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M: Food Microbiology
& Safety
Microbial Quality of Industrial Liquid Egg White:
Assumptions on Spoiling Issues in Egg-Based
Chilled Desserts
Clarisse Techer, Amina Daoud, Marie-No¨
elle Madec, Michel Gautier, Sophie Jan, and Florence Baron
Abstract: As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French egg
processing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally
found efficient on mesophilic contaminants (1.7 ±1.6 and 0.8 ±0.9 log CFU/mL in raw and pasteurized samples,
respectively), including for the control of Salmonella.However,Gram-positiveenterococci were still detected in the pasteur-
ized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based
chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French
chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage
of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream,
on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities
were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further
selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates
were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This study
emphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi,in
order to guarantee the shelf life of egg-based chilled desserts.
Keywords: bacterial contamination, chilled desserts, enzymatic activities, liquid egg white, spoilage
Practical Application: This study provides new insights on the bacterial contamination of industrial liquid egg white
and represents the starting point for the control of spoilage issues in the sector of chilled egg-based desserts potentially
hosting egg white bacterial contaminants.
Introduction
Over the past few decades, the production of egg products has
been constantly increasing. In 2012, France was among the 1st
European producer, with approximately 290000 tons in liquid
equivalent (Itavi 2013). Egg products, defined as eggs removed
from their shells, are sold in the form of whole egg, egg yolk, or
egg white products, either in liquid, dried, or frozen forms. They
are used for the production of a wide range of foods such as pastry,
sea-food products, meat products, or dairy products, including raw
or poorly cooked finished products that are particularly sensitive
from a microbiological point of view.
While the content of shell eggs is generally sterile under safe
conditions of breeding, the average level of contamination of the
eggshell surface is comprised between 3.8 and 6.3 log CFU/egg for
the aerobic mesophilic flora, depending on the studies (Baron and
Jan 2010 for review). Eggshells are therefore a likely source of cross-
contamination at the step of industrial egg breaking (Baron and Jan
2010 for review). In order to ensure the microbial stabilization of
the egg products, heat treatments are almost systematically carried
out. In the French egg processing industry, pasteurization is carried
out for 2 to 6 min at 65 °Cto68°C for whole egg and egg
MS 20141105 Submitted 6/27/2014, Accepted 12/4/2014. Authors are with
Equipe Microbiologie de l’Oeuf et des Ovoproduits (Micov), UMR1253 Science et
Technologie du Lait et de l’Œuf, Agrocampus Ouest, INRA, F-35042, Rennes,
France. Direct inquiries to author Baron (E-mail: florence.baron@agrocampus-ouest.fr).
yolk and at lower temperatures (55 to 57 °C) for egg white, due
to the higher heat-sensitivity of the constitutive proteins of this
later. After this pasteurization step, the liquid egg products are
rapidly cooled and they are conditioned, stored, and delivered at
refrigeration temperature. Their shelf life ranges from few weeks
to 3 mo at 4 °C.
There are numerous studies relating to egg contamination with
Salmonella Enteritidis, due to the strong involvement of this mi-
croorganism in food-poisoning outbreaks in relation to egg con-
sumption (EFSA 2014). By contrast, the contamination with
heat-resistant bacteria has been poorly investigated in this sector,
whereas they can shorten the shelf life of the egg products them-
selves or of food products containing egg products as ingredients,
with damaging economic consequences for the egg processing
industry. On the few studies reporting the contamination of pas-
teurized liquid egg products by heat resistant bacteria, Enterococcus
sp. (Hidalgo and others 2008; Miller and others 2010) and bac-
teria of the Bacillus cereus group (Baron and others 2007; Techer
and others 2014) were already highlighted in pasteurized liquid
whole egg products. However, little is known on the microbial
quality of industrial egg white. This issue is of great importance
when foams of egg white are used for the production of highly
perishable products, including chilled egg-based desserts such as
the French floating island. This chilled dessert comprises a sug-
ared egg white foam deposited onto custard cream. Contrary to
egg white, which is recognized as not readily supporting bacterial
growth (Baron and Jan 2010 for review), the custard cream is rich
C2015 Institute of Food Technologists R
doi: 10.1111/1750-3841.12764 Vol. 80, Nr. 2, 2015 rJournal of Food Science M389
Further reproduction without permission is prohibited
M: Food Microbiology
& Safety
Bacteria from liquid egg white . . .
in various substrates such as proteins, lipids, and carbohydrates.
Since it is sterilized at 132 °C for a few minutes, this ingredient
is unlikely to host bacteria, provided that postcontaminations are
avoided. The spoilage events recorded along storage of the dessert
may then rather involve a bacterial population provided by the
pasteurized egg white foam. While weakly allowed to grow in this
medium, this bacterial population may find in the custard cream
all the nutrients for growth. In order to test this assumption, the
present study aimed at characterizing the microbial quality of in-
dustrial liquid egg white and at testing, at low temperature, the
spoilage potential of egg white bacterial contaminants inoculated
in custard cream. This later was used as such or in an agar test
allowing the quantification of specific bacterial enzymatic activi-
ties, which are recognized as being fairly induced in food spoilage
(Pirttijarvi and others 1996; Cosentino and others 1997).
Materials and Methods
Microbiological analyses of the egg white samples
Sixty-three samples of liquid egg white were provided by a sin-
gle egg processing industry from Western France for a 1-y period.
The egg white products came from clean and uncraked eggs.
They contained no ingredients, no stabilizers, no whipping or
processing aids, and no preservatives. For the batches 1 to 19,
the samples were analyzed both before (raw egg white products)
and after (pasteurized egg white products) pasteurization at 57 °C
for 6 min. The batches 20 to 30 were exclusively analyzed be-
fore pasteurization, while the batches 31 to 44 were exclusively
analyzed after pasteurization.
