach the

final state of staling (Amigo et al., 2016). Both parameters

revealed big differences between waxy wheat and standard

bread wheat. This behavior was expected, as slower retro-

gradation for breads with the addition of waxy wheat has

been shown by Bhattacharya et al. (2002) previously.

4. Conclusions

The main goal of the current study was to set up a micro-

baking procedure, which allows the measurement of bread

crumb texture parameters. The repeatability of this easy-

to-use baking test was sufficient to identify differences

among specific loaf volume, crust color and crumb firm-

ness of different wheat flours. An additional strength of

this test is that we also defined a procedure for bread made

from frozen dough, which is an important product today.

This study has also shown that it is possible to evaluate

staling kinetics of bread on a micro-scale. Notwithstanding

the relatively limited sample, this method has the poten-

tial to be used in further research on bakery ingredients,

to understand their influence on staling kinetics. Because

of the use of RVA cans as baking pans and as a crumb

Bread type Avrami equation parameters

n k R²

Fresh bread

Flour A 1.85 0.184 0.934

Flour B 3.42 0.030 0.998

Flour C 5.66 0.002 0.999

Frozen dough bread

Flour A 0.95 0.314 0.999

Flour B 4.92 0.005 0.999

Flour C 3.14 0.048 0.999

Table 2. Staling kinetic parameters according to the Avrami equationTabelle 2. Kinetische Parameter des Altbackenwerdens laut Avrami-Gleichung


Angemeldet





measuring stand, this method offers simple handling and

easily accessible materials. If this method owns enough se-

lectivity for determination of differences in gluten qualities

remains unclear, as this work is aimed at using flours with

different starch compositions. Another aspect is that the

mixing process of dough has not really been performed on

micro scale yet. In pre-trials, the Promylograph E3 (Ap-

paratebau Egger, St. Blasen, Austria) was tested for dough

preparation, which uses only 10 g of flour for mixing. The

resulting doughs were satisfactory, but it is only possible

to perform one dough/bread from one batch in this way.

As the main aim of this study was the determination of

crumb firming during storage of more breads from one

batch, therefore it was decided to switch to a larger dough

Figure 4. Changes of bread crumb firmness during storage at 20°C, 50% relative humidityAbbildung 4. Veränderungen der Krumenfestigkeit während der Lagerung bei 20°C, 50 % relative Luftfeuchtigkeit


Angemeldet




mixing equipment. Another reason to apply mixing on a

rather medium scale was that it was intended to imple-

ment a two-step fermentation process, which includes first

a fermentation, then a dividing step and then a second fer-

mentation, in order to simulate as close as possible to the

usual baking practices. We are aware that 100 g is still not a

suitable sample size for, for example, early generation grain

breeding.

Since this was the first approach to determine the texture

properties of bread crumb on a micro-scale with only 15 g

of dough, further studies should be performed to evaluate

correlations with standard-sized procedures.

Acknowledgment

This work was financially supported by the Austrian Re-

search Promotion Agency (FFG Project No. 844234).

Original seeds of Waximum were kindly provided by Pas-

cal Giraudeau (Secobra Recherches, Maule, France).

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Angemeldet


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NOT E

Effect of different lipases on bread staling in comparison with

Diacetyl tartaric ester of monoglycerides (DATEM)

Johannes Frauenlob | Marlies Scharl | Stefano D’Amico | Regine Schoenlechner

Institute of Food Technology, Department

of Food Science and Technology,

BOKU-University of Natural Resources

and Life Sciences, Vienna, Austria

Correspondence

Regine Schoenlechner, Institute of Food

Technology, Department of Food Science

and Technology, BOKU-University of

Natural Resources and Life Sciences,

Vienna, Austria.

Email: [email protected]

Funding information

€Osterreichische

Forschungsf€orderungsgesellschaft, Grant/

Award Number: FFG Project No. 844234

Background and objectives: Lipases are applied in breadmaking as an enzy-

matic replacement for typical bakery surfactants like Diacetyl tartaric ester of

monoglycerides (DATEM) (diacetyl tartaric ester of monoglycerides). In this

study, the influence of six commercially available lipases and DATEM on bread

staling and bread height was investigated. Therefore, a microbaking experiment

was conducted, where 15 g of dough was used for each bread. Microbaking

breads were analyzed for height, baking loss, and crumb firmness. Evolution of

crumb firmness was measured seven times during a storage period of up to

10 days, under a controlled atmosphere (20°C).

Findings: All additives increased bread height significantly in comparison with

the control (without DATEM and lipase). Maximum height was achieved by addi-

tion of DATEM. All additives had a significant antistaling effect in comparison

with the control. The lowest bread firmness throughout the whole storage period

was achieved by addition of DATEM. Between the six lipases significant differ-

ences existed, suggesting a diverse influence of their lipid products on bread stal-

ing and bread height.

Conclusions: Taken together, none of the lipases was able to replace DATEM

without the occurrence of reduced bread height and worsened staling behavior at

the applied concentrations.

Significance and novelty: Although lipase addition in general had a positive

effect on increasing bread height and retarding staling, it has to be considered in

future work that none of the studied lipases was able to replace DATEM and

great differences between the single lipases existed.

KEYWORD S

bread staling, lipase, microbaking, DATEM

1 | INTRODUCTION

In breadmaking, application of emulsifiers is widely

applied to strengthen the dough, increase the bread volume,

soften the crumb, and retard bread staling (Goesaert et al.,

2005). The mechanism is based on the amphiphilic nature

of emulsifiers, which favors complex formation with starch

and gluten proteins, leading to increased dough strength

(G�omez et al., 2004). Diacetyl tartaric ester of

monoglycerides (DATEM) is one of the most widely

applied emulsifiers, in particular, in the baking industry

(Moayedallaie, Mirzaei, & Paterson, 2010). Due to the cur-

rent legislation, enzymes can be used for clean label prod-

ucts as a replacement of conventional bakery additives,

because complete inactivation during baking is assumed

(Smith, Daifas, El-Khoury, Koukoutsis, & El-Khoury,

2004). Lipases are utilized to replace emulsifiers in bread-

making (Colakoglu & €Ozkaya, 2012). Lipases are enzymes

Received: 24 January 2018 | Accepted: 9 March 2018

DOI: 10.1002/cche.10047

Cereal Chemistry. 2018;1–6. wileyonlinelibrary.com/journal/cche © 2018 AACC International | 1

http://wileyonlinelibrary.com/journal/CCHE

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that hydrolyze lipids and form surface-active lipids in the

dough (Gerits, Pareyt, & Delcour, 2014), that then exert

functional effects in breadmaking, mostly due to the prod-

ucts formed from the polar lipids (Schaffarczyk, Østdal,

Matheis, & Koehler, 2016). Their improving effects on

bread volume (Moayedallaie et al., 2010) and dough rheol-

ogy (Colakoglu & €Ozkaya, 2012) are comparable with

DATEM. However, increasing bread volume is one impor-

tant property of emulsifiers, retarding of staling is the other

important one (Goesaert et al., 2005). According to Agus,

Tanaka, and Mority (1999), also lipase addition retards

staling. However, as different available lipases produce

diverse hydrolysis products (Gerits et al., 2014), a different

impact on staling is most likely, as the different lipid

classes produced by lipases have different impact on loaf

volume (Schaffarczyk, Østdal, & Koehler, 2014).

