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Effect of preservatives on the shelf-life and sensory characteristics of

pasteurized liquid whole egg stored at 4°C

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DOI: 10.3382/ps/pez378

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Effect of preservatives on the shelf-life and sensory characteristics
of pasteurized liquid whole egg stored at 4°C

Lenka Necidová,∗ Šárka Bursová,∗,1 František Ježek,† Danka Haruštiaková,‡,§ Lenka Vorlová,∗
and Jozef Golian¶
∗
Department of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of
Veterinary and Pharmaceutical Sciences Brno, 61242 Brno, Czech Republic; † Department of Meat Hygiene and
Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences
Brno, 61242 Brno, Czech Republic; ‡ Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk
University, 62500 Brno, Czech Republic; § Research Centre for Toxic Compounds in the Environment, Faculty of
Science, Masaryk University, 62500 Brno, Czech Republic; and ¶ Department of Food Hygiene and Safety, Faculty
of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, 94976 Nitra, Slovak Republic

ABSTRACT The study focused on the effects of
traditional (benzoate-sorbate and triphosphates) and
alternative (nisin, Laktocid, Defence JB, and Gali-
max Flavor) food preservatives on the microbiological
quality and sensory properties of pasteurized liquid
whole eggs (LWE). The LWE samples with the addi-
tion of a test preservative and a control were stored at
4°C for 45 D. The selected microbiological parameters,
sensory attributes, and color space parameters were
determined. The results were statistically analyzed by
means of factorial ANOVA, followed by the Tukey post
hoc test. The multivariate method of principal compo-
nent analysis based on the correlation matrix was em-
ployed to assess the relationships between pH and sen-
sory characteristics of LWE. Total plate counts at the
Key words: total plate count, lactic acid bacteria, nisin, organic acid, benzoate-sorbate
end of storage differed significantly, being the lowest
in samples with benzoate-sorbate (1.69 ± 0.12 log
cfu.g−1 ) and Laktocid (2.12 ± 0.12 log cfu.g−1 ). The
final counts of lactic acid bacteria on day 45 were lower
in the samples with benzoate-sorbate, triphosphates,
nisin, and Laktocid (maximum 1.01 ± 1.44 log cfu.g−1 ).
The amounts of yeasts at the end of storage did not
differ among samples with different preservatives; none
were detected in any samples with the exception of De-
fence JB (1.28 ± 1.90 log cfu.g−1 ). The lowest final
mold count was observed in LWE with Laktocid (0.92 ±
1.29 log cfu.g−1 ). In conclusion, Laktocid showed great
potential as a preservative; it had however a nega-
tive impact on pH value and the sensory properties of
LWE.
2019 Poultry Science 0:1–9
http://dx.doi.org/10.3382/ps/pez378

INTRODUCTION temperature pasteurization, usually for several minutes

Eggs are among the most complex foodstuffs be-
cause they are rich in vitamins, trace minerals, fatty
acids, and high-quality proteins (Ruxton et al., 2010).
In recent years, pasteurized egg products such as liquid
whole egg (LWE), liquid egg white, and liquid yolk have
been preferred in various branches of the food indus-
try and catering (Ratu et al., 2017). When compared
to shell eggs, LWE and other egg-based products are
easier to handle and store. On the other hand, LWE
is an excellent medium for microbial growth (Nistor
et al., 2016). Liquid whole egg is produced by homoge-
nization and pasteurization of broken whole eggs, which
extends the shelf-life when stored chilled or frozen. Low-
© 2019 Poultry Science Association Inc.
Received March 6, 2019.
Accepted June 10, 2019.
1 Corresponding author: bursovas@vfu.cz
at about 60°C, along with the short shelf-life, how-
ever, implies that some heat-resistant bacteria can sur-
vive the heat treatment (Miller et al., 2010; Németh
et al., 2011). The storage temperature for chilled
pasteurized LWE products should not exceed 4°C
(Wareing, 2009). However, storage in the refrigeration
regime only slightly changes the chemical composition
and nutritive value of pasteurized LWE and, implicitly,
it does not stop the biochemical processes responsible
for the degradation of quality (Nistor et al., 2016).
The hygienic quality and the physical characteristics
of the raw egg material are highly variable, depending
on shell soundness, storage temperature, and storage
time before breaking. Liquid whole egg may also be
contaminated in other ways, including failure of the
time-temperature binomial during pasteurization, or
post-processing contamination (Rossi et al., 2010; Rêgo
et al., 2014). The initial microflora of LWE consists
of a mixture of Gram-positive and Gram-negative

