International Journal of Food Properties

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Multiparametric investigation of mould‐free shelf

life of bread via factorial design

D. Doulia , F. Rigas & G. Katsinis

To cite this article: D. Doulia , F. Rigas & G. Katsinis (2000) Multiparametric investigation of
mould‐free shelf life of bread via factorial design, International Journal of Food Properties, 3:3,
363-374, DOI: 10.1080/10942910009524641

To link to this article: https://doi.org/10.1080/10942910009524641

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INTERNATIONAL JOURNAL OF FOOD PROPERTIES, 3(3), 363-374 (2000)

MULTIPARAMETRIC INVESTIGATION OF MOULD-FREE SHELF LIFE OF

BREAD VIA FACTORIAL DESIGN

D. Doulia*, F. Rigas, and G. Katsinis

Chemical Engineering Department, National Technical University of Athens

Zografou Campus, GR-157 80, Athens, Greece
*Corresponding author (Tel: +30 1 7723122, Fax: +30 1 7723163, and E-mail:
ddoulia@orfeas.chemeng.ntua.gr)

ABSTRACT

The investigation of a complex process, such as the bread making process, can be
greatly favored by the approach of statistical design of experiments. Using simple first
order factorial designs, reliable models were constructed investigating the effects of
salt, sugar, sorbic acid, calcium propionate, vegetable fibers, fat and emulsifier on the
shelf life of bread (optimization parameter). These effects were in general linear. An
exception was observed in the case of use of calcium propionate where an interaction
between salt and sugar was determined. The effects of all significant factors on
optimization parameter were reasonable. Sorbic acid and calcium propionate proved to
be more effective preservatives than salt and sugar by an order of magnitude.
Antifungal efficiency of sorbic acid was found to be about three times that of calcium
propionate. Prolongation of shelf life reached 117% for sorbic acid and 63 % for
calcium propionate, when salt and sugar were fixed to their basic levels. Vegetable
fibers had a detrimental effect on shelf life greatly decreasing it by as much as 65 % of
the control samples (without fibers). Addition of fat and emulsifier in the dough showed
a slightly negative effect on the mould-free shelf life of bread.

INTRODUCTION

Increased shelf life of bread and other baked products can improve the productivity and
profitability of a company allowing important expansion of bread lines with
simultaneous economy in production, stocking and long distance distribution of higher

363

Copyright © 2000 by Marcel Dekker, Inc. www.dekker.com

364 DOULIA, RIGAS, AND KATSINIS

quality products. Also, the baker is liable to prosecution under the Food Regulations
when his products show microbiological deterioration. Bread, as most bakery products,
is subject to deterioration and so has a limited shelf life. Staling, moisture loss and
microbiological spoilage are the most common forms of this deterioration (Legan and
Voisey, 1991).
Particularly, mould growth is considered frequently the major limiting factor
and its prevention or delay leads to an extension of the shelf life of bread (Seiler, 1983;
Islam, 1982). The estimates of bread losses attributable to mould range between 1-5 %,
while the dominant spoilage flora varies with the type of bread and the storage
conditions, with Penicillium species being by far the most common (Legan, 1994).
During bread making process mould spores are usually destroyed. However,
mould spoilage may occur after baking process (even if a sanitation program is
adopted) and is aggravated by high temperature and humidity (Horner and
Anagnostopoulos, 1973). This deterioration can be delayed by the use of chemical
preservatives such as, acid ingredients and their salts (Grundy, 1996; King, 1981) or by
packaging in modified atmospheres (Smith and Simpson 1996). Calcium propionate
and sorbic acid are among the most common mould inhibitors used in bread (Legan,
1994; Grundy 1996). Ascorbic acid has also proven to be a suitable preservative
(Granito and Guerra, 1997), as well as sodium acetate or vinegar in combination with
calcium propionate (McNaughton et al., 1998).
In some recent Japanese patents the inventors have proposed di-Me fumarate,
Me fumarate or Di-Et fumarate and oxygen absorbers (Hoshino, 1998), or a mixture
containing anhydrous acetic acid, adipic acid, fumaric acid and salts of these organic
acids or glycerin fatty acid esters (Uetaka et al., 1999) or isothiocyanic acid derivatives
(Okada et al., 1999) as effective preservatives for bread. A more exotic antimicrobial
agent comprise sintered pearl or shell supported on natural substances having cell
membranes (Matsuura et al., 1999).
In addition to chemical preservatives, certain biopreservatives are found in the
literature including some strains of lactic acid bacteria and propionic acid bacteria
(Suomalainen and Mayra-Makinen, 1999), an antimycotic propionibacteria metabolite
(King et al., 1998) and xylanase which makes insoluble pentosans partially soluble,
binding in this way water ten to twenty times their weight and thus increasing the shelf
life of bread (Sprossler, 1996).
With regard to packaging in modified atmospheres, new oxygen scavengers
have been developed that can be incorporated easily in multiplayer food packaging
systems (Cernak and Chiang, 1997). Ethanol emitters in combination with oxygen
scavengers have been investigated (Salminen et al., 1996), as well as low-density
polyethylene films in which benzoic anhydride has been incorporated as an antimycotic
agent (Weng and Hotchkiss, 1993). Another treatment of baked goods concerns the
bacteriocide power of intense pulsed light to extend the shelf life of foods (Dunn et al.,
1995).
Since bread is a multicomponent mixture made by dough and additional
optional ingredients (such as other cereals flour, emulsifiers, fat, sugar, milk products,
gluten, preservatives, etc.), it is very difficult to investigate the influence of each
component on the shelf life in combination with the behaviour of other components of
the mixture.
MOULD-FREE SHELF LIFE OF BREAD 365