The concentration of total mesophilic bacteria was assessed after
serial decimal dilutions in Tryptone salt (TS; 0.1% Tryptone, AES
laboratoire, Combourg, France; 0.85% NaCl, Labogros, Buchs,
France), plating onto Brain Heart Infusion-Yeast Extract agar
(BHI-YE) (3.7% BHI (Merck, Darmstadt, Germany); 0.1% Yeast
extract (Merck); 1.5% agar (Merck) and incubation under aerobic
and anaerobic (Anaerocult A, Merck) conditions for 48 h at 30 °C.
Each sample was analyzed in triplicate under both aeration con-
ditions. After incubation, the colony forming units (CFU) were
counted and the results were expressed as the mean of 3 replicates
in log CFU/mL ±standard deviation (SD) for each sample.
Identification of the bacteria and construction of the
bacterial collection
For each egg white sample, 5 to 10 colonies were randomly
collected in the BHI-YE agar counting plates. In order to avoid
redundancy, 3 to 5 purified colonies coming from the same egg
white sample were selected, based on macroscopic and micro-
scopic criteria. This selection resulted in the creation of a bacterial
collection comprising 166 isolates. All these isolates were (i) iden-
tified by 16S rDNA sequencing and (ii) tested in liquid custard
cream for assessing their growth potential and the type of spoilage
they induced. A 43-isolate subset was further selected inside the
collection in order to guarantee the best representativeness of each
genus and of each spoilage type. These 43 isolates were then sub-
mitted to enzymatic assays on custard cream-based agar media.
16S rDNA sequencing
Three hundred microliters of a 25% (m/v) sterile suspension
of Chelex beads (Grosseron, Saint-Herblain, France) prepared in
sterile Milli-Q water (Sigma Aldrich, Saint-Quentin Fallavier,
France) was added to 5 mL of each bacterial frozen culture
previously thawed and incubated overnight in BHI-YE at 30 °C.
The mixture was vortexed and centrifuged at 5700 ×gfor 7 min
at 4 °C. The cell pellet was resuspended in 200 µL sterile Milli-Q
water (Sigma Aldrich) and lyzed by heating at 100 °C for 10 min.
After centrifugation at 5700 ×gfor 7 min at 4 °C, 150 µLsuper-
natant was collected and recentrifuged under the same conditions.
The supernatant was then used for 16S rDNA gene amplification.
The final PCR mixture contained 50 ng DNA template, 0.18
mM dNTPs (Eurogentec, Seraing, Belgium), 0.7 µM of each 16S
primers (5’- GCCAGCAGCCGCGGTAA -3’ as forward degen-
erate primer and 5’- GACGGGCGGTGTGTAC -3’ as reverse
primer), 53.6 U/mL AmpliTaq polymerase and 0.9X AmpliTaq
buffer (Biolabs, Evry, France). Thermal cycling was carried out in
a PCR thermocycler (iCycler optical module 584BR, BioRad,
Marnes-la-Coquette, France) with the following run: a starting
cycle of 4 min at 95 °C, followed by 30 cycles of 30 s at 95 °C, 30 s
at 50 °Cand30sat72°C, and a final extension of 7 min at 72 °C.
The PCR products were verified after migration on 1% agarose gel
(80V, 30 min) in TBE 1X (Eurobio, Courtaboeuf, France) together
with the DNA Smart Ladder SF molecular weight marker (Euro-
gentec). Gels were stained with ethidium bromide (Sigma Aldrich)
and digitized using a gel imager (Bioblock, Illkirch, France).
The 16S rDNA amplified fragment was identified according to
its apparent length (about 800 bp). Purification and sequencing
were performed by GATC Biotech AG (Konstanz, Germany)
using the following primer: 5’- GCCAGCAGCCGCGGTAA-
3’. DNA sequences were compared with available sequences
deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank)
using nucleotide-nucleotide Basic Local Alignment Search Tool
(BLAST) for nucleotides (blastn) alignments accessed from the
website (http://www.ncbi.nlm.nih.gov/BLAST/).
Spoilage experiments
Inoculum. For each selected isolate, a purified colony was
transferred into 10 mL BHI-YE and the mixture was incubated
at 30 °C for 18 h under aerobic conditions. Aliquots of each
culture were frozen at –20 °C after glycerol addition at a final
concentration of 25%. Before each experiment, frozen aliquots
were thawed and twice propagated in BHI-YE at 30 °Cfor18h
under aerobic conditions.
Culture media. For liquid-state experiments, the culture
medium consisted in commercial UHT custard creams provided
by a local supermarket. According to the manufacturer, the creams
contain 80% milk, 12% sucrose, 7% whole egg, around 1% starch
and unknown quantities of vanilla aroma and stabilizer.
For solid-state experiments, the custard cream-based agar
medium was prepared with 10% sterile commercial custard cream
added to a previously sterilized (121 °C for 21 min) medium con-
sisting in 1.5% agar in distilled water (pH 6.9). Twelve milliliters
of this mixture were poured into each Petri dish. For the detection
of the lipolytic activity, 2% of rhodamine B (Sigma Aldrich) was
added before pouring. This later becomes fluorescent when com-
plexed with the free fatty acids released by the bacterial lipases,
according to the studies of Kouker and Jaeger (1987), Feng and
others (2005), and Yan and others (2007).
Growth in liquid custard cream. A collection of 166 iso-
lates was tested for growth experiments in sterile liquid custard
cream (see the section Identification of the bacteria and construction
of the bacterial collection). Each inoculum was previously diluted in
tryptone salt in order to reach an initial concentration of 3 to 5 log
CFU/mL in sterile commercial custard cream (10 mL in Falcon
tubes). The contaminated custard cream samples were incubated
at 10 °C for 21 d under aerobic conditions. Before and after this
M390 Journal of Food Science rVol. 80, Nr. 2, 2015
M: Food Microbiology
& Safety
Bacteria from liquid egg white . . .
incubation period, enumeration was carried using a plate-counting
micro-method (Baron and others 2006). Cells counts were deter-
mined after 24 h growth at 30 °ConBHI-YEagar.
Experiments were repeated for visual confirmation of bacterial
growth. Enumerations were carried out only once, and the results
were expressed in log CFU/mL for each tested isolate.
Spoilage characteristics of the liquid custard cream.