1

2 NECIDOVÁ ET AL.
bacteria similar to the indigenous microflora of shell
eggs (Miller et al., 2010). Wareing (2009) refers the typ-
ical initial bacterial counts of 4 to 6 log cfu per g in com-
mercial unpasteurized LWE (there may be however con-
siderable variation between sites), while the bacterial
counts in pasteurized LWE were typically less than 2 cfu
per g. The presence of yeasts was negligible (Wareing,
2009). However, Németh et al. (2011) have reported
that the main group of microorganisms contaminating
the egg products is bacteria of the family Enterobacte-
riaceae entering the liquid egg from the shell. A study
of Miller et al. (2010) has demonstrated that the pre-
dominant spoilage microorganisms in pasteurized LWE
are lactic acid bacteria (LAB), particularly enterococci
that survive pasteurization and have the potential to
produce undesirable flavor compounds. The EU legisla-
tion establishes that the pasteurized egg products shall
not contain Salmonella spp. (not detected in 25 g) and
limits the presence of Enterobacteriaceae to a maximum
of 2 log cfu per g (European Commission, 2005).
Another approach to LWE shelf-life extension is the
use of chemical stabilization. Using chemical reagents
for food preservation is among the most ancient and tra-
ditional methods. However, the use of chemical reagents
as food additives and preservatives is a sensitive is-
sue because of health concerns (Amit et al., 2017).
Sodium benzoate, sodium and calcium propionate, sor-
bic acid, ethyl formate, and sulfur dioxide are exam-
ples of commercially used food preservatives (Sancho-
Madriz, 2003). In the European Union, approved food
additives, including preservative materials and condi-
tions for their use, are listed in the Commission Regula-
tion (EU) No 1129/2011 (European Commission, 2011).
Liquid egg products may include additives such as
stabilizers (salt, benzoate, sorbate, phosphates, etc.),
emulsifiers and thickeners, anti-coagulants, and acids
or bases (to modify pH) (Wareing, 2009). Besides, the
addition of preservatives (EDTA, polyphosphates, lac-
tic acid, hydrogen peroxide, or nisin) increases the
heat sensitivity of the microorganisms in egg products
(Doyle and Mazotta, 2000).
Due to toxicological concerns about synthetic preser-
vatives (e.g., sorbate, benzoate, or phosphates) and
market trends that show increased popularity of foods
with no artificial substances (Sancho-Madriz, 2003),
producers have been recently focusing on the applica-
tion of naturally occurring preservatives such as mi-
crobial products with antimicrobial activity, organic
acids, hydrogen peroxide, and bacteriocins (Fialová
et al., 2008).
Bacteriocins are generally defined as peptides pro-
duced by bacteria that inhibit or kill other related and
unrelated microorganisms. They can be divided into 4
classes. The main representative of lantibiotics (class I)
is nisin produced by some strains of Lactococcus lactis
subsp. lactis. Nisin, available as a commercial product,
has been largely used in the food industry as an anti-
botulinic agent in cheese and liquid eggs, sauces, and
canned foods. Nisin inhibits the growth of a wide range
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of Gram-positive bacteria, including Listeria monocyto-
genes, Staphylococcus aureus, Bacillus cereus, and LAB
species. It is listed in regulations as a food additive
and is approved for use in most major food-producing
countries. In addition, nisin was also included in the
European food additive list as a biopreservative ingre-
dient (Miller et al., 2010; European Commission, 2011;
Balciunas et al., 2013).
Among organic acids, the lactic acid, acetic acid,
and their salts (individually or in various combinations)
have also demonstrated the potential to be used for
preservation (Fialová et al., 2008). For example, Lakto-
cid (a blend of lactic acid, acetic acid, and sodium lac-
tate), Galimax Flavor V50 (low sodium buffered vine-
gar), and Defence JB (natural fermentate of LAB and
yeast starter culture) are commercially available prepa-
rations permitted by the Czech law.
The aim of this study was to evaluate the ef-
fect of different types of traditional and alternative
preservative materials on the microbiological quality
and sensory characteristics of pasteurized LWE stored
at 4°C. The present study responds to the require-
ments of producers whose customers are increasingly
demanding the use of alternative, naturally occurring
preservatives. We hypothesized that there would be
differences in the (1) growth of microorganisms in pas-
teurized LWE preserved by individual preservatives, (2)
different bacterial/mold/yeast counts at the end of stor-
age in LWE preserved by individual preservatives, and
(3) different sensory characteristics of LWE preserved
with individual preservatives.
MATERIALS AND METHODS
Liquid Whole Egg Samples
Pasteurized LWE was used as a test medium. The
LWE samples with the addition of the test preservatives
were prepared by a Czech producer. First, the LWE was
pasteurized at 65°C for 3.5 min and then immediately
cooled down to 2 to 3°C. Six selected traditional and
alternative preservatives were tested; the preservatives,
their characteristics, and final concentrations are listed
in Table 1. Each preservative was tested separately in
5 replicates (where replicates are separate sample col-
lections from a single batch of pasteurized LWE). With
each preservative and for each replicate, six 30-mL sam-
ples were prepared in sterile screw cap plastic bottles.
One sample collection (6 bottles) of pasteurized LWE
without any added preservative was used as a control.
The prepared LWE samples were transported in a cool
box to the laboratory and subsequently stored at 4°C
for 45 D.
Microbiological Parameters
Microbiological analysis of the samples was per-
formed on days 1, 15, 22, 29, 36, and 45 of storage.
The experiment was carried out in 5 replicates, i.e.,
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PRESERVATION OF PASTEURIZED LIQUID WHOLE EGG 3
Table 1. Food preservatives tested, their characteristics, and final concentrations in pasteurized
LWE samples.