In this work, we applied the factorial experiment design (Biles and Swain, 1980)
in order to investigate the effect of some selected common ingredients of bread and
their interactions with other factors involved, on the mould-free shelf life during the
bread making process. This method facilitates the investigation by reducing
significantly the number of required experiments, thus saving time and money. In
addition, the constructed models could be used as prediction models of the probable
mould-free shelf life of bread. To the best of our knowledge no similar statistical
approach has been used till now to investigate such industrial problems.

STATISTICAL DESIGN OF EXPERIMENTS

A first order factorial design (Biles and Swain, 1980) was applied in this study with
three design factors in each statistical experiment (N= 23). The coded and natural values
of design factors are shown in Table 2. The coded values of the design factors are
defined as:

_ xj ~xjo
X =

where x> is coded value of the factor, 3cy is natural value of the factor, 3c,-0 is natural
value of the basic level, Jj is variation interval, and j is number of the factor. Three
repetitions were performed for each experiment trial (design point). For control
purposes, bread samples with the composition shown in Table 1 were prepared in
triplicate and were simultaneously baked in the same oven with the samples of the
experimental designs.
The optimization parameter selected (yj) was the relative shelf life (in relation to
the corresponding control) of design bread samples, tested for time of appearance of
mould colonies on their crust. Faulty observations were determined with the aid of
"Student's" t-test. The homogeneity of variances of trials was tested with Cochran's
test and Fischer ratio. After calculating the factors coefficients from the results
obtained, the insignificant factors were discarded using the estimated confidence
interval. Then, the adequacy of the models was tested by the aid of the Fischer ratio.

MATERIALS

Food grade ingredients were used (wheat flour, salt and sugar). The fat was palm oil
refined and hydrogenated with melting point 40 °C. Pure encapsulated sorbic acid
(Panosorb Hoechst, Germany) and calcium propionate of 95 % purity (Probake, Ducoa,
Holland) were used as preservatives. The emulsifier (Imwitor 171, Hulls) was a self-
emulsifying monoglyceride with a monoester content of minimum 85 %. The dietary
fibers used (Fibrex) were made from sugar beets (Brochure, 1972).
366 DOULIA, RIGAS, AND KATSINIS

Table 1. Composition % of Bread Samples in Experimental Designs

Number of Experimental Design

Ingredient 1st 3 rd 4th Control

content %

Flour 100 100 100 100 100

Water 55 55 55 55 55
Yeast 3 3 3 3 3
Salt variable* variable* variable* variable* 1.5
Sugar variable* variable* variable* 3.5 3.5
Sorbic Acid variable* 0 0 0 0
Calcium propionate 0 variable* 0 0 0
Fibre 0 0 variable* 0 0
Fat 3 3 3 variable* 3
Emulsifier 0.5 0.5 0.5 variable* 0.5
' The content of this ingredient varied according to the levels shown in Table 2.

