Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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DISINFECTION OF TABLE EGGS USING LEMON JUICE AS A
NATURAL BIOCIDE
Alaa T. Abdulwahid* , Hiba A. Nasear*, Samar S. Ghazi*
*Veterinary Public Health Department, Veterinary Medicine College, University of Basrah, Iraq
Corresponding Author: alaa.alsandaqchi@uobasrah.edu.iq
Key words: egg shell surface, egg table contamination, VITEK® 2 system, lemon juice.
ABSTRACT
Bacterial contamination of table eggs is a serious public health problem around the world
due to increase the risks of food-borne illness. Disinfection of table eggs is essential to minimize
the possibility egg contamination from shells. In the current study, 100 samples (table eggs)
were collected from different supermarkets of Basrah city. Identification and disinfection of
bacteria on shell of table eggs were done in Veterinary Medicine College, Public Health
Laboratory / University of Basrah. Samples were cultured on blood agar, mannitol salt agar,
macConkey agar, salmonella-shigella agar, eosin methylene blue agar, and tryptic soy agar to
differentiate different types of bacteria before and after processing with lemon juice depend on
its morphology and Gram's staining. The detection of organisms for genus and species were then
done based on biochemical characteristics using VITEK® 2 system. The present study revealed
that the major contaminant of table eggs was with Gram-negative bacteria and the minor
contaminant was with Gram-positive bacteria. Gram-positive bacteria detected on shell of table
eggs (Leuconostoc species and Gemella bergeri) were resistant to lemon juice. However, Gramnegative
bacteria identified on shell of table eggs (Cronobacter sakazakii, Raoultella
ornithinolytica, Klebsiella oxytoca, Enterobacter aerogenes, Moraxella group, and Serratia
plymuthica) were sensitive. In conclusion, table eggs collected from supermarkets were
contaminated with pathogenic bacteria. Lemon juice was suitable to be used as an antiseptic
agent to minimize the contamination of eggshells with Gram-negative bacteria.
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INTRODUCTION
Eggs are one of the most important nutrition sources for sustenance and growth of human
being. They are a highly nutritious product containing high levels of fat soluble, vitamins,
minerals, and protein, In addition, eggs are a highly quality of saturated and monounsaturated
fatty acids. Furthermore, eggs are a highly calories and consumption of eggs at the breakfast is
recommended to start the day with a high energy sources, However, contamination of egg shell
makes them a serious public health problem around the world due to increase the risks of foodborne
disease (1).
Nutrient elements in hen’s egg create an excellent environment for the development of
both microfloral and pathogenic bacteria. Eggs can be infected vertically (prior- shell formation)
or horizontally (post- shell formation). Through vertical transmission, pathogens
(namely Salmonella) are introduced from the infected ovary or oviduct tissue to eggs prior- shell
formation. Contamination via horizontal transmissions usually happens from cloaca to shell egg,
which the excretion of faeces takes place. In addition, several factors have been responsible in
contamination of egg table including cloths and hands of poultry workers and the environment
vectors (dust, pets and rodents). Package, storage, transporting and marketing are other factors
responsible in contamination of table egg. The common pathogenic to human beings that isolated
from shell egg are Staphylococcus, Micrococcus, Sarcinia, Arthrobacter, Bacillus,
Pseudomonas, Achromobacter, Alcaligenes, Flavobacterium, Cytophaga, Escherichia,
Aerobacter, Proteus, Serratia, Aeromonas (2, 3).
The environmental condition combined with the poor hygiene is suitable area for the
survival and proliferation of micro-organisms in table eggs (4).
To ensure table eggs are safe for consumers, several methods have been used to minimize
the egg contamination from shells. Chlorine, ultraviolet, hydrogen peroxide, peracetic acid,
quaternary ammonium compounds have been used to reduce the microbial contamination from
shell egg. Chlorine and ultraviolet combined, peracetic acid and ultraviolet combined, and
ultraviolet and hydrogen peroxide combined have also been used to reduce the microbial
contamination from shell egg (5).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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However, all these methods unable to reduce microbial contamination on egg shells
completely. In addition, these disinfectants are high cost, toxic, and unfriendly to the
environment. Therefore, others effective methods are required to ensure that shell egg is strong,
internal egg quality is good, and eggs are sanitary as possible when they collect by consumers.
Lemon juice is an alternative agent has been used a disinfectant (destroyed the bacteria on nonliving
objects) to clean hard surfaces (6).
In addition, the acid in lemons has been used as antiseptic (destroyed the bacteria on
living skin) to eliminate a variety types of Gram- negative bacteria from war wounds, antiinflammatory,
antiparasitic (destroyed the parasites within the body) for treating malaria
(plasmodium parasites), anti-oxidant, and antibacterial (destroyed the bacteria within the body)
for treating cholera (Vibrio cholera), diphtheria (Corynebacterium diphtheria), typhoid
(Salmonella typhi), phague (Yersinia pestis), Leptospirosis (Leptospira interrogans), and internal
hemorrhage by the vitamin p (bioflavinoids) in it (7,8,9).
However, there is no study has been carried out to examine the effect of lemon juice as
antibacterial to food surfaces. Thus far, this work aimed to use lemon juice as antibacterial to
minimize the possibility egg contamination from shells.
MATERIALS AND METHODS
Study design
A total of 100 samples (table eggs) were collected from different supermarkets of Basrah
city. Detection and disinfection of bacteria were done in Veterinary Medicine