As there are indications that antistaling properties of

lipases are different compared to DATEM, a comprehen-

sive storage study was performed to study these properties.

Therefore, DATEM and a range of six different commer-

cially available lipases were chosen, and a microbaking

experiment was conducted followed by repeated bread

firmness determinations over 10 days.

2 | MATERIALS AND METHODS

2.1 | Materials

Commercial bread wheat flour type W700 (GoodMills Aus-

tria GmbH, Schwechat, Austria) was used in this study. Six

different commercially available lipases were applied in

this experiment: Lipase 1 (Alphamalt EFX Mega, M€uhlen-

chemie GmbH & Co. KG, Ahrensburg, Germany), Lipase

2 (POWERBake� 4090, Danisco A/S, Copenhagen, Den-

mark), Lipase 3 (LipopanTM F BG, Novozymes A/S, Bags-

værd, Denmark), Lipase 4 (Panamore� Golden, DSM,

Heerlen, Netherlands), Lipase 5 (BrennBake LIP 112,

Brenntag AG, M€ulheim, Germany), Lipase 6 (VERON�

Hyperbake-ST, AB Enzymes GmbH, Darmstadt, Germany).

As a reference, the widely applied emulsifier DATEM

(Panodan� A2020, Danisco A/S, Copenhagen, Denmark)

was applied. Dry yeast (saf-instant, Lesaffre Austria AG,

Wiener Neudorf, Austria), sucrose, and salt (NaCl, iodized)

were obtained locally.

2.2 | Lipase activity assay

Specific lipase activity was determined using the para-

nitrophenyl palmitate (pNPP) assay according to Gupta,

Rathi, and Gupta (2002). Lipase solutions were pre-

pared dissolving 1 mg in 1 ml phosphate puffer (pH

7.5). The substrate solution contained 1 mM pNPP dis-

solved in 2-Propanol. The assay was carried out using

200 ll phosphate puffer, 20 ll substrate solution, and

50 ll lipase solution. Microtiter plates were used, and

incubation time was 20 min at 20°C. The reaction pro-

duct, p-nitrophenol was detected spectrophotometrically

at 410 nm. Analysis was performed in triplicate, and

enzyme activity was expressed as U/g, defined as

amount of enzyme releasing 1 lmol of p-nitrophenol

per minute.

2.3 | Microbaking experiment

Breadmaking procedure was performed according to our

recently developed microbaking procedure, which allows

accurate determination of crumb firmness on a microscale

(Frauenlob et al., 2017). The basic bread formulation con-

tained 100 g wheat flour, 1.5 g sucrose, 2.0 g NaCl, 2.0 g

dry yeast and 63 ml water. As recommended from the

application range in the data sheets, for all lipases 15 ppm,

except lipase 1 (50 ppm), were added to the bread formula-

tion. The concentration chosen for DATEM was 0.5%; all

concentrations were based on flour weight. A control bread

was baked without DATEM or lipases. Dough was pro-

duced by mixing all ingredients on a flourgraph E6 (Hau-

belt Laborgeraete GmbH, Berlin, Germany) for 5 min.

Mixing resulted in a final dough temperature between 29°C

and 30°C. The dough was removed, rounded, and a first

proofing step was performed for 30 min at 30°C and 85%

rh. Then, dough was divided into 11 portions of

15 � 0.5 g each, rounded by hand, and placed into a

Rapid Visco Analyzer (Perten Instruments, Haegersten,

Sweden) sample canister. After a second proofing step

(45 min at 30°C and 85% rh), doughs were baked in a

standard bakery oven (60/3 W, Manz Backtechnik,

Creglingen, Germany) for 20 min. Breads were cooled for

10 min at room temperature, then removed from the pans

and equilibrated for further 50 min within a controlled

atmosphere (20°C, 50% rh). After that breads were

weighed for calculation of baking loss, and bread height

was determined using a caliper. Two breads were then ana-

lyzed for crumb firmness, all others were packaged, each

in a single airtight plastic bag (ZIPPER� 1 l, Toppits�,

Minden, Germany) and stored at 20°C until further crumb

firmness analysis was performed. Dough was prepared in

duplicate, resulting in 22 microbaking breads for each for-

mulation.

2.4 | Crumb firmness determination

After a storage time of 1, 2, 3, 4, 7, and 10 days breads

were analyzed for crumb firmness according to the previ-

ously developed procedure (Frauenlob et al., 2017).

Breads were cut at a height of 2 cm from the bottom,

and a compression test was performed using a TA-XT2i

2 | FRAUENLOB ET AL.

texture analyzer (Stable Micro SystemTM Co., Godalming,

UK). The recorded peak force was reported as crumb

firmness. For each storage duration, 3 breads were ana-

lyzed in total.

2.5 | Statistical analysis

One-way ANOVA was performed by using SPSS 21 for

Windows (IBM, Armonk, NY, USA) to analyze any signif-

icant effect of the formulation on bread height, baking loss,

and crumb firmness. To determine individual differences

between groups, the Tukey test was applied at p > .05.

2.6 | Avrami curve fitting

Bread staling parameters (k and n) were fitted to the Avrami

equation by using the website www.mycurvefit.com

(accessed 2017, October 31st). Average values of crumb

firmness for each storage duration were used for curve

fitting.

FðtÞ ¼ ð1� e�k�tnÞ � Fmax � F0ð Þ þ F0 (1)

where Fmax and F0 were measured crumb firmness at the

beginning and final stage (10 days) of bread staling, and F

(t) corresponds to firmness at time t (Cornford, Axford, &

Elton, 1964).