Bread Making Procedure

Bread dough samples were prepared by mixing the chosen required and optional
ingredients at proportions included in the formulae given in Table 1. The values of
variables (design factors) are given in Table 2. Mixing was performed for 2 minutes at
low speed and 5 minutes at high speed, until the dough temperature rose to 29 °C.
Resting of films dough samples covered by plastic followed at ambient temperature for
10 minutes. Dough samples were divided in four pieces of 500 g each, which are
manually moulded in a cylindrical loaf, 30 cm long. Then, the dough pieces were
fermented for 45 min in a proofing box at 36 °C and relative humidity 85 %, and baked
in a preheated deck oven at 220 °C for 40 minutes. Cooling followed at ambient
temperature for 30 min (time judged good enough for a temperature of 30 °C to be
achieved in the center of the loaf). Finally, each loaf, packaged in a polypropylene bag
and stored at ambient temperature, was tested daily by visual observation for the
appearance of mould colonies on its crust. The spoiled bread was discarded and its
mould-free shelf life was recorded. As mould-free shelf life was defined the period in
days from baking to the first observation of mould on the surface of the samples.

RESULTS AND DISCUSSION

Four different factorial experiments were designed and carried out to investigate the
effects of the design factors (xj) on the optimization parameter (yj):

= f(x], = f(x]> X2. x$) and y4 = f(xjt X& xj)

MOULD-FREE SHELF LIFE OF BREAD 367

Table 2. Coded and Natural Values of Design Factors

Design Factors Coded (-l,0,+l) and Natural Levels (content %)

Symbol Ingredient ^1 0 +1
content

x. Salt 1 1.5 2
x2 Sugar . 2 3.5 5
x3 Sorbic Acid 0.05 0.125 0.2
x4 Calcium Propionate 0.05 0.125 0.2
x5 Vegetable Fibers 4 5 6
x6 Fat 2 3 4
x7 Emulsifier 0.3 0.5 0.7

The results of these experiments are shown in Tables 3-6. After processing these results
discarding the insignificant factors, the following models were derived, expressed in
natural values of significant factors:

y, = -0.15 + 034xl + 0.2 lx2 + 5.33x3

y2 = L18-0.36xj-0.09x2 +1.95x4 + 0.18xjx2
y3 = 1.02- 0.06x5
y4 =0.68+ 0.19xj

The first model (yj) is a linear correlation of all the factors engaged (content of salt,
sugar and sorbic acid). Their signs are all positive which means that increasing any of
these factors, the relative shelf life increases, too. As expected, the effect of the
antifungal agent sorbic acid in this case, on the optimization parameter is an order of
magnitude stronger than the salt or sugar contents, which act mainly by decreasing
water activity of bread and thus retarding the microbial growth by leading to the
dehydration of microorganisms (Grundy, 1996). This preservative increased shelf life
of bread by 37 to 117%, when its concentration ranged from 0.05 to 0.2% and the other
two factors were fixed to their basic levels, confirming its inhibiting function by
blocking specific metabolic reactions (King, 1981). By fixing the less significant factor,
namely the sugar content, to its basic level and constructing a contour plot of the other
two factors versus optimization parameter we obtain Fig.l. In this figure the
dependence of the relative shelf life of bread (with regard to control) on salt and sorbic
acid contents is shown.
In the second model (y2), in which the preservative was replaced by calcium
propionate, the situation is somewhat more complicated, since an interaction of the
factors xj (salt content) and X2 (sugar content) appears. In this case, it is not clear
368 DOULIA, RIGAS, AND KATSINIS

Table 3. First Design Matrix and Results of Experimentation (x,=salt, x2=sugar,

x3=sorbic acid)

Shelf Life* Shelf Life (y,)

Trial No Xj *2 *3
(days) (relative to control)