Spoilage criteria were based on visual observations (thickening,
coagulation, production of holes), on a hearing test (sound detec-
tion at the opening of the tube) and on pH measurements (pH
meter 315i, WTW, Weilheim, Germany). Results were expressed
as ࢞pH, corresponding to the difference between the pH of the
custard cream at time zero of inoculation and after 21 d of incu-
bation at 10 °C.
Enzymatic activities in custard cream agar-based
media. A collection of 43 isolates was selected (see the section
Identification of the bacteria and construction of the bacterial collection).
Two microliters of each inoculum was spotted onto the agar
plates. Incubation was carried out for 48 h at 30 °C under aerobic
conditions. After this incubation period and according to the
protocol of Loperena and others (2012), proteolytic activities
were evaluated by the measure of the clearing zone surrounding
the colonies (diameter of the clearing zone – diameter of the
colony)/2, expressed in mm. Results were expressed as the mean
of 2 replicates. According to the protocol of Hankin and others
(1975), amylolytic activities were evaluated after addition of an
iodine solution on the agar plates (lugol, Sigma Aldrich) and mea-
sure of the clearing zone surrounding the colonies (diameter of the
clearing zone – diameter of the colony)/2. Results were expressed
as the mean of 2 replicates. According to Feng and others (2005),
lipolytic activities were detected under Rhodamine B fluorescent
illumination (Olympus BX51TF, Olympus, Hamburg, Germany).
Wavelengths used were 553 and 627 nm for excitation and emis-
sion, respectively. Experiments were performed in duplicate.
Controls. The sterility of the custard cream was systematically
checked before each experiment, as follows: 1 mL of custard cream
was poured into a Petri dish containing BHI-YE agar. The ab-
sence of growth was checked after overnight aerobic incubation at
30 °C.
For the characterization of the type of spoilage of the custard
cream, a noninoculated sample was systematically incubated under
the same conditions as those for the assays.
For the enzymatic tests, both negative and positive controls were
carried out. Positive controls were implemented independently
by deposit of 2 µL of a pur ified protease from Bacillus licheniformis
(ref. P5459, Sigma Aldrich), a purified lipase from Pseudomonas
fluorescens (ref. 95608, Sigma Aldrich), and an amylase from
Bacillus sp. (ref. A7720, Sigma Aldrich) for proteolytic, lipolytic,
and amylolytic activities, respectively. The absence of interference
between the 3 enzymatic tests was also checked by deposit of
each purified enzymes onto each performed medium.
Results and Discussion
Microbial quality of liquid egg white
Regardless of the type of sample analyzed, the bacterial counts
were of the same order of magnitude under aerobic and anaerobic
conditions of enumeration (Table 1). It can therefore be con-
cluded that egg white contaminants were aero-anaerobic bacteria.
This observation allowed interpreting the results regardless of the
aeration conditions of enumeration.
Considering the raw egg products, the bacterial concentrations
were fairly low with, on average, less than 2 log CFU/mL. These
results are in accordance with the intrinsic properties of egg white.
This fluid is known as unlikely supporting microbial growth due
to the cumulative effect of alkaline pH, iron deficiency, and the ex-
pression of antimicrobial molecules, such as lysozyme, ovotransfer-
rin, proteinase inhibitors (cystatin, ovomucoid, and ovoinhibitor),
and vitamin-binding proteins (riboflavin-, avidin-, and thiamin-
binding proteins) (Baron and Jan 2010 for review). However,
2 raw samples were highly contaminated, exhibiting more than
5 (Nr 29) and more than 8 log (Nr 30 in Table 1) CFU/mL.
These high levels of contamination may be ascribed to the use
of endogenously contaminated eggs or to the release of bacte-
ria from a biofilm present in the processing equipment. Another
explanation should be that a poor separation of egg white from
egg yolk would have resulted in the driven of egg yolk into these
egg white samples, resulting in the impairment of the egg white
antimicrobial activities. The presence of as low as 0.5% egg yolk
in egg white is recognized as sufficient to allow bacteria to grow
in egg white at permissive temperatures (Baron 1998).
Despite the globally low bacterial concentrations, the contam-
ination concerned 93% of the analyzed raw samples (Table 1).
Events of cross-contaminations with eggshells represent the most
likely explanation for this result. Indeed, the contact of the egg
content with eggshells is unavoidable at the step of egg break-
ing. Contrary to the egg content, which is generally sterile under
safe conditions of breeding (Baron and Jan 2010 for review), the
eggshells are systematically contaminated (Musgrove and others
2004, 2005; De Reu and others 2005, 2006). This systematic
cross-contamination confirms the need to stabilize industrial egg
products, including liquid egg white. The European regulation
describes specific requirements for the manufacture of egg prod-
ucts. Considering the microbiological criter ia, the regulation Nr
2073/2005 defines (i) a food safety criterion for Salmonella in “egg
products, excluding products where the manufacturing process or
the composition of the product will eliminate the Salmonella risk”
for egg products placed on the market during their shelf life (ab-
sence in 25 g) and (ii) a process hygiene criterion for Enterobacteri-
aceae at the end of the process of egg product manufacturing (less
than 2 log CFU/g).
In the present study, pasteurization was found efficient since
the percentage of positive samples was reduced between 64% and
50% after pasteurization under aerobic and anaerobic conditions,
respectively (Table 1). The efficiency of pasteurization was globally
confirmed regarding the average bacterial concentrations which
were reduced to less than 1 log CFU/mL in the positive samples
(Table 1). The range of concentrations was narrower than in the
raw samples, that is, from less than 1 CFU/mL to around 2 log
CFU/mL (Table 1).