Preservative Composition, E number
Benzoate-sorbate Sodium benzoate E211 and
potassium sorbate E202
Triphosphates Sodium triphosphate E451i
Nisin Nisin E234
Laktocid Blend of lactic acid E270,
acetic acid E260, and
sodium lactate E325
Defence JB Natural fermentate of
lactic acid bacteria and
yeast starter culture
Galimax Flavor V50 Low sodium buffered
vinegar
Final concentration1
[g of pres. per kg of LWE] Supplier
2 + 2.5 Hages Ltd, Rudná u Prahy,
Czech Republic
10 Hubka-Petrášek and
Grandsons Ltd, Praha,
Czech Republic
0.25 Barentz Czech Republic,
Klatovy, Czech Republic
20 Zeelandia Ltd, Malšice,
Czech Republic
2 Novali JSC, Hluboká nad
Vltavou, Czech Republic
14 Barentz Czech Republic,
Klatovy, Czech Republic

1
Final concentrations of preservatives were chosen by the LWE producer.

Table 2. The evaluated microbiological parameters; used media, incubation conditions, and references.

Method Nutrient medium Incubation References

Total aerobic plate count Plate Count Agar 30°C, 72 h, aerobic ČSN EN ISO 4833–1:2014
Family Enterobacteriaceae Violet Red Bile Glucose Agar 37°C, 24 h, aerobic ČSN ISO 21528–2:2006
Mesophilic lactic acid bacteria De Man, Rogosa and Sharp Agar 30°C, 72 h, aerobic ČSN ISO 15214:2000
Yeast Dichloran Rose Bengal Chloramphenicol Agar 25°C, 5 D, aerobic ČSN ISO 21527–1:2009
Molds Dichloran Rose Bengal Chloramphenicol Agar 25°C, 5 D, aerobic ČSN ISO 21527–1:2009

Table 3. Evaluated sensory characteristics of pasteurized LWE; description of the best and the worst scores on a 9-point scale.

Parameter Score 9 (the best)1 Score 1 (the worst)

Appearance Well blended, opaque blend Non-homogeneous liquefied blend with diverse particles
Color Yellow to orange Very light or unnatural (e.g., red)
Consistency Homogenous consistency, air bubbles are not a defect Non-homogeneous liquefied blend with diverse particles
Odor Smell of egg mass, free of foreign odors Very strong foreign odor
1
Typical sensory characteristics of LWE according to ČSN 57 2301:1992.

35 samples (6 preservatives + control) were analyzed
at each time point. The evaluated microbiological pa-
rameters and methods are listed in Table 2.
Sensory Analysis
The sensory analysis was performed on the same
day as the microbiological examination, i.e., on days
1, 15, 22, 29, 36, and 45 of storage. Three random sam-
ples (20 mL) from each preservative or control group
were taken for sensory assessment on each experimental
day. For sensory assessment, LWE samples were placed
in glass beakers and tempered to room temperature.
During the evaluation, fresh LWE (20 mL in glass
beaker) was available for comparison by assessors. All
sensory assessments were performed by a panel of 5
trained assessors under the same laboratory conditions
(according to ČSN EN ISO 8589:2008). The ability of
sensory perception of color, smell, and taste was verified
for all assessors (according to ČSN EN ISO 8586:2015).
A 9-point scale was used (Lawless and Heymann, 1998;
Badr, 2006) to assess the following sensory parameters:
appearance, color, consistency, and odor. The descrip-
tion of the best (scoring 9) and the worst (scoring 1)
levels of a 9-point scale is shown in Table 3.
Color
The color of each LWE sample was determined us-
ing the CIELab system of coordinates after the sen-
sory assessment was completed. Samples were measured
immediately after sensory assessment (3 random sam-
ples from each preservative/control group, same as for
sensory evaluation). Each LWE sample (20 mL) was
placed in a Petri dish of 45 mm in diameter. The mea-
surement procedure was performed according to Chen
et al. (2010). The color space parameters L—lightness,
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4 NECIDOVÁ ET AL.
Table 4. Total plate counts (log cfu g−1 ) in pasteurized LWE samples supplemented with different preservatives.