3 | RESULTS AND DISCUSSION

3.1 | Lipase activity

Table 1 provides the results of the determination of the

specific lipase activity. Lipase activity ranged between 8.32

and 12.02 U/g. No significant difference between Lipase 4,

5, and 6 was found. Enzyme activities of lipases 3, 2, and

1 were significantly lower; nevertheless, lipase 1 was

applied at 50 ppm in the baking experiments as recom-

mended. A note of caution has to be paid regarding a pos-

sible different pH dependency of the lipases, because the

actual pH in bread dough is clearly lower than 7.5 as in

this procedure. This fact limits the transferability on the

actual activity in the dough, therefore the concentrations in

the baking experiments were chosen similar to each other

and within the range recommended by the manufacturers.

Additionally, the work of Colakoglu and €Ozkaya (2012)

demonstrated that lipase effects are almost dosage-indepen-

dent within the recommended range.

3.2 | Influence of lipase on bread height andbaking loss

Figure 1 provides the results obtained for bread height and

weight loss during baking. The measured bread height can

be used as an estimate of the loaf volume, as pan breads

were produced in this study. With all applied additives,

bread height was significantly increased in comparison with

the control. Bread height was highest by using 0.5%

DATEM, followed by lipase 6. Bread height was signifi-

cantly lower for all lipases in comparison with DATEM,

however, individual performance of lipases varied also

noticeably. These differences among the loaf volumes were

not found in the study of Moayedallaie et al. (2010) among

loaf volume, the different test baking setup could be a pos-

sible explanation for that. The differences between bread

heights between the six lipases were most likely related to

their different enzymatic reaction products and especially

due to the ratios between lipase-treated polar and nonpolar

lipids as described by Schaffarczyk et al. (2016). An influ-

ence of the applied concentrations cannot be excluded defi-

nitely as they were different. Lipase 6 with the highest

activity did also result in the highest bread height, how-

ever, a significant correlation between the measured

enzyme activities and the bread height was not found (data

not shown).Interestingly, the highest baking loss was

observed for DATEM. The reason for that might have been

that due to the larger surface of the bread, higher loss of

water occured during baking.

3.3 | Bread storage evaluation

Evolution of crumb firmness during storage is reported in

Table 2. The lowest initial crumb firmness was found for

the formulation containing DATEM, not significantly dif-

ferent from lipase 4 and 6. The highest initial crumb firm-

ness was found for the control breads. Crumb firming

kinetics is highly influenced by the initial crumb structure

(Armero & Collar, 1998), which can be seen by the further

evolution of crumb firmness during storage. Throughout

TABLE 1 Specific activity of lipases determined by the pNPP

assay at pH 7.5 calculated as amount of p-nitrophenol released per

minutea

Lipase activity, U/g

Applied concentration

in the baking trialsb,c

Lipase 1 8.32 � 0.10d 50 ppm (0.041 U)

Lipase 2 9.26 � 0.06 cd 15 ppm (0.014 U)

Lipase 3 10.26 � 0.58bc 15 ppm (0.015 U)

Lipase 4 11.35 � 1.15ab 15 ppm (0.017 U)

Lipase 5 11.87 � 0.57a 15 ppm (0.018 U)

Lipase 6 12.02 � 0.16a 15 ppm (0.018 U)

aMean values with the same following letter in the same column are not

significantly different (p > .05).bFlour basis.cValues in brackets represent the calculated specific lipase activities in the

baking trials.

FRAUENLOB ET AL. | 3

http://www.mycurvefit.com

the complete storage time, the lowest firmness was always

determined for breads containing DATEM. After 1 and

2 days of storage, no significant differences were found

between the six lipases, but during further storage

(3-10 days), differences became apparent between the six

lipases. The lowest crumb firmness was measured at each

measurement point for the bread containing lipase 6. This

suggests that the reaction products obtained after addition

FIGURE 1 Bread height and baking

loss of breads obtained by microbaking

procedure supplemented with lipases and

Diacetyl tartaric ester of monoglycerides

(DATEM). Mean values with the same

letter on top of the bar are not significantly

different (p > .05)

TABLE 2 Crumb firmness and staling kinetic parameters of microbaking supplemented with different lipases and Diacetyl tartaric ester of

monoglycerides (DATEM) during 10 days of storage at 20°C

Sample

Crumb firmness (N)a Avrami parameters

1 hr 1 d 2 d 3 d 4 d 7 d 10 d n 1/k R²

Control 4.1d 7.6b 13.1c 18.4d 19.5d 24.9c 24.7c 1.50 4.92 .99

DATEM 1.6a 3.5a 5.4a 6.6a 8.1a 9.0a 13.9a 1.10 6.29 .94

Lipase 1 3.1bc 4.7a 7.2ab 10.8abc 12.6abc 17.4bc 18.7ab 1.57 7.93 .99

Lipase 2 3.4cd 5.1a 8.4b 13.2c 14.7bcd 17.6bc 18.0ab 1.81 7.64 .99

Lipase 3 3.3cd 5.4a 9.6b 13.9 cd 16.3bcd 17.2bc 25.1c 1.16 6.36 .95

Lipase 4 2.3abc 4.1a 7.3ab 11.6bc 11.8abc 15.2ab 16.7ab 1.45 5.01 .98

Lipase 5 2.8bc 4.7a 8.4b 11.3abc 16.8cd 15.6ab 20.5bc 1.28 5.82 .94

Lipase 6 1.9ab 3.5a 6.7ab 8.0ab 10.1ab 13.8ab 14.2a 1.48 6.70 .99

aMean values with the same following letter in the same column are not significantly different (p > .05).

FIGURE 2 Evolution of crumb

firmness during 10 days of storage at 20°C,

as affected by Diacetyl tartaric ester of

monoglycerides (DATEM) and six different

lipases. The fitted staling curve according

to Equation. 1 is shown as dashed line and

compared with the measured values for

crumb firmness (◆)


of lipase 6 were obviously more suitable to slow down

starch retrogradation, although this phenomenon would

need to be confirmed by detailed measurements.