Comparison with literature is difficult since, to our knowledge,
the microbial quality of industrial egg white has so far been
poorly investigated in Europe. Correa and others (2008) have
reported a bacterial concentration of 2 log CFU/mL in Spanish
pasteurized liquid egg white products, corroborating the average
low concentrations highlighted in the present study. The literature
is more abundant regarding the contamination of pasteurized
whole egg products. Whole egg products are more likely to
be contaminated due to the fact that, unlike egg white, they
represent optimal media for bacterial growth (Baron and Jan
2010 for review). However, the pasteurization of whole egg
products is possible under stronger conditions than those used
for egg white, due to the higher heat-resistance of egg yolk
proteins. The resulting level of contamination reported in the
literature is nearly similar as that found in pasteurized liquid egg
Vol. 80, Nr. 2, 2015 rJournal of Food Science M391
M: Food Microbiology
& Safety
Bacteria from liquid egg white . . .
Table 1–Plate-counting of bacteria in raw and pasteurized (57 °C for 6 min) liquid egg white samples in Brain Heart Infusion-Yeast
extract (BHI-YE) agar under aerobic and anaerobic conditions at 30 °C for 48 h. Results are expressed as the mean of 3 technical
replications in log CFU/mL ±standard deviation.
Before pasteurization After pasteurization
Batch number Aerobic flora Anaerobic flora Aerobic flora Anaerobic flora
11.4±0.03 1.3 ±0.2 1.8 ±0.0 1.8 ±0.0
21.4±0.1 1.7 ±0.0 <LD <LD
30.7±0.3 0.9 ±0.1 1.6 ±0.2 1.5 ±0.2
41.3±0.1 1.1 ±0.1 <LD <LD
52.4±0.1 2.3 ±0.1 <LD <LD
60.7±0.2 0.2 ±0.1 <LD <LD
71.5±0.1 1.8 ±0.1 1.5 ±0.2 1.5 ±0.1
82.3±0.0 1 ±0.1 0.1 ±0.2 0.2 ±0.2
91.1±0.2 1 ±0.04 0.4 ±0.3 0.5 ±0.0
10 1.2 ±0.1 1.3 ±0.2 <LD 0.1 ±0.2
11 1.6 ±0.1 1.7 ±0.2 0.8 ±0.2 0.9 ±0.3
12 1.1 ±0.1 0.7 ±0.2 1 ±0.8 <LD
13 0.3 ±0.3 0.6 ±0.2 0.2 ±0.3 <LD
14 3.1 ±0.1 3 ±0.1 0.3 ±0.3 0.2 ±0.2
15 1.9 ±0.1 1.5 ±0.2 0.2 ±0.3 <LD
16 3.6 ±0.1 2.3 ±10.3±0.2 0.4 ±0.1
17 0.3 ±0.0 <LD 1 ±0.0 1 ±0.0
18 1.3 ±0.1 1.1 ±0.12 1.2 ±0.1 1.3 ±0.2
19 1.6 ±0.2 1.3 ±0.2 <LD <LD
20 3.2 ±2.8 2.8 ±0.2 NA NA
21 1.7 ±0.1 1.6 ±0.1 NA NA
22 1.1 ±0.3 0.8 ±0.3 NA NA
23 <LD <LD NA NA
24 2.7 ±0.1 2.9 ±0.2 NA NA
25 1.4 ±0.6 0.5 ±0.5 NA NA
26 1.4 ±0.3 1.4 ±0.1 NA NA
27 1.2 ±0.1 1.3 ±0.1 NA NA
28 1.2 ±0.2 1.3 ±0.1 NA NA
29 5.2 ±0.3 5.3 ±0.4 NA NA
30 8.3 ±0.3 8.3 ±0.3 NA NA
31 NA NA 0.9 ±0.1 1.0 ±0.1
32 NA NA 2.1 ±0.3 <LD
33 NA NA <LD <LD
34 NA NA <LD <LD
35 NA NA 0.7 ±0.3 0.8 ±0.1
36 NA NA 1.8 ±0.1 1.8 ±0.0
37 NA NA 0.2 ±0.3 <LD
38 NA NA <LD <LD
39 NA NA <LD <LD
40 NA NA 0.3 ±0.5 0.1 ±0.2
41 NA NA 3.3 ±1.0 2.7 ±2.4
42 NA NA <LD <LD
43 NA NA <LD <LD
44 NA NA 0.86 ±0.8 <LD
Mean 1.7 ±1.6 1.6 ±1.6 0.8 ±0.9 0.6 ±0.8
Number (percentages) of contaminated samples 29 (93%) 28 (93%) 21 (64%) 16 (50%)
NA, not analyzed; LD, limit of detection of the plate-counting method (1 CFU/mL).
white, that is, around 2.3 log CFU/mL of mesophilic aerobic
bacteria for pasteurized whole egg products processed in France
(Protais and others 2006) and also in Canada (Miller and others
2010).
Identification of bacterial contaminants in liquid egg white
A total of 166 isolates, including 100 and 66 isolates coming
from raw and pasteurized egg white samples, respectively, were
further identified by 16SrDNA sequencing. Regardless the type
of sample (raw or pasteurized), 64 (38.5%), 33 (19.9%), 31
(18.7%), 19 (11.5%), 10 (6%), 3 (1.8%), 1 (0.6%), 1 (0.6%), 1
(0.6%), 1 (0.6%), 1 (0.6%), 1 (0.6%) of the recovered isolates
were of the genera Enterococcus,Escherichia,Enterobacter,Serratia,
Shigella,Klebsiella,theBacillus cereus group, and the genera Rah-
nella,Salmonella,Pantoea,Pseudomonas,andYersinia, respectively
(Figure 1). Several sequencing allowed identification at the
species level. For example, in the Enterococcus and Escherichia
genera, the main species identified were Enterococcus faecalis (33%
of Enterococcus sp.) and Escherichia coli (75% of Escherichia sp.)
(Figure 1). These results confirmed the contamination of egg
white products with aero-anaerobic bacteria (Figure 1).