Control 1.5 3 0 8.3 ±0.6 . 1

1 2 5 0.2 24.7 ± 2.5 2.96 ±0.30
2 2 5 0.05 15.0 ±1.7 1.80 ±0.21
3 2 2 0.2 15.7 ±0.6 1.88 ±0.07
4 2 2 0.05 9.3 ± 1 . 5 1.12± 0.18
Control 1.5 3 0 8.3 ±0.6 1
5 1 5 0.2 19.0 ±1.0 2.28+0.12
6 1 5 0.05 11.0± 1.0 1.32 ±0.12
7 1 2 0.2 13.0 ±1.7 1.56 + 0.21
8 1 2 0.05 10.3 ±0.6 1.24 ±0.07
Basic Level 1.5 3.5 0.125 16.3 ±1.5 1.96 ±0.18
*Mean±SD

Table 4. Second Design Matrix and Results of Experimentation (x,=salt,

x2=sugar, x4= calcium propionate)

Shelf Life* Shelf Life (y2)

Trial No Xj *2 x4 (days) (relative to
control)

Control 1.5 3 0 8.3 ± 0.6 1

1 2 5 0.2 21.7 ±2.9 2.60 + 0.35
2 2 5 0.05 12.3 ±1.5 1.48 ±0.18
3 2 2 0.2 12.7 ±0.6 1.52 ±0.07
4 2 2 0.05 10.7 ±1.5 1.28 ±0.18
Control 1.5 3 0 8.3 ±0.6 1
5 1 5 0.2 15.3 ±3.2 1.84 ±0.39
6 1 5 0.05 13.3 ±1.5 1.60 ±0.18
7 1 2 0.2 9.3 ± 0.6 1.12 ±0.07
8 1 2 0.05 9.0 ±1.0 1.08 ±0.12
Basic Level 1.5 3.5 0.125 13.7 ±1.5 1.64 ±0.18
'Mean±SD
MOULD-FREE SHELF LIFE OF BREAD 369

Table 5. Third Design Matrix and Results of Experimentation (x,=salt,

x2=sugar, xs= vegetable fibers)

Trial No Shelf Life* Shelf Life (y3)

Xj (days) (relative to
control)

Control 1.5 3 0 7.7 ± 0.6 1

1 2 5 6 5.3 ±0.6 0.70 ± 0.08
2 2 5 4 5.0 ± 0.0 0.65 ± 0.00
3 2 2 6 5.0 + 0.0 0.65 ± 0.00
4 2 2 4 4.7 ±0.6 0.61 ± 0.08
5 1 5 6 5.7 ± 0.6 0.74 + 0.08
Control 1.5 3 0 6.3 ±0.6 1
6 1 5 4 5.0 ±0.0 0.79 ± 0.00
7 1 2 6 5.3 ±0.6 0.84 + 0.09
8 1 2 4 4.7 ± 0.6 0.74 ± 0.09
Basic Level 1.5 3.5 5 5.3 ±0.6 0.84 ± 0.09
*Mean±SD

Table 6. Fourth Design Matrix" and Results of Experimentation(Mean ± SD)

(x,=salt, x6=fat, x7= emulsifier)

Trial No Shelf Life* Shelf Life (y4)

x6 x7 (days) (relative to
control)

Control 0 0 1.5 8.3 ±0.6 1

1 4 0.7 2 9.71 ±0.6 1.16 ±0.07
2 4 0.7 1 7.0 ±1.0 0.84 ±0.12
3 4 0.3 2 7.3 ± 0.6 0.88 + 0.07
4 4 0.3 1 7.0 ±0.0 0.84 ± 0.00

Control 0 0 1.5 8.5 ± 0.7 1

5 2 0.7 2 9.0 ±1.0 1.06 ±0.12
6 2 0.7 1 8.0 ±1.0 0.94 ±0.12
7 2 0.3 2 9.7 ±1.2 1.14 + 0.14
8 2 0.3 1 7.3 ± 0.6 0.86 ± 0.07
Basic Level 3 0.5 1.5 7.3 ± 0.6 0.98 ± 0.07
"Mean±SD
370 DOULIA, RIGAS, AND KATSINIS