The measured values for crumb firmness during

10 days of storage in comparison with the fitted staling

curve are presented in Figure 2. Avrami parameters

derived from the curve fitting were the Avrami exponent

n, indicating the type of nucleation, and the rate 1/k,

describing the firming rate (Amigo, del Olmo Alvarez,

Engelsen, Lundkvist, & Engelsen, 2016). Avrami expo-

nents ranging from 1.10-1.81 were found. The lowest

exponent was found with DATEM, suggesting that the

crumb firmness is slowly converging toward the final

limiting firmness. The highest exponent was found with

lipase 2, resulting in a more sigmoidal shape (Armero &

Collar, 1998). However, as it is shown in Figure 2, the

initial and the limiting firmness are highly influencing

the characteristics of the fitted staling curve. In this

study, the firmness after 10 days of storage was applied

as the limiting firmness for curve fitting, although it

must be noted that bread firmness can increase over

much longer storage times (He & Hoseney, 1990). To

improve the fitting model, limiting firmness can also be

estimated (Aguirre, Osella, Carrara, S�anchez, & Buera,

2011), nevertheless, fewer data points are necessary to

perform a comprehensive curve fitting. This discrepancy

highlights the problem of applying Avrami model on

bread staling, although if this is noted; it is still a useful

mathematical model (Armero & Collar, 1998). The val-

ues for 1/k were ranging between 4.92, suggesting the

fastest firming process and 7.93, suggesting the slowest

firming process (Amigo et al., 2016).

Taken together, continuous measurement of crumb

firmness and calculation of Avrami parameters showed

that the slowest bread firming occurred when DATEM

was applied. Antistaling effect of lipases was existent, but

different among them, the most substantial effect occurred

with lipase 6. Same as for the bread height, it is most

likely that the different lipase products induce different

antistaling effects (Schaffarczyk et al., 2016). However,

the different enzyme activities might also have influenced

this effect.

4 | CONCLUSIONS

Although lipase addition, in general, had a positive effect

on increasing bread height and retarding staling, it has to

be considered in future work that none of the studied

lipases was able to replace DATEM and great differences

between the single lipases existed. For future studies on

baking lipases, the antistaling effect of the individual

lipase-produced lipid products should be studied in detail.

ACKNOWLEDGMENTS

This work was financially supported by the Austrian

Research Promotion Agency (FFG Project No. 844234).

REFERENCES

Aguirre, J. F., Osella, C. A., Carrara, C. R., S�anchez, H. D., & Buera,

M. D. P. (2011). Effect of storage temperature on starch retrogra-

dation of bread staling. Starch/Staerke, 63, 587–593. https://doi.

org/10.1002/star.201100023

Agus, S. T., Tanaka, N., & Mority, N. (1999). Effect of lipase com-

bined with a-amylase on retrogradation of bread. Food Science

and Technology Research, 5, 356–361.

Amigo, J. M., del Olmo Alvarez, A., Engelsen, M. M., Lundkvist,

H., & Engelsen, S. B. (2016). Staling of white wheat bread

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tribution and kinetic modeling of hardness and resilience. Food

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Armero, E., & Collar, C. (1998). Crumb firming kinetics of wheat

breads with anti-staling additives. Journal of Cereal Science, 28,

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Colakoglu, A. S., & €Ozkaya, H. (2012). Potential use of exogenous

lipases for DATEM replacement to modify the rheological and

thermal properties of wheat flour dough. Journal of Cereal

Science, 55, 397–404. https://doi.org/10.1016/j.jcs.2012.02.001

Cornford, S., Axford, D., & Elton, G. (1964). The elastic modulus of

bread crumb in linear compression in relation to staling. Cereal

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Frauenlob, J., Nava, M., D’Amico, S., Grausgruber, H., Lucisano, M.,

& Schoenlechner, R. (2017). A new micro-baking method for

determination of crumb firmness properties in fresh bread and

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suches zur Evaluierung der Krumeneigenschaften von frischen

Broten und Broten aus vorgegarten Tiefk€uhlteiglingen. Die

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Gerits, L. R., Pareyt, B., & Delcour, J. A. (2014). A lipase based

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Caballero, P. A. (2004). Functionality of different emulsifiers on

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pean Food Research and Technology, 219, 145–150. https://doi.

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Gupta, N., Rathi, P., & Gupta, R. (2002). Simplified para-nitrophenyl

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FRAUENLOB ET AL. | 5



https://doi.org/10.1016/j.foodchem.2016.02.162


https://doi.org/10.1006/jcrs.1998.0190

https://doi.org/10.1016/j.jcs.2012.02.001



https://doi.org/10.1016/j.tifs.2004.02.011

https://doi.org/10.1016/j.tifs.2004.02.011

https://doi.org/10.1007/s00217-004-0937-y

https://doi.org/10.1007/s00217-004-0937-y

https://doi.org/10.1016/S0003-2697(02)00379-2

emulsifier. Food Chemistry, 122, 495–499. https://doi.org/10.

1016/j.foodchem.2009.10.033

Schaffarczyk, M., Østdal, H., & Koehler, P. (2014). Lipases in wheat

breadmaking: Analysis and functional effects of lipid reaction

products. Journal of Agricultural and Food Chemistry, 62, 8229–

8237. https://doi.org/10.1021/jf5022434

Schaffarczyk, M., Østdal, H., Matheis, O., & Koehler, P. (2016).

Relationships between lipase-treated wheat lipid classes and their

functional effects in wheat breadmaking. Journal of Cereal

Science, 68, 100–107. https://doi.org/10.1016/j.jcs.2016.01.007

Smith, J. P., Daifas, D. P., El-Khoury, W., Koukoutsis, J., & El-

Khoury, A. (2004). Shelf life and safety concerns of bakery prod-

ucts—a review. Critical Reviews in Food Science and Nutrition,

44, 19–55.

How to cite this article: Frauenlob J, Scharl M,

D’Amico S, Schoenlechner R. Effect of different

lipases on bread staling in comparison with Diacetyl

tartaric ester of monoglycerides (DATEM). Cereal

Chem. 2018;00:1–6. https://doi.org/10.1002/

cche.10047




https://doi.org/10.1021/jf5022434

https://doi.org/10.1016/j.jcs.2016.01.007

https://doi.org/10.1002/cche.10047

https://doi.org/10.1002/cche.10047

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This work was financially supported by the Austrian Research Promotion Agency (FFG Project No. 844234).