All positive raw samples (100%) were contaminated with
Enterobacteriaceae, and mainly with Enterobacter sp., Escherichia
sp., Serratia sp., Shigella sp., and Pantoea sp., detected in 78, 50,
33, 28, and 11% of the positive samples, respectively. Other
Enterobacteriaceae were rarely detected (in a single batch, corre-
sponding to 6% of the analyzed samples), including Klebsiella sp.,
Yersinia sp., Rahnella sp., and Salmonella sp. (Table 2). These results
support the assumption that eggshells may have represented a
likely source of contamination of the raw products at the step
M392 Journal of Food Science rVol. 80, Nr. 2, 2015
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Bacteria from liquid egg white . . .
of industrial egg breaking. Indeed, the eggshells are described as
being contaminated with 1.2 to 4.9 log CFU Enterobacteriaceae
per egg (Musgrove and others 2005; De Reu and others 2005,
2006), including with Escherichia sp. and Enterobacter sp., and, to a
lower extent, with Salmonella sp., Serratia sp., Yersinia sp., Klebsiella
sp., and Pantoea sp. (Musgrove and others 2004). In addition
to the contamination with Enterobacteriaceae,Enterococcus sp. was
shown to be highly occurring (39% of the positive raw samples).
Finally, Pseudomonas sp. and the Bacillus cereus group were detected
in a single raw sample each (6% of the positive samples). This
result does not exclude their presence in the other samples, but
at concentrations too low for allowing their collection under
our experimental conditions. The B. cereus group was also found
poorly occurring in raw whole egg products (Protais and others
2006), as well as on the eggshell surface (Kon´
e and others 2013).
The flora of the pasteurized samples was significantly differ-
ent and less diversified, confirming the efficiency of the pasteur-
ization toward Enterobacteriaceae, whose presence was reduced to
41% of the positive samples. It is important to note that pas-
teurization was also found efficient on Salmonella sp. which was
not detected after pasteurization (batch Nr 19, Table 2). The
genera Enterobacter,Serratia,Shigella,andKlebsiella were each de-
tected in a single batch after pasteurization (7% of the pasteurized
samples for each) (Table 2). However, Escherichia sp. was still de-
tected in 27% of the pasteurized samples. To our knowledge, these
latter had never been described as contaminants of European in-
dustrial pasteurized egg products, according to the few studies
available in the literature (Protais and others 2006; Hidalgo and
others 2008). The enterobacteria are not recognized as particularly
heat-resistant, except for some Enterobacter spp. However, th e heat
treatments usually carried out for egg white stabilization (from 55
to 57 °C for 2 to 6 min in France) may be too mild for their
eradication. The D55°Cvalues reported in water are from 3.72
to 6.68, 5.08 to 5.9, 1.18, and 0.37 for E. coli,Shigella,Serratia,
and Klebsiella, respectively (Spinks and others 2006). If one takes
into consideration the batch Nr. 29, the bacterial concentra-
tion of raw egg white was around 5.2 log CFU/mL (Table 1)
and the identified bacteria were E. coli together with Enterobacter
sp. and Shigella sp. (bolded characters in Table 2). Considering
Escherichia, the time necessary for the reduction by around 5 log of
the population would be of 19 to 33 min, far more than the 6 min
currently applied for egg white pasteurization. Unfortunately, it
was not possible to assess the level of reduction in this egg white
batch since it was not analyzed after pasteurization. Otherwise,
Escherichia sp. remained after pasteurization in the batches Nr. 16
and Nr. 35, even if at low concentration (less than 1 log CFU/mL,
Table 1) and in the batch Nr. 36 (bolded in Table 2), display-
ing a higher concentration (1.8 log CFU/mL, Table 1). Even if
Enterobacteriaceae are recovered after pasteurization in the liquid egg
white batches analyzed in the present study, their low levels may
Figure 1–Identification of bacteria in industrial samples of raw and pasteurized liquid egg white. Bacteria are identified at the genus (whole pie-chart)
or at the species level (exploded pie-chart) by 16SrDNA sequencing.
Vol. 80, Nr. 2, 2015 rJournal of Food Science M393
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Bacteria from liquid egg white . . .
Table 2–Distribution of the recovered aerobic bacterial genera in industrial raw and pasteurized liquid egg white samples. Results
are expressed as percentage of each genus/group for each raw (total analyzed samples =18) and pasteurized (total analyzed
samples =15) egg white samples. The batch number (Nr.) and the number of identified isolates (indicated in brackets) are
highlighted in light-gray and dark-gray for the raw and the pasteurized samples, respectively. Bolded characters highlight the
batches specifically discussed in the text. The number of isolates for each specific batch is given between brackets.
Frequency (%) in
Genus/group Raw egg white Pasteurized egg white Batch number (number of isolates)
Enterobacter 78 7 Nr.2 (2) ; Nr.4 (3) ; Nr.6 (1) ; Nr.7 (1) ;
Nr.9 (2); Nr.10 (5) ; Nr.15 (1) ;
Nr.17 (1) ; Nr.19 (2) ; Nr.20 (2) ;
Nr.21 (1) ; Nr.25 (1) ; Nr.27 (1) ;
Nr.29 (1) ; Nr.36 (9)
Escherichia 50 27 Nr.5 (6) ; Nr.14 (3) ; Nr.16 (4) ;
Nr.16 (1) ; Nr.20 (1) ; Nr.21 (2) ;
Nr.25 (1) ; Nr.27 (2) ; Nr.29 (2) ;
Nr.30 (2) ; Nr.35 (4) ;Nr.36 (2)
Enterococcus 39 93 Nr.2 (9) ; Nr.3 (9) ; Nr.8 (4) ; Nr.9 (4) ;
Nr.11 (2) ; Nr.12 (1) ; Nr.13 (3) ;
Nr.16 (3) ; Nr.16 (1) ; Nr.17 (2) ;
Nr.18 (5) ; Nr.19 (3) ; Nr.20 (3);
Nr.21 (1) ; Nr.25 (1) ; Nr.32 (6) ;
Nr.35 (2) ; Nr.36 (3) ; Nr.37 (1) ;
Nr.40 (1) ; Nr.44 (1)
Serratia 33 7 Nr.5 (4) ; Nr.6 (3) ; Nr.7 (2) ; Nr.12 (4) ;
Nr.16 (2) ; Nr.17 (1) ; Nr.25 (2) ;
Nr 44 (1)
Shigella 28 7 Nr.5 (4) ; Nr.16 (1) ; Nr.19 (1) ;
Nr.27 (1) ; Nr.29 (1) ; Nr.35 (2)
Pantoea 11 - Nr.21 (1) ; Nr.27 (1)
Klebsiella 67
Nr.4 (1) ; Nr.9 (1)
Yersinia 6-
Nr.27 (1)
Rahnella 6-
Nr.15 (1)
Salmonella 6-
Nr.19 (1)
Pseudomonas 6-
Nr.16 (1)
Bacillus cereus group 6-
Nr.2 (1)
Table 3–Types of spoilage of the custard cream regarding thickening, coagulation, production of holes, and production of gas;
(+) positive/presence and (–) negative/absence; ࢞pH (initial pH of the custard cream – pH after 21 d of incubation at 10 °C) ±
standard deviation (SD).