Total plate count (log cfu g−1 )

Preservative Day 1 Day 15 Day 22 Day 29 Day 36 Day 45

Benzoate-sorbate 2.59 ± 0.10a,A

2.46 ± 0.10a,A
2.34 ± 0.09a,A
2.41 ± 0.10 a,A
2.47 ± 0.19a,A
1.69 ± 0.12a,A
Triphosphates 2.58 ± 0.08a,A 2.36 ± 0.14a,A 2.69 ± 0.29a,A 3.05 ± 0.87a,A 4.08 ± 2.22a,A,B 6.57 ± 2.04b,B
Nisin 1.57 ± 0.97a,A 1.65 ± 2.25a,A 2.21 ± 1.05a,A 3.76 ± 1.56a,A 6.09 ± 2.06b,B,C 7.34 ± 1.09b,B
Laktocid 2.71 ± 0.07a,A 2.54 ± 0.04a,A 2.56 ± 0.10a,A 2.68 ± 0.35a,A 2.39 ± 0.06a,A 2.12 ± 0.12a,A
Defence JB 2.88 ± 0.05a,A 2.67 ± 0.42a,A 3.33 ± 0.84a,A 4.63 ± 0.32a,b,A 6.87 ± 0.61b,c,C 7.98 ± 0.31c,B
Galimax Flavor V50 2.76 ± 0.05a,A 2.56 ± 0.06a,A 2.63 ± 0.07a,A 3.30 ± 0.58a,b,A 5.36 ± 2.09b,c,B,C 7.46 ± 1.00c,B
Control group 2.98 ± 0.05a,A 2.68 ± 0.18a,A 3.19 ± 0.85a,A 4.21 ± 1.14a,b,A 6.36 ± 1.37b,B,C 5.67 ± 0.79b,B

Mean ± standard deviation, n = 5 per day and preservative/control group.

Total plate count on days followed by the same small letter in the row did not differ significantly (Tukey post hoc test in factorial ANOVA).
Total plate count in samples supplemented with different preservatives followed by the same capital letter in the column did not differ significantly
(Tukey post hoc test in factorial ANOVA).

a—redness, and b—yellowness were measured 3 times
for each sample using a Konica Minolta CM-5 Spec-
trophotometer (Konica Minolta Sensing, Inc., Japan)
and reflectance method. The instrument was calibrated
to white point, and a setting with a D65 light source
and measurement slit of 30 mm was used.
Determination of the pH Value
Along with the microbiological and sensory analyzes,
the pH value was measured with a pH610 digital pH-
meter (EUTECH Instruments, Singapore) immediately
after the measurement of the color (3 random samples
from each preservative/control group, same as for sen-
sory evaluation).
Statistical Analysis
All microbial counts (cfu g−1 ) were logarithmically
transformed to log10 scale (log cfu g−1 ). The results
of total plate count (TPC) and LAB, yeast, and mold
counts are reported as the mean values ± standard devi-
ations. Differences in the bacterial/yeast/mold counts
between preservatives during the culture period were
analyzed using factorial ANOVA, including both the
preservative and time and their interaction as inde-
pendent factors. Factorial ANOVA was followed by the
Tukey post hoc test. Special attention was paid to the
final bacterial/yeast/mold counts after 45 D of culture.
In all tests, P < 0.05 was regarded as significant.
The multivariate method of principal component
analysis (PCA) based on the correlation matrix was
employed to assess the relationships among lightness,
redness, yellowness, pH, and sensory characteristics of
LWE and to demonstrate differences in characteristics
between LWE samples preserved with different preser-
vatives. As the lightness (L), redness (a), yellowness
(b), and pH were measured in 3 independent samples
per day and preservative, and sensory characteristics
(appearance, color, consistency, and odor) yielded 12
to 15 independent values per testing day and preserva-
tive, all values were aggregated by averaging the values,
resulting in 42 cases entering the PCA (6 preservatives
+ 1 control group × 6 days).
Statistical analysis was performed in IBM SPSS
Statistics, version 23 (IBM Corp., Armonk, NY) and
Statistica, version 13 (Dell Inc., Tulsa, OK).
RESULTS AND DISCUSSION
The initial TPC in pasteurized LWE (day 1) did
not exceed 3 log cfu g−1 in any of the preserva-
tive/control samples (Table 4). We can assume that
this level of microbial contamination of pasteurized
LWE is relatively frequent, as shown by the results of
other studies (Wareing, 2009; Miller et al., 2010; Rossi
et al., 2010; Cwiková and Nedomová, 2014). Selected
preservatives affected the TPC differently in individ-
ual time points (factorial ANOVA, preservative F6,168
= 26.409, P < 0.001, time F5,168 = 67.771, P < 0.001,
time*preservative F30,168 = 7.625, P < 0.001). Total
plate counts were very similar in all samples within the
first 3 wk (days 1, 15, and 22) but differed over the next
weeks. Within 45 D of storage, the TPCs in pasteurized
LWE preserved by Laktocid and benzoate-sorbate still
did not exceed 3 log cfu g−1 , whereas LWE sam-
ples with other preservatives and the control without
any preservative exhibited bacterial growth to up to
7–8 log cfu g−1 (Table 4). Total plate counts at the end
of observation (day 45) differed significantly between
samples, being the lowest in LWE samples preserved
with benzoate-sorbate and Laktocid (comparison of
samples with benzoate-sorbate and Laktocid against all
other preservatives and control group by the Tukey post
hoc test: P < 0.001 in all comparisons).
The initial counts of LAB (day 1) ranged, on average,
from 0.83 ± 1.14 to 2.38 ± 0.53 log cfu g−1 (Table 5).
Miller et al. (2010) report the average total LAB counts
of 1.9 ± 0.6 log cfu g−1 in samples of pasteurized LWE
from various Czech producers, which is in agreement
with our results.
Selected preservatives in pasteurized LWE affected
the amount of LAB in time differently (factorial
ANOVA, preservative F6,168 = 20.107, P < 0.001,
time F5,168 = 7.867, P < 0.001, time*preservative
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PRESERVATION OF PASTEURIZED LIQUID WHOLE EGG 5
Table 5. Lactic acid bacteria counts (log cfu g−1 ) in pasteurized LWE samples supplemented with different preservatives.