Influence of freezing technology on quality of pre-fermented frozen dough

Department of Food Science and Technology (DLWT)

Institute of Food Technology

MATERIALS & METHODS

As shown in Fig. 1, a standardized frozen dough

breadmaking experiment was done. Nine different

freezing configurations (Tab. 1) were used to obtain

different freezing curves (Fig. 2). Evaluation of final

bread quality included determination of specific bread

volume, crumb firmness, crumb porosity and crust

color. Proteins from thawed dough were extracted with

guanidine-hydrochloride (GuHCl). Protein content

content of extract was determined with bradford

method. Content of free sh-groups was measured with

ellmanns reagent.

Johannes Frauenlob1, Elisabeth Tatschl1, Stefano D‘Amico1 and Regine Schoenlechner1 1 Institute of Food Technology, Department of Food Science and Technology (DLWT), BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. [email protected]

INTRODUCTION & AIM

In comparison to conventional bread making, the use of

frozen dough technology needs stricter control of

processing conditions. Only detailed process control

can lead to products with quality comparable to breads

from conventional breadmaking. The most important

step is the freezing procedure, which has great

influence on bread quality (1).

Therefore different freezing rates and techniques,

including cryogenic freezing with CO2 and N2, were

applied. After 1, 4 and 12 weeks of frozen storage. the

final bread and dough quality was evaluated.

References 1. ROSELL CM, GOMEZ M. Frozen Dough and Partially Baked Bread: An Update. Food Rev Int 2007; 23: 303–319.

CONCLUSIONS

• Freezing is a quality-determining process step in

production of pre-fermented frozen dough

• In comparison to a conventional air blast freezing

at -40°C, freezing duration can be reduced by

40% with usage of CO2 at -75°C

• Temperatures lower than -75°C lead to inferior

bread quality

• After 4 weeks of frozen storage positive effects of

fast freezing were overcome by storage effects

Tab. 1: Applied freezing procedures for freezing of pre-

fermented wheat dough to -15°C core temperature.

Fig. 3: Specific loaf volume of breads made from frozen

dough produced with different freezing processes

(shown in Tab.1) after frozen storage at -18°C over 1, 4

and 12 weeks. Values with the same letter and the

same storage duration are not significantly different (P

< 0.05).

RESULTS

The different freezing configurations resulted in freezing

rates from 0.25 to 2.00°C/min (Fig. 2 and Tab. 1). After

1 week of frozen storage, highest specific bread volume

was achieved by CO2 cryogenic freezing at -40°C,

followed by air blast freezing at -40°C. Temperatures

lower than -75°C resulted in very low volume and

inferior quality (Fig. 5). After 4 and 12 weeks of frozen

storage only slight differences in bread quality were

found. Results were similar for both wheat flours.

Higher freezing rates resulted also in lower weight loss,

due to reduced time without packaging. It can be

assumed that freezing rates around 0.8 to 1.2°C/min

are preferable for production of frozen dough. Yet, for

prolonged frozen storage, lower freezing rates are not

detrimental.

One of the key advances in bread-making technology in the last decades was the use of frozen storage

for preservation of bread and dough. Freezing technology can be applied at several process steps of

bread production. Common practices are freezing of fully baked bread, partially baked bread, pre-

fermented dough or unfermented dough. Although the quality of frozen dough has increased markedly

since its first implementations, still quality drawbacks occur, such as decreased bread volume, poor

texture, crust fissures, worsened crumb structure or splitting of crust. Factors that influence frozen dough

quality are the quality of raw materials, use of additives and processing conditions, ranging from dough

preparation to freezing and thawing. One of the most important process steps is the freezing process

itself. To study the suitability of different freezing technologies for the production of pre-fermented frozen

dough, a standardized baking experiment using two different types of wheat flour (wet gluten content

32% and 27%) was performed. Freezing technologies investigated were a standard blast air freezer and

an experimental cryogenic freezer, using either N2 or CO2 as cooling gas. Processing temperatures

ranging from -20°C to -120°C were used to freeze dough pieces of 200 g each to a core temperature of

-18°C, which took between 20 min and 3 h. After a frozen storage period of 1, 4 and 12 weeks, breads

were baked and analyzed. After 1 week of frozen storage, highest specific bread volume was achieved

by CO2 cryogenic freezing at -40°C, followed by air blast freezing at -40°C. Temperatures lower than

-75°C resulted in very low volume. After 4 and 12 weeks of frozen storage only slight differences in bread

quality were found. Results were similar for both wheat flours. These findings indicate that there is an

ideal freezing rate or freezing temperature, which should not be too fast or too slow. Nevertheless for

frozen storage times of a few weeks the influence of the freezing process itself is decreased and

overlapped by storage effects.

ABSTRACT

No. Freezing Procedure

Freezing

Rate [°C/min]

Duration

+26 to -15°C [min]

1 -20°C non circulating air 0.25 166.3

2 -30°C non circulating air 0.58 70.7

3 -40°C air blast freezer 0.99 41.5

4 -40°C cryogenic CO2 0.86 47.7

5 -60°C cryogenic CO2 1.04 39.3

6 -75°C cryogenic CO2 1.76 23.3

7 -75°C cryogenic N2 1.70 24.3

8 -100°C cryogenic N2 2.00 20.5

9 -120°C cryogenic N2 1.84 22.5

Fig. 2: Temperature evolution during freezing, in the

center of a 200 g pre-fermented wheat dough.

Fig. 5: Formation of fissures during thawing of dough

frozen with N2 at 120°C.

-15

0

15

30

0 30 60

T [

°C]

t [min]

-30°C non circulating air

-40 °C conventional blast freezer

-75 °C cryogenic CO2

-100°C cryogenic N2

bc abc cd

d

bc bc bc

a ab

1,5

2,0

2,5

3,0

3,5

1 2 3 4 5 6 7 8 9

sp

ecif

ic lo

af

vo

lum

e [

cm

³/g

]

1 week frozen storage

b b b b

b b

a a a

1,5

2,0

2,5

3,0

3,5

1 2 3 4 5 6 7 8 9

sp

ecif

ic lo

af

vo

lum

e [

cm

³/g

]

4 weeks frozen storage

d cd d d

ab cd

d

a bc

1,5

2,0

2,5

3,0

3,5

1 2 3 4 5 6 7 8 9

sp

ec

ific

lo

af

vo

lum

e

[cm

³/g

]

Freezing Procedure No.

12 weeks frozen storage

Fig. 1: Breadmaking procedure for production of pre-

fermented frozen dough.

Fig. 4: Cryogenic freezing cabinet IBF14675 (Packo,

Belgium), after freezing with -75°C CO2.

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Diese Arbeit wird finanziell von der Österreichische Forschungsförderungsgesellschaft (FFG Projekt Nr. 844234) unterstützt.