Type of spoilage Thickening Coagulation Production of holes Production of gas ࢞pH ±SD Number (%) of isolates
1++ + − 2±0.2 64 (39)
2++ + +1.8 ±0.4 50 (32)
3++ − − 2±0.4 19 (11)
4+− − − 2±0.2 10 (6)
5+− + +1.6 ±0.4 10 (6)
6+− + −1.6 ±0.5 9 (5)
7++ − +1.8 ±0.7 2 (1)
8+− − +2.2 ±0.6 2 (1)
Number (%) of isolates 166 (100) 135 (81) 133 (80) 64 (39) 166 (100) 166 (100)
be in accordance with the European regulation requiring less than
2 log CFU/g for Enterobacteriaceae at the end of the process of egg
product manufacturing. The absence of positive samples hosting
Salmonella may also meet the legal requirements (absence in 25 g).
Considering the contamination of the pasteurized egg white
products with bacteria which are not part of the Enterobacteriaceae
family, Pseudomonas sp. was not detected after pasteurization, in
accordance with the low heat-resistance of these bacteria. This
result corroborates the observations of Protais and others (2006)
in whole egg products. The B. cereus group was also absent in
the pasteurized samples analyzed. However, knowing the heat-
resistance of these bacteria due to their spore-forming ability, one
could not conclude on the effect of pasteurization on this specific
flora. It is important to keep in mind that B. cereus group bacteria
were shown to be highly occurring in samples of pasteurized
whole egg products, provided that an enrichment step was carried
out before identification (Jan and others 2008). The experimental
conditions of analysis of the present study may have failed to collect
B. cereus group colonies on the plates concomitantly exhibiting
high levels of other bacteria.
Otherwise, B. cereus group bacteria were shown to be sensitive
to egg white (Baron and others 2014): at the natural alkaline pH
of egg white, ovotransferrin was shown to induce the death of B.
cereus group bacteria through membrane perturbations.
M394 Journal of Food Science rVol. 80, Nr. 2, 2015
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Bacteria from liquid egg white . . .
Table 4–Types of spoilage of the custard cream related to each of the bacterial genus tested. Results are expressed as number (%)
of isolates for each genus.
Type of spoilage
Genus Number of isolates 1 2 3 4 5 6 7 8
Enterococcus 64 29 (46) 20 (31) 4 (6) 4 (6) 5 (8) 1 (1.5) 1 (1.5)
Escherichia 33 13 (39) 7 (21) 6 (18) 5 (14) 2 (6)
Enterobacter 31 8 (26) 17 (56) 3 (9) 1 (3) 1 (3) 1 (3)
Serratia 19 6 (32) 4 (20) 3 (16) 3 (16) 3 (16)
Shigella 10 3 (30) 2 (20) 2 (20) 2 (20) 1 (10)
Klebsiella 3 2 (67) 1 (33)
Bacillus cereus group 1 1 (100)
Rahnella 1 1 (100)
Salmonella 1 1 (100)
Pantoea 1 1 (100)
Pseudomonas 1 1 (100)
Yersinia 1 1 (100)
Total 166 64 (39) 50 (31) 19 (12) 9 (5) 10 (6) 9 (5) 2 (1) 2 (1)
Finally, the remaining of enterococci concerned 93% of the
contaminated pasteurized liquid egg white samples analyzed, cor-
roborating the observations of Protais and others (2007), Hidalgo
and others (2008), and Miller and others (2010) in whole egg
products. Enterococci are widely distributed in the environment,
principally inhabiting the human and animal gastrointestinal
tract (Moreno and others 2006 for review). They are also highly
occurring in soil, in surface waters and on plants, vegetables, and
insects (Deibel and Silliker 1963; Mundt 1963). Even if widely
used as probiotics for their ability to produce bacteriocins (ente-
rocins) in fermented foods, some enterococci are also recognized
of safety concern because they carry potential virulence factors
and can display pathogenic traits (De Vuyst and others 2003).
They are also among the most thermo-tolerant of the nonspore
forming bacteria (Sanz Perez and others 1982; Magnus and others
1988) and they are able to adapt to various substrates and growth
conditions (low and high temperatures, extreme pH and salinities)
(Moreno and others 2006 for review). Since populations around
3.2 log CFU/eggshell have been reported on the eggshell surface
by Mallet and others (2004, 2006), their presence in pasteurized
egg white may also be ascribed to cross-contaminations at the step
of egg breaking and to their remaining after the pasteurization
process. The risk of postprocess contamination should not be
underestimated since they are also known as able to form biofilms,
in which bacteria can better resist the disinfection and cleaning
procedures than under their planktonic lifestyle (Moreno and
Figure 2–Percentage of isolates belonging to each recovered genus exhibiting proteolytic, lipolytic, and/or amylolytic activity(ies). The numberof
isolates belonging to each genus is indicated between brackets.
Vol. 80, Nr. 2, 2015 rJournal of Food Science M395
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& Safety
Bacteria from liquid egg white . . .
others 2006 for review). An example of a possible postprocess con-
tamination by Enterococcus isshowninthebatchNr.17(Table1),
where Enterococcus was detected in the pasteurized sample (bolded
characters in Table 2), and not in the raw one. Considering these
whole statements, our results highlight the need to better control
their development in the egg processing environment.