2.0G

1.00
0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
Sorbic acid content (%)

Figure 1. Dependence of relative shelf life of bread on salt and sorbic acid contents
with sugar fixed at its basic level. Contour lines stand for the relative shelf life in
days.

whether the effects of salt and sugar on optimization parameters are reasonable. To
clarify this confusion, a contour plot of these two factors against the optimization
parameter was constructed (Figure 2). It is obvious from this plot that the relative shelf
life of bread increases with both salt and sugar contents, as expected, when calcium
propionate was fixed to 0.125% concentration. This preservative increased the
optimization parameter from 34 to 63%, when its concentration ranged from 0.05 to 0.2
% (that is the same region with sorbic acid) and the two other factors were fixed to their
basic levels. Thus, sorbic acid was proved to be a more effective antifungal agent than
calcium propionate (2.7 times by comparison of their coefficients). This event is in
accordance with the relevant literature, where it is mentioned that sorbates are most
effective at inhibiting mould growth than propionates (Legan, 1994), since unsaturated
acids have been proved more effective fungicides than saturated acids (Chichester and
Tanner, 1972). This behaviour could be attributed to the toxic form of the undissociated
sorbic acid compared to the dissociated propionate anion of calcium propionate (King,
1981). By fixing the less significant factor (sugar) to its basic level and constructing a
contour plot of the two other factors, the dependence of the relative shelf life of bread
on salt and calcium propionate can be depicted (Figure 3).
In the third experimental design, the dependence of the optimization parameter
(y3) on vegetable fibers was investigated, in addition to salt and sugar. The model
MOULD-FREE SHELF LIFE OF BREAD 371

2.0a

1.80-

1.60-

I
™ 1.40-

1.20-

1.0&
2.00 2.50 3.00 3.50 4.00 4.50 5.00
Sugar content (%)

Figure 2. Dependence of relative shelf life of bread on salt and sugar contents with
calcium propionate fixed at its basic level. Contour lines stand for the relative
shelf life in days.

obtained showed a negative dependence of the shelf life on vegetable fibers content
only. Although the factors salt and sugar were not significant, at least in the variation
intervals of factors investigated, the presence of fibers had a deleterious effect on the
preservation of bread samples. Thus, relative shelf life fell down to 65 - 78% of the
control samples by adding vegetable fibers in the dough in the range 6 to 4%,
respectively. This negative effect could be attributed among others to the rough bread
crust caused by fibers, which results in both, increased contact surface with funguses'
spores and more effective anchoring of spores.
In the fourth model, the effects of fat and emulsifier were investigated in
addition to the salt content. The model obtained was a simple linear model of only salt
content. The coefficients of the two other factors were insignificant, yet an overall
slight negative effect of the presence of the fat and the emulsifier is observed. Thus, the
relative shelf life ranged from 0.87 to 1.06 in this case, when salt content ranged from 1
to 2%. This deterioration of bread quality could be attributed mainly to the adsorption
of fat and emulsifier on flour, rendering it less hydrophilic and, thus, increasing water
activity. In addition the emulsifier increasing the degree of dispersion of fat facilitates
this effect.
372 DOULIA, RIGAS, AND KATSINIS

2.00

1.80-

1.60-

1-40-

1.20-

1.00
0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
Calcium propionate content (%)

Figure 3. Dependence of relative shelf life of bread on salt and calcium propionate
contents with sugar fixed at its basic level. Contour lines stand for the relative
shelf life in days.

CONCLUSIONS

Among the antifungal agents of bread used in this study sorbic acid proved to be the
most effective prolonging the mould-free shelf life of bread by as much as 117 %.
Calcium propionate was a less effective preservative than sorbic acid prolonging the
shelf-life of bread only up to 63 % in the same concentration range as sorbic acid.
Vegetable fibers had a detrimental effect on shelf life of bread, decreasing its shelf life
by as much as 78 %. Fat and emulsifier showed a slightly negative effect on mould-free
shelf life of bread decreasing its shelf life by as much as 13 %.

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(Received revised December 20, 1999; February 10, 2000; accepted February 23, 2000)