Qualitätsverbesserung von teilgebackenen tiefgekühlten Broten durch Pflanzenfasern

Department für Lebensmittelwissenschaften und Lebensmitteltechnolgie (DLWT)

Institut für Lebensmitteltechnologie

MATERIAL UND METHODEN

Folgenden Hydrokolloide wurden eingesetzt: Weizenfaser WF200, WF600-30 und WF600, Psyllium P95, Bambusfaser BAF90, BBL100 (WF600, P95 und KF401) und BBL500 (WF600 + P95), CMC und Guarkernmehl (J. Rettenmaier & Söhne, Rosenberg, Deutschland)

RVA-Analyse von Mehl/Faser-Mischungen

3,5 g Weizenmehl W700 und je 175 mg Pflanzenfaser (5%) bzw. 17,5 mg (0,5%) CMC oder Guarkernmehl wurden in einen RVA-Kanister eingewogen und mit dem Verkleisterungsprogramm „Standard 1“ analysiert. Die verkleisterte Probe wurde mit Frischhaltefolie umwickelt und tiefgekühlt. Nach 24 h aufgetaut und erneut eine mit den selben Einstellungen am RVA analysiert.

Backversuch – Herstellung teilgebackener Brote:

Grundrezeptur: 1,5% Trockenhefe, 2% Salz, 1,5% Zucker, Teigausbeute 158. Zusatz von 3% Fasern, 1% Guarkernmehl oder 0,5% CMC. Wassermenge wurde laut Herstellerempfehlung angepasst. Das Teiggewicht betrug 200 g. Der erste Backvorgang dauerte 14 min, anchließend wurden die Brote bei -18°C gelagert und nach 4 Wochen Lagerung 14 min bei 230°C aufgebacken.

RVA-Analyse der gefriergetrockneten Brotkrume:

Die Brotkrume der fertigen Brote wurde nach der jeweiligen Lagerzeit gefriergetrocknet und anschließend vermahlen. Von den hergestellten Pulvern wurden 3,5 g mit 25 g Wasser vermengt und eine RVA-Analyse mit dem Profil „Standard 1“ durchgeführt.

Johannes Frauenlob1, Philipp Niederschick1, Stefano D‘Amico1 und Regine Schoenlechner1 1 Institut für Lebensmitteltechnologie, Department für Lebensmittelwissenschaften und Lebensmitteltechnologie (DLWT), BOKU - Universität für Bodenkultur, Muthgasse 18, 1190 Wien, Österreich.

EINLEITUNG UND ZIEL

Teilgebackenen Brote und Gebäcke sind oft von beschleunigtem Altbackenwerden betroffen (1). Möglicherweise resultierten die Qualitätseinbußen aus einer geschwächten Wasserbindung nach der Tiefkühlung (2). Eine verbesserte Wasserbindung kann durch den Einsatz von Hydrokolloiden erzielt werden. Neben häufig eingesetzten deklarationspflichtigen Hydrokolloiden (z.B. Guarkernmehl oder Carboxymethylcellulosen), kommen auch Pflanzenfasern zum Einsatz, welche nicht als Zusatzstoffe deklariert werden müssen. Diese werden aus Verarbeitungsnebenprodukten durch Feinstvermahlung hergestellt. Vorrangig werden diese Pflanzenfasern heutzutage zur Ballaststoffanreicherung eingesetzte. In der Fleischwirtschaft werden Pflanzenfasern bereits häufig zur erhöhten Wasserbindung eingesetzt. Das Potential zum Einsatz in teilgebackenen Backwaren soll in der vorliegenden Studie untersucht werden. Dazu wurden rheologische Untersuchungen und Backversuche mit anschließenden Lagertests durchgeführt.

Literatur 1. ASGHAR A, ANJUM FM, ALLEN JC. Utilization of Dairy Byproduct Proteins, Surfactants, and Enzymes in Frozen Dough. Crit Rev Food Sci Nutr 2011; 51: 374–382. 2. ALMEIDA EL, STEEL CJ, CHANG YK. Par-baked Bread Technology: Formulation and Process Studies to Improve Quality. Crit Rev Food Sci Nutr 2016; 70–81.

SCHLUSSFOLGERUNGEN

• Die technologische Wirkung ist stark von Faserart

und Faserlänge abhängig

• BBL500 (Weizenfaser, Psyllium) für Frischhaltung

am besten geeignet

• Bei Zugabe von 3% keine negativen Auswirkung auf

Brotvolumen, Geschmack und Farbe festgestellt

• RVA-Analyse der Brotkrume zeigt charakteristische

Muster je nach Faserart

Tab.1: Veränderung der Krumenfestigkeit während kontrollierter Lagerung (20°C, 50% RF)

Abb.2: Spez. Volumen von teilgebackenen Broten mit Zusatz von Pflanzenfasern (STD = ohne Zusatz)

ERGEBNISSE

Die RVA-Analyse von verkleisterten Proben die einem Gefrier-/Tauzyklus unterzogen wurden zeigen eine gleichbleibende bzw. zunehmende Viskosität (Abb. 1). Die höchsten Viskositäten werden durch die Zugabe von P95 erzielt. Brote mit der geringsten Krumenfestigkeit wurden bei Zugabe von BBL500 produziert (Tab. 1). Das Brotvolumen wurde durch mehrere Fasern im Vergleich zur Referenz erhöht, eine Reduktion wurde nicht beobachtet (Abb. 2). Die RVA-Messung der Brotkrume (Abb. 3) zeigte große Unterschiede zwischen den Fasern. Bei mehreren Fasern konnte ein „Bump“ bei rund 600s beobachtet werden. Es wird davon ausgegangen dass dieser Bump durch eine Amylose-Lipid Wechselwirkung zustande kommt. Somit ist eine Auswirkung der Fasern auf diesen Mechanismus sehr wahrscheinlich.