Spoilage potential of egg white bacterial contaminants in
custard cream
Regarding the use of egg white foams in the production of
desserts containing highly nutritive substrates, the bacteria isolated
from industrial egg white products were further tested for their
spoilage ability in custard cream. This food was chosen as a model
of refrigerated egg-based dessert, which is covered with sugared
egg white foams in the French floating island dessert.
Samples of sterile commercial custard creams were inoculated
with each isolate and incubated for 21 d at 10 °C in order to mimic
the conditions of the ageing tests usually carried out in the chilled
dessert industry. The whole bacterial collection tested (166 iso-
lates) found optimal growth conditions in custard cream, reaching,
in average, 9.3 ±0.4 log CFU/mL at the end of the incubation
period. The custard cream used in this study was composed of 80%
milk, 10% sucrose, 9% whole egg, and around 1% starch, probably
providing all the nutrients needed for bacterial growth. Moreover,
the whole collection was able to grow at low temperature, corrob-
orating our previous studies on the psychrotolerance of B. cereus
group bacteria coming from pasteurized liquid whole egg prod-
ucts (Baron and others 2006; Techer and others 2014) and the one
of Protais and others (2006) who detected the same total counts of
bacteria (5.4 log CFU/mL in average) in raw whole egg products
analyzed on plate-count agar media incubated at medium (30 °C)
and low (8 °C) temperature for 3 and 8 d, respectively. Unlike the
egg processing industry, the literature concerning food contamina-
tion by psychrotrophic bacteria is particularly abundant regarding
the European dairy industry. In the early 1980s, psychrotrophic
bacteria were shown to account for 10% to 90% of the whole
flora in chilled raw milk, under safe and poor hygiene conditions,
respectively (Cousin 1982). Although the hygiene conditions of
food processing have been considerably improved during the last
decades, they still remain an important source of food spoilage
in the dairy industry. According to a recent study, the modern
European dairy industry may lose up to 30% of the whole produc-
tion due to the expression of spoiling activities by psychrotrophic
bacteria (Samarzija and others 2012). Regarding the results of our
study, one could make an analogy between the spoilage issue in
dairy and in egg products. Both chilled milk and egg products may
be source of psychrotrophic bacteria, which may be responsible of
spoilage issues in egg-and dairy-based chilled desserts. Consider-
ation should hence be given to the control of the flora that may
be conveyed either by milk or by egg products in a finish product
such as egg- and milk-based desserts.
In addition to the fact that optimal growth was observed in
custard cream at 10 °C, several physico-chemical modifications
of the custard cream were observed throughout the incubation
period (Table 3). The acidification of the custard cream, expressed
in ࢞pH values, was, in average, of 1.9 ±0.4 (Table 3), probably
due to the production of organic acids such as acetic and lactic
acids from the available carbon sources. The production of organic
acids is recognized as leading to the development of abnormal
tastes in the food product, such as sourness and astringency (Neta
and others 2009; Lawless and others 1996). Moreover, the texture
of the custard cream was affected by bacterial growth. All the
isolates of the collection (100%) induced the thickening of the
custard cream. In addition, 81%, 80%, and 39% of the isolates were
Figure 3–Proteolytic activity of the isolates recovered from raw and pasteurized liquid egg white products. The activity is evaluated through measure
of the thickness of the clearing zone (mm) developed after 48 h incubation at 30 °C on custard cream-based agar. Results are given as the mean of 2
technical experiments. Bars represent standard deviation. The isolates are clustered as weakly (0<thickness of the clearing zone ࣘ2 mm; soft gray
bars), moderately (2<thickness of the clearing zone ࣘ4 mm; dark gray bars), and highly (thickness of the clearing zone >4 mm; black bars) proteolytic.
M396 Journal of Food Science rVol. 80, Nr. 2, 2015
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Bacteria from liquid egg white . . .
able to induce the coagulation of the cream, to produce holes, and
to produce gas, respectively (Table 3). According to these spoilage
criteria, 8 types of spoilage were highlighted (Table 3). The
spoilage types 1 and 2 were mainly represented in the bacterial
collection with 39% and 32% of the isolates analyzed, respectively.
The spoilage types 3 to 8 were less represented (less than 20% of
the analyzed isolates, Table 4). Due to the too low occurrence
of several genera in the collection, the types of spoilage were
further analyzed regarding only the genera Enterococcus,Escherichia,
Enterobacter,Serratia,andShigella, corresponding to around 95%
of the collection. The isolates of these genera mainly led to the
type 1 spoilage, characterized by the thickening and coagulation
of the custard cream and the production of holes. Enterobacter
isolates mainly led to type 2 spoilage, which, in addition to the
spoilage criteria of type 1 spoilage, led to the production of gas.
The psychrotrophic members of the Enterobacteriaceae family and
Enterococcus are frequent members of the spoilage microflora of
vacuum-packed meats and lightly preserved fish products like
cold-smoked salmon or shrimps (Gram and others 1999; Daalgard
and others 2003; Mejlholm and others 2008; Jaffr`
es and others
2009; Alfaro and Hernandez 2013; S¨
ade and others 2013). In
the meat sector, Enterobacteriaceae have also been described as
being potential causative agents of the “blown pack” spoilage of
meats, characterized by abundant production of gas, off-odors,
and exudates together with proteolysis and modifications of pH
and color (Brightwell and others 2007; Adam and others 2010;
Chaves and others 2012). Concerning Enterococcus sp., this genus
is described as positively contributing to the pH decrease and to
the modification of the texture and/or the sensorial characteristics
of some fermented food. However, the production of organic
acids, carbon dioxide, and volatile compounds by these bacteria
may be detrimental for nonfermented foods, such as heat-treated
packaged processed meats (Moreno and others 2006 for review).
In vitro determination of enzymatic activities of egg white
bacterial contaminants in custard cream-based agar media
Considering the high contents in proteins, lipids, and starch of
the custard cream, 43 isolates of the collection were further tested
regarding their proteolytic, lipolytic, and amylolytic activities in
custard cream-based-agar media. These enzymatic activities may
contribute to the modifications of the texture of the cream and
also to the development of off-flavors and/or off-odors.