Lactic acid bacteria count (log cfu g−1 )

Preservative Day 1 Day 15 Day 22 Day 29 Day 36 Day 45

Benzoate-sorbate 1.70 ± 0.95a,A

0.68 ± 0.93a,A
1.65 ± 0.96a,A
0.34 ± 0.76 a,A 1,a,A
0 01,a,A

Triphosphates 2.38 ± 0.53a,A 0.44 ± 0.97a,A 1.02 ± 0.93a,A 0.68 ± 0.93a,A 01,a,A 1.01 ± 1.44a,A
Nisin 0.84 ± 1.16a,A 01,a,A 0.34 ± 0.76a,A 0.34 ± 0.76a,A 01,a,A 01,a,A
Laktocid 0.83 ± 1.14a,A 1.92 ± 0.21a,A 0.68 ± 0.93a,A 0.68 ± 0.93a,A 0.83 ± 1.14a,A,B 01,a,A
Defence JB 1.83 ± 1.11a,A 0.40 ± 0.89a,A 1.58 ± 0.93a,A 2.38 ± 1.41a,A 1.20 ± 1.75a,A,B 5.50 ± 0.59b,B
Galimax Flavor V50 1.69 ± 0.97a,b,A 0.74 ± 1.02a,A 1.14 ± 1.05a,A 2.12 ± 0.64a,b,A 2.54 ± 1.28a,b,B 4.19 ± 2.35b,B
Control group 2.35 ± 0.39a,b,A 1.80 ± 1.12a,b,A 1.24 ± 1.86a,A 1.76 ± 1.06a,b,A 2.54 ± 0.83a,b,B 4.23 ± 1.55b,B

Mean ± standard deviation, n = 5 per day and preservative/control group.

1
No colonies of lactic acid bacteria were detected in any of the replicates by spreading 0.2 mL of 10−1 sample dilution.
Lactic acid bacteria count on days followed by the same small letter in the row did not differ significantly (Tukey post hoc test in factorial ANOVA).
Lactic acid bacteria count in samples supplemented with different preservatives followed by the same capital letter in the column did not differ
significantly (Tukey post hoc test in factorial ANOVA).

Table 6. Yeast counts (log cfu g−1 ) in pasteurized LWE samples supplemented with different preservatives.

Yeast count (log cfu g−1 )

Preservative Day 1 Day 15 Day 22 Day 29 Day 36 Day 45
1,a,A 1,a,A 1,a,A 1,a,A 1,a,A 1,a,A
Benzoate-sorbate 0 0 0 0 0 0
Triphosphates 01,a,A 0.80 ± 1.79a,A 0.79 ± 1.77a,A 0.67 ± 1.50a,A 0.80 ± 1.79a,A 01,a,A
Nisin 0.40 ± 0.55a,A 01,a,A 01,a,A 01,a,A 01,a,A 01,a,A
Laktocid 01,a,A 01,a,A 01,a,A 01,a,A 01,a,A 01,a,A
Defence JB 0.40 ± 0.89a,A 0.32 ± 0.72a,A 01,a,A 0.73 ± 1.64a,A 01,a,A 1.28 ± 1.90a,A
Galimax Flavor V50 01,a,A 01,a,A 01,a,A 01a,A 01,a,A 01,a,A
Control group 01,a,A 01,a,A 01,a,A 01,a,A 0.73 ± 1.63a,A 01,a,A

Mean ± standard deviation, n = 5 per day and preservative/control group.