Das Tiefgefrieren zur Konservierung von Brot und Teig wird heute flächendeckend von Bäckereibetrieben angewendet und findet sich in der wachsenden Zahl von Aufbackstationen in Supermärkten wider. Ein großer Teil der dort erhältlichen Produkte wird zentral hergestellt, vorgebacken, tiefgefroren gelagert, an Filialen ausgeliefert und dort aufgebacken. Diese teilgebackenen Brote und Gebäcke sind oft von beschleunigtem Altbackenwerden betroffen. Diese Qualitätseinbußen resultieren aus einer geschwächten Wasserbindung nach der Tiefkühlung. Eine verbesserte Wasserbindung kann durch den Einsatz von Hydrokolloiden erzielt werden. Hydrokolloide aus Pflanzenfasern werden aus Verarbeitungsnebenprodukten wie Stroh oder Schalen von verschiedensten Nutzpflanzen gewonnen und werden aktuell häufig zur Ballaststoffanreicherung verwendet. Im Fokus dieser Arbeit stand jedoch ihr technologischer Nutzen auf teilgebackene

gefrorene Backwaren. RVA-Analysen von Mehl-Faser Mischungen zeigten, dass sich die Faserzugabe auf die Viskosität während und nach der Verkleisterung auswirkte. In Backversuchen mit anschließenden Lagertests konnte gezeigt werden, dass Pflanzenfasern die Frischhaltung von teilgebackenen Broten signifikant verlängern können. Die Auswirkungen sind jedoch stark abhängig von Faserlänge und -typ. Beispielsweise hatten Brote bei Zugabe von 2 % Fasern (Mischung aus Bambus-, Weizenfaser und Psyllium) nach 6 Tagen kontrollierter Lagerung (20 °C, 50 % R.F.) geringere Krumenfestigkeiten wie Brote ohne Zusatz nach 2 Tagen. Außerdem konnten mit rheologischen Analysen der gefriergetrockneten Brotkrume unterschiedliches Verhalten während der Retrogradation gezeigt werden.

ABSTRACT

200

225

250

275

300

325

350

spez

ifisc

hes

Bro

tvol

umen

[c

m³/

100g

]

0

1000

2000

3000

4000

5000

Fin

al V

isco

sity

[cP

]

Standard + Gefrier/Tau Schritt

Fmax [N]

Lagerdauer [d] 0 ± 2 ± 6 ±

STD 5,0 1,7 11,5 2,1 19,7 7,1

BBL500 2,3 0,3 5,2 1,6 7,7 1,8

BBL100 4,3 0,0 8,9 0,1 17,3 5,4

WF200 4,2 0,1 10,3 0,3 31,0 4,3

WF600-30 4,4 0,0 14,3 1,0 30,9 8,5

WF600 4,7 0,4 8,3 0,0 19,0 7,1

BAF90/P95/WF600 4,2 0,0 7,3 0,5 17,5 1,1

CMC 4,4 0,8 7,2 1,0 25,5 7,7

Guarkernmehl 2,8 0,5 7,6 0,0 19,0 1,0

Abb.1: RVA-Final Viscosity von Weizenmehl mit Zusatz von Hydrokolloiden mit und ohne Gefrier-/Tau-Zyklus

0

1500

0 800

STD

0

1500

0 800

BBL500

0

1500

0 800

BBL100

0

1500

0 800

WF200

0

1500

0 800

WF600-30

0

1500

0 800

WF600

0

1500

0 800

BAF90/P95/WF600

0

1500

0 800

CMC

0

1500

0 800

Guarkernmehl

Abb.3: RVA-Analyse von gefriergetrockneter Brotkrume. Y-Achse: Viskosität [cP], X-Achse: Zeit [s]

frisch

2 Tage

6 Tage

8 Tage

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Stärkemutanten bei Weizen

Stärke stellt die Hauptkomponente

des Weizenkorns (ca. 60 %) bzw.

des Weizenmehls (ca. 75 %) dar. Die

Weizen stärke besteht aus Amylose

und Amylopektin. Während Amylose

vorwiegend aus linear verbundenen

Glukoseeinheiten besteht, weist das

wesentlich größere Molekül Amy-

lopektin zusätzlich auch zahlreiche

Verzweigungen auf. Die unterschied-

liche Morphologie der beiden Kompo-

nenten beeinflusst die Eigenschaften

und folglich auch das Verhalten in

ihrer Verarbeitung.

Herkömmliche Weizensorten enthalten etwa 25 – 28 % Amylose und 72 – 75 % Amylopektin.

Waxy-Weizen für TieSühlteiglinge

Die Bildung von Stärke aus, im Zuge der Photosynthese entstandener Saccharo-se, wird durch verschiedene Enzyme gesteuert. Ein Mangel an bestimmten Enzymen kann zu einer veränderten Stärkezusammensetzung in Samen führen. Die Stärkesynthase GBSS I wird vom sogenannten Waxy-Gen codiert. Liegt eine Mutation vor, bei der GBSS I nicht ausgebildet wird, so kommt es zu keiner Bildung von Amylose, das heißt die Stärke besteht nur aus verzweigten Molekülen (Amylopektin). Die dadurch bedingte größere Oberfläche der Mole-küle ermöglicht eine höhere Wasserauf-nahme und die Stärkekörner schwellen schneller an.

Waxy-Mutanten werden von der ös-terreichischen Stärkeindustrie derzeit bei Mais genutzt. Bei Weizen ist die Züchtung von Waxy-Sorten durch die hexaploide Genetik erschwert. Um in diesem Fall einen Amylose-freien

Weizen zu erzeugen, muss die Mutation dreifach vorliegen. Bei nur teilwei-sem Ausbleiben des Enzyms wird der Amylosegehalt lediglich verringert und es entstehen sogenannte Partial Waxy-Typen. Waxy-Weizen zeigen deutlich veränderte Verkleisterungs-eigenschaften der Stärke. Dadurch ergeben sich gravierende Änderungen in der Verarbeitung und Verwendung, aber auch in der Getreideanalytik. Bei-spielsweise kann bei Waxy-Weizen die Fallzahl zur Bestimmung von Auswuchs nicht verwendet werden. Hier wird der Stärkekleister durch die hohe Tempera-tur bereits bei Messbeginn thermisch zerstört und dadurch erneut flüssig, weshalb sich immer Messwerte unter

70 Sekunden ergeben. Eine Viskositäts-bestimmung mittels Amylogramm oder ähnlichen Geräten ist hingegen möglich. Dabei zeigen sich für Weizen unübliche, sehr niedrige Temperaturen (65 – 67°C) beim Verkleisterungsmaximum. Die Bestimmung der Amylogrammfläche ermöglicht auch bei Waxy-Weizen eine Abschätzung der Amylaseaktivität. Die Vermahlung von Waxy-Weizen ist unproblematisch und unterscheidet sich nicht von herkömmlichen Sorten.