The bacterial collection tested comprised 43 isolates of the
B. cereus group (1 isolate), Enterobacter sp. (5 isolates including
1 isolate of the Enterobacter aerogenes species), Enterococcus sp.
(14 isolates including 2, 4, and 1 isolates of the Enterococcus avium,
E. faecalis,andPseudomonas avium species, respectively), Escherichia
sp. (7 isolates including 6 and 1 isolates of the E. coli and Escherichia
ferqusonii species, respectively), Klebsiella sp. (1 isolate), Pantoea sp.
(2 isolates), Pseudomonas sp. (1 isolate), Serratia sp. (6 isolates includ-
ing 3, 1, and 1 isolates of Serratia liquefasciens,Serratia plymuthica,
and Serratia proteomaculans species, respectively), and Shigella sp.
(6 isolates including 2 isolates of each Serratia dysenteria, Serratia
flexenri,andSerratia sonnei species). The whole collection exhibited
proteolytic activities and 60% and 79% also expressed lipolytic and
amylolytic activities, respectively (Figure 2), after 48 h incubation
at optimal temperature (30 °C) under aerobic conditions.
Whereas all the isolates were proteolytic, the level of this
activity varied depending on the isolate (Figure 3). At the end
of the incubation period, the thickness of the clarification halo
varied from 1 to 6.2 mm, with an average value of 3.9 ±1.3 mm
(Figure 3). Three groups were distinguished: a weakly, a
moderately, and a highly proteolytic group, each containing 11.3,
45.4, and 43.2% of the tested isolates, respectively (Figure 3). The
level of the proteolytic activity was shown to be strain-dependent.
Enterococcus sp. isolates were represented in the 3 groups. However,
the 4 Enterococcus faecalis isolates were highly proteolytic. Although
performed under different conditions regarding the type (liquid or
dried milk, casein) and concentration of the substrates, the type of
culture (liquid or solid), and the type of detection of the activity
(spectrophotometry for liquid cultures), the data presented in
the present study confirmed those of Veljovic and others (2009),
Miller and others (2010), and Moreno and others (2006). These
authors have reported that the level of the proteolytic activity
depended on the strain inside a same genus. They also detected
low proteolytic activities for enterococci, except for the strains of
the species E. faecalis.
The lipolytic activity was expressed by 60%, 50%, 85.7%, 66.7%,
and 66.7% of Enterobacter sp., Enterococcus sp., Escherichia sp., Serratia
sp., and Shigella sp. isolates, respectively (Figure 2). This activity was
lacking for B. cereus group and Pseudomonas sp. isolates (Figure 2).
These later are frequently associated with milk spoilage due to their
ability to express phospholipase activities (Champagne and others
1994; Sorhaug and Stepaniak 1997; Dogan and Boor 2003). How-
ever, this property was not investigated in the present study, due
to the fact that phospholipids are slightly concentrated in custard
cream (<0.5%). However, one could not exclude the involvement
of phospholipases in the different types of spoilage observed in the
present study. Except the study of Miller and others (2010), who
reported that around 87% of the strains isolated from whole liquid
egg products presented a lipolytic activity, few data are available in
the literature on this type of activity in the sector of egg process-
ing. Subsequent works will be directed toward the quantification
of lipase and phospholipase activities in custard cream.
The amylolitic activity was expressed by all the Enterobacter sp.,
Serratia sp., Pantoea sp., B. cereus group, and Pseudomonas sp. isolates
tested, and by 57% and 71% of the Enterococcus sp. and Escherichia
sp. isolates, respectively (Figure 2). The measure of the diameter of
the clarification halo after lugol addition did not allow significantly
distinguishing the level of this activity from an isolate to another in-
side the collection (data not shown). Coton and others (2011) also
found that 80% of spore-forming bacteria isolated from surimi-
based products (40 isolates studied) expressed amylolytic activities.
A similar frequency (more than 70%, 174 spore-forming isolates
studied) was reported by Valerio and others (2012) for bacter ia
collected in the bakery industry. In this sector, amylolytic bacte-
ria, including Bacillus sp., are often associated to the deterioration
process of bread texture, described as “rope spoilage.” This later is
characterized by slime formation as a result of the combined ex-
pression of amylolytic and proteolytic bacterial enzyme activities
(Rosenkvist and Hansen 1995; Viedma and others 2011; Valer io
and others 2012). Such combining effects of bacterial enzymes is
probably of interest in custard cream since bacteria are often pro-
ducer of more than one enzyme, catalyzing the hydrolysis of the
numerous custard cream substrates.
Conclusions
The present study is the 1st report on the microbial quality of in-
dustrial liquid egg white in France and on its possible involvement
in spoilage issues in the sector of chilled egg-based desserts. Our
data demonstrate the efficiency of the industrial pasteurization
process for the eradication of Gram-negative bacteria. However, it
also highlights the need to control the quality of the raw material
regarding the spoiling potential of egg white contaminants, and
Vol. 80, Nr. 2, 2015 rJournal of Food Science M397
M: Food Microbiology
& Safety
Bacteria from liquid egg white . . .
especially Gram-positive Enterococcus bacteria, which were as-
sumed to resist pasteurization in several analyzed samples and were
showntobeabletospoilcustardcreamandtoexpressproteolytic,
lipolytic and amylolytic activities in this type of food. A better
control of enterococci in egg products would reduce spoiling
issues, and particularly in egg-based desserts supporting further
bacterial growth at low temperature. Subsequent works will be
directed toward the study of the correlation between the bacterial
enzymatic activities and specific characteristics of spoilage. In
addition to food safety issues, a better control of spoiling bacteria
may allow the egg processing companies to reach optimal customer
satisfaction and to reduce wastes. The commitment to this ap-
proach would represent, for the egg processing industry, a tangible
competitive advantage in the food sectors using eggs as ingredients
in particularly sensitive foods such as refrigerated desserts.
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