1
No colonies of yeasts were detected in any of the replicates by spreading 0.1 mL of sample.
Yeast count on days followed by the same small letter in the row did not differ significantly (Tukey post hoc test in factorial ANOVA).
Yeast count in samples supplemented with different preservatives followed by the same capital letter in the column did not differ significantly
(Tukey post hoc test in factorial ANOVA).

F30,168 = 4.749, P < 0.001), although the LAB count
was rather the same within the first 3 wk (day 1–22, see
Table 5). The final counts of LAB on day 45 were lower
in the samples with benzoate-sorbate, triphosphates,
nisin, and Laktocid, but they were higher in the sam-
ples with Galimax Flavor V50 and Defence JB as well as
in the control group; the difference was significant (the
Tukey post hoc test: P < 0.001 in all above-mentioned
comparisons).
In previous studies, the preservatives such as sorbic
acid, benzoic acid, and/or their combination have not
proved very effective in inhibiting the growth of lacto-
bacilli (Matsuda et al., 1994; Míková et al., 2003). Our
results, however, did not confirm their findings. Fialová
et al. (2008) stated that the bacteriocin nisin inhibits
the growth of a wide range of Gram-positive bacteria
including LAB. Miller et al. (2010) demonstrated, con-
sidering the LAB to be the major spoilage bacteria of
pasteurized LWE, that the addition of nisin to LWE
prior to pasteurization appears to be a promising option
for extending the shelf-life by at least 5 wk at 4°C com-
pared with pasteurization alone. Although nisin was
added after pasteurization of LWE in our study, its
good preservative effect regarding LAB was confirmed.
Further, Fialová et al. (2008) studied the preservative
effect of an organic acid based preparation Laktocid
in mayonnaise-based products. The authors assumed
that Laktocid could efficiently prevent the growth of
undesirable lactobacilli and extend the shelf-life of these
products.
In most cases, the initial counts of yeasts and molds
(day 1) were below the detection limit of the plate
method used, i.e., no colonies were detected by spread-
ing 0.1 mL of sample (Tables 6 and 7). Likewise,
Cwiková and Nedomová (2014) have reported very low
counts of molds and yeasts in 24 samples of pasteurized
LWE collected over a period of 1 yr (April to Febru-
ary). Molds and yeasts were detected only in January
at an amount of 1 log cfu g−1 .
The amount of yeasts was affected by the preservative
but not by time of storage (factorial ANOVA, preser-
vative F6,168 = 2.788, P = 0.013, time F5,168 = 0.128,
P = 0.986, time*preservative F30,168 = 0.780, P =
0.789). A detailed analysis of the final amounts of yeasts
did not confirm differences between samples with dif-
ferent preservatives (the Tukey post hoc test P > 0.05).
Not even the slightly higher yeast count in the samples
with Defence JB was statistically significant (Table 6).
The amount of molds was affected by the time of
storage and preservative but not by their interaction
 aded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez378/5536755 by The University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic user on 13 Augu
6 NECIDOVÁ ET AL.
Table 7. Mold counts (log cfu g−1 ) in pasteurized LWE samples supplemented with different preservatives.

Mold count (log cfu g−1 )

Preservative Day 1 Day 15 Day 22 Day 29 Day 36 Day 45

Benzoate-sorbate 01,a,A
0 1,a,A
01,a,A
0.98 ± 1.34 a,b,A
1.72 ± 0.99 a,b,A
2.26 ± 0.33b,A
Triphosphates 01,a,A 01,a,A 01,a,A 0.73 ± 1.62a,b,A 0.98 ± 1.35a,b,A 2.26 ± 1.28b,A
Nisin 01,a,A 01,a,A 01,a,A 01,a,A 0.64 ± 0.91a,b,A 2.32 ± 0.51b,A
Laktocid 01,a,A 01,a,A 01,a,A 01,a,A 01,a,A 0.92 ± 1.29a,A
Defence JB 01,a,A 0.40 ± 0.89a,A 01,a,A 01,a,A 0.43 ± 0.96a,A 1.33 ± 1.22a,A
Galimax Flavor V50 01,a,A 01,a,A 01,a,A 01,a,A 0.73 ± 1.01a,A 1.65 ± 1.12a,A
Control group 01,a,A 01,a,A 0.37 ± 0.83a,b,A 0.26 ± 0.58a,b,A 0.70 ± 1.09a,b,A 1.75 ± 0.99b,A

Mean ± standard deviation, n = 5 per day and preservative/control group.

1
No colonies of molds were detected in any of the replicates by spreading 0.1 mL of sample.
Mold count on days followed by the same small letter in the row did not differ significantly (Tukey post hoc test in factorial ANOVA).
Mold count in samples supplemented with different preservatives followed by the same capital letter in the column did not differ significantly
(Tukey post hoc test in factorial ANOVA).

Table 8. pH values of pasteurized LWE samples supplemented with different preservatives.

pH value
Preservative Day 1 Day 15 Day 22 Day 29 Day 36 Day 45
Benzoate-sorbate 7.89 ± 0.02 7.92 ± 0.06 7.98 ± 0.06 7.98 ± 0.08 7.98 ± 0.15 7.93 ± 0.14
Triphosphates 8.08 ± 0.03 8.13 ± 0.08 8.27 ± 0.15 8.10 ± 0.07 8.18 ± 0.10 7.80 ± 0.09
Nisin 7.71 ± 0.04 7.77 ± 0.04 7.73 ± 0.05 7.76 ± 0.01 7.67 ± 0.03 7.60 ± 0.04
Laktocid 6.14 ± 0.01 6.18 ± 0.00 6.15 ± 0.01 6.15 ± 0.02 6.13 ± 0.03 6.14 ± 0.02
Defence JB 7.64 ± 0.02 7.73 ± 0.21 7.73 ± 0.05 7.65 ± 0.19 7.61 ± 0.04 7.13 ± 0.38
Galimax Flavor V50 7.25 ± 0.06 7.41 ± 0.12 7.57 ± 0.07 7.32 ± 0.03 7.56 ± 0.14 7.43 ± 0.10
Control group 7.73 ± 0.03 7.81 ± 0.08 7.79 ± 0.06 7.91 ± 0.11 7.86 ± 0.23 7.48 ± 0.20

Mean ± standard deviation, n = 3 per day and preservative/control group.

as the increase in the mold counts showed the same
pattern in all samples supplemented with various
preservatives (factorial ANOVA, preservative F6,168 =
2.900, P = 0.010, time F5,168 = 34.556, P < 0.001,
time*preservative F30,168 = 1.010, P = 0.460). Molds
rarely occurred within the first 4 wk (days 1 to 29) but
their presence increased in the next weeks, neverthe-
less not exceeding 3 log cfu g−1 (Table 7). The lowest
final mold count was observed in pasteurized LWE pre-
served with Laktocid, although the difference between
samples preserved with different preservatives was not
significant (the Tukey post hoc test, P > 0.05).
During the whole storage period, the observed counts
of Enterobacteriaceae were below the detection limit
of the plate method used in all samples (preserva-
tive/control group). Gram-negative bacteria including
members of the Enterobacteriaceae family very often
contaminate shell eggs and can enter the liquid egg
while breaking the shell (De Reu et al., 2005; Németh
et al., 2011). On the other hand, they are relatively
easy to destroy by heat treatment (Miller et al., 2010).
In accordance with our results, Miller et al. (2010) have
reported the counts of Enterobacteriaceae to be below
the detection limit of the plate method used (<1 log
cfu g−1 ) in 13 samples of pasteurized LWE, with the
remaining 2 samples containing 1.2 log cfu g−1 and
1.6 log cfu g−1 .
Our results show great potential of the preservative
Laktocid in reducing TPC and suppressing the growth
of LAB and molds. The shelf-life of pasteurized LWE
can potentially reach up to 45 D at 4°C.
Another aspect we evaluated was the effect of the
preservatives on the sensory quality of pasteurized
LWE. The measured pH values are listed in Table 8.
Principal component analysis was used to explore gen-
eral relationships among lightness, redness, yellowness,
pH, and sensory characteristics (appearance, color, con-
sistency, and odor) of LWE samples supplemented with
different preservatives. The first 2 principal components
accounted for 88.3% of the total variance in data. The
projection of the variables on the factor plane confirms
strong positive correlation of pH and redness (a value),
strong negative correlation of redness and yellowness
(b value), and their independence on sensory character-
istics. Appearance and consistency correlated positively
(Figure 1).
Samples’ projection into the ordination space is
determined by the measured characteristics. The
highest redness and pH were found in LWE samples
preserved with benzoate-sorbate, and high pH val-
ues were observed in samples preserved with triphos-
phates. At the same time, the samples treated with
benzoate-sorbate showed the lowest values for lightness,
appearance, color, and consistency. The highest yellow-
ness along with the lowest redness, pH, and odor was
found in LWE samples preserved with Laktocid. Liq-
uid whole egg samples preserved with nisin, Defence
JB, and Galimax Flavor V50 did not differ from the

PRESERVATION OF PASTEURIZED LIQUID WHOLE EGG
1.0
0.5
Principal component 2: 35.8%
0.0
-0.5
pH
a thin) causing the bleaching of LWE color due to higher
Odor
-1.0
-1.0 -0.5
Principal component 1: 52.5%
Figure 1. Positions of lightness (L), redness (a), yellowness (b), pH,
and sensory characteristics of pasteurized LWE samples supplemented
with different preservatives in the first 2 principal components of PCA
(λ1 = 4.197, λ2 = 2.862, sum of all eigenvalues 8.0). Position of cases
in the ordination space in Figure 2.
5 addition of sodium diphosphate and trisodium phos-
4
3
Benzoate-sorbate
Principal component 2: 35.8%
2
1 Triphosphates
0 acceptability of many food products; therefore, cus-
-1
-2
-3
-4
-5
-5 -4 -3 -2 -1
Principal component 1: 52.5%
Figure 2. Positions of pasteurized LWE samples preserved with 6
different preservatives and of control group in the first 2 principal
components of PCA. ●—benzoate-sorbate, —triphosphates, —nisin
—Laktocid, —Defence JB, ♦—Galimax Flavor V50, +—control
group. Positions of LWE characteristics in the ordination space are
presented in Figure 1.
control group in the measured characteristics, as sam-
ple points overlap in the ordination space (Figure 2).
Our results correspond to the findings of Chan and
Chen (2000) who discovered strong correlations be-
tween the pH and Hunter L, a, and b values of LWE.
They have reported that the L and b values of LWE
were decreasing (P < 0.05) and the a values were in-
creasing (P < 0.05) with increasing pH. The Hunter b
value was one of the indicators to determine the changes
in the texture and color with pH changes.
aded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pez378/5536755 by The University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic user on 13 Augu
7
The success of many cooked foods is related to pro-
tein coagulation, especially the coagulation of egg pro-
teins, which are capable of forming gels with desirable
nutritional and textural characteristics (Mine, 1995).
b The pH of shell eggs and egg products is an essential
attribute for achieving adequate LWE quality proper-
ties, such as foaming, emulsifying, gelling, and color
(Monfort et al., 2012). The use of preservatives chang-
L
ing pH value can therefore negatively affect the texture
characteristics of the LWE.
The paler color has been associated with the oxi-
dation of pigments (xanthophylls lutein and zeaxan-
Consistency
Appearance temperatures (Koc et al., 2011). Badr (2006) has re-
ported that the visual color of liquid egg yolk samples
Color
was significantly (P < 0.05) affected after 6 D of cold
0.0 0.5 1.0
storage but they still scored as acceptable. Besides,
samples of untreated liquid egg white and of liquid
egg yolk showed significant (P < 0.05) changes in their
odor acceptability and scored unacceptable on day 6 of
storage. Similarly, Ratu et al. (2017) have stated that
pasteurized LWE suffered certain sensorial modification
during storage at 4°C for 28 D, especially in terms of
consistency and color. According to Li et al. (2018), the
phate significantly affected the solubility, surface hy-
drophobicity, z-average particle size, and pH of LWE
Laktocid
dispersions. Moreover, the phosphate dimer (sodium
diphosphate) has a stronger ferrous chelating capac-
ity than the phosphate monomer (trisodium phosphate)
and thus can significantly improve the color character-
istics. At last, the color from the egg yolk affects the
Nisin tomers may not accept discoloration and color changes
Defence JB
Galimax Flavor V50
due to processing, considering them detrimental to the
Control group egg quality (De Souza and Fernandez, 2011).
CONCLUSION
In recent years, an increased customer pressure to
0 1 2 3 4 5 limit the use of traditional chemical food preservatives
and to replace them with natural alternatives has been
observed. At the same time, the egg-based products are
required to have as long shelf-life as possible.
This study demonstrated that a mixture of organic
acids (acetic acid, lactic acid, and sodium lactate) in
the preservative Laktocid has a great potential for ex-
tending the shelf-life of pasteurized LWE to up to 45
days when stored at 4°C. On the other hand, Laktocid
had a significant effect on the pH value and sensory
characteristics of LWE, as compared with the control
sample. The pH decrease could potentially lead to de-
terioration of technological properties of LWE. Simi-
larly, the lighter color could be perceived as a mark
of lower quality or possible product spoilage by the
customers.
Other alternative preservatives tested, i.e., those
based on bacteriocins (nisin), natural fermentate of
starter cultures, or vinegar, are effective in controlling

8 NECIDOVÁ ET AL.
the undesirable development of microorganisms in pas-
teurized LWE for a limited period of 3 wk while having
a minimum impact on their sensory characteristics.
In conclusion, the results of our study indicate that
none of the tested traditional/alternative preservatives
has been capable of substantially extending the shell-
life of LWE while preserving its optimum properties.
From this perspective, we cannot make any definite
recommendation of an alternative to the commonly
used preservatives sodium benzoate and potassium sor-
bate. The selection of an alternative LWE preservative
should therefore be performed in view of the subse-
quent technological usage of the LWE and of customers’
requirements.
ACKNOWLEDGMENT
This work was supported by the Internal Grant
Agency of the University of Veterinary and Pharma-
ceutical Sciences Brno (IGA VFU Brno, grant number
212/2017/FVHE).
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