Ein großer Teil der Backwaren wird heute über den Einsatz von Tiefkühlung hergestellt. Dabei werden rohe Teiglinge oder vorgebackene Brote und Gebäcke tiefgekühlt und in Supermärkten,

Mikro-Backversuche mit 100 % Weichweizen (links) bzw. Waxy-Weizenmehl (rechts). Reine Waxy-Mehle zeigen niedrigeres Backvolumen, schlechte Porung, aber bessere Haltbarkeit. Beimengungen von bis zu 20 % Waxy-Mehl zeigen keine negativen Auswirkungen auf das Backen bei verbesserter Frischhaltung.

6 | inform 2-2017

PFLANZENZÜCHTUNG

Bäckereifilialen bzw. in der Gastrono-mie aufgebacken. Die so hergestellten Backwaren sind im Vergleich zu frischen Produkten von einer schnelleren Alte-rung (altbacken) betroffen und werden oft bereits nach wenigen Stunden hart. Am Institut für Lebensmitteltechnologie der Universität für Bodenkultur Wien (BOKU) wurde kürzlich der Einsatz von Waxy-Weizen in Backversuchen erprobt. Waxy-Weizen zeichnete sich durch eine höhere Wasseraufnahme (ca. 68 %) als Standard-Weichweizen aus. Ein Mikro-backversuch zeigte, dass Brote aus 100 % Waxy-Weizen nicht backfähig sind, auch wenn die Teigverarbeitung unproblema-tisch ist. Beim Backvorgang kommt es zuerst zu einer großen Volumenzunahme (Ofentrieb), ab einem gewissen Punkt fällt das Brot jedoch in sich zusammen und es resultiert daraus eine „runzelige“ Oberfläche. Eine sehr grobe Porung der Krume ist ebenfalls charakteristisch für Brote aus Waxy-Weizen. Es konnte jedoch bereits bei diesen Broten fest-gestellt werden, dass sie länger frisch bleiben, das heißt die Brotkrume war nach mehreren Tagen Lagerung noch deutlich weicher als bei Vergleichsbroten aus Standard- Weichweizen.

Diese ersten Versuche zeigten, dass in Backwaren nur ein gewisser Anteil der Mehlmenge durch Waxy-Weizen ersetzt werden kann, um optimale Ergebnisse zu erzielen. In der Folge bewährte sich bei Standard-Backversuchen eine Zumischung von 10 – 20 % Waxy- Weizen (bezogen auf die Mehlmenge) in vorgegarten sowie vorgebackenen Tiefkühlbackwaren. Dabei trat keine der oben genannten negativen Auswir-kungen mehr auf, vielmehr konnte der erwünschte Effekt, eine bessere Frisch-haltung, festgestellt werden. Dadurch stellt Waxy-Weizen eine potenzielle Alternative zu üblichen haltbarkeits-verlängernden Zusatzstoffen, wie z.B. Diacetylweinsäureester (E472e) oder Natriumstearoyl-2-lactylat (E481), in Backwaren dar. Ein Einsatz als Ersatz bzw. zur Reduktion von Lebensmittel-zusatzstoffen ist auch in weiteren Pro-dukten denkbar. Aktuell werden weltweit

nur geringe Mengen an Spezialmehlen aus Waxy- Weizen hergestellt, beispiels-weise für das Eindicken von Soßen in der Lebensmittelindustrie. Der Vorteil dabei ist, dass sich beim Erkalten der Soße weniger oder keine Haut bildet, da die Stärke bei der Temperaturverringerung nicht bzw. nur gering dickflüssiger wird. Weitere Produkte, bei denen über eine Qualitätsverbesserung durch den Einsatz von Waxy-Weizen berichtet wurde, sind Waffeln und asiatische Weizennudeln. Modifizierte Waxy-Stärke wird auch als Verdickungsmittel für Füllungen, Soßen und Salatdressings genutzt. Außerhalb des Lebensmittelbereichs findet Waxy-Stärke beispielsweise für Biokunststoffe und in der Papierleimung Anwendung.

Hoch-Amylose Weizen

Weitere Mutationen betreffen die Enzy-me, die für die Verzweigung der Stärke, das heißt die Bildung von Amylopekt-in, verantwortlich sind. In diesem Fall werden weniger dieser Enzyme her-gestellt und so wird mehr Amylose als üblich gebildet. High-Amylose-Mutanten wurden bisher vor allem bei Mais und Erbse verwendet um Futter- oder Lebens-mittel mit einem höheren Ballaststoff-gehalt zu erzeugen. Bei entsprechender Verarbeitung wird durch Retrogradation die Amylose zu resistenter Stärke. Diese

wird im Dünndarm nicht verdaut und wirkt deshalb wie ein Ballaststoff. High-Amylose- Mehle können mehr Wasser aufnehmen und bilden einen zähflüs-sigeren Teig als normale Weizenmehle. Die Stärke geliert bereits bei etwas niedrigeren Temperaturen und ähnlich wie Waxy-Teige sind sie weniger stabil während des Knetens. Das gebackene Brot zeichnet sich durch eine verbes-serte Textur und Knusprigkeit aus, das Volumen fällt jedoch geringer aus. Bei einer Beimengung von bis zu 50 % zu herkömmlichem Mehl bleiben das Back-volumen und die Festigkeit im akzeptab-len Bereich und der Anteil an resistenter Stärke wird deutlich gesteigert.

Eva ZAND schließt ihre Masterarbeit zum teigrheologischen Verhalten von Waxy-Weizen am Department für Nutz-pflanzenwissenschaften ab. Johannes FRAUENLOB arbeitet an seiner Disserta-tion zum Thema „Tiefkühlbackwaren“ am Institut für Lebensmitteltechnologie (Prof. Henry JÄGER). Prof. Heinrich GRAUSGRUBER arbeitet an der Züch-tung von Waxy- Weizen und High-Amylo-se-Weizen für österreichische Produkti-onsbedingungen.

eva zand b.sc., di johannes frauenlob,

prof. dr. heinrich grausgruber,

universität für bodenkultur

Verkleisterungskurven unterschiedlicher Weizenmehle in einem Rapid-Visco-Analyser: QWBQG 7 = Qualitätsweizen BQ 7; A54 und A11 = Partial-Waxy Weizenlinien aus dem BOKU Zuchtprogramm

VERKLEISTERUNGSKURVEN

inform 2-2017 | 7

PFLANZENZÜCHTUNG

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DISSERTATION Quality improvement of bread made from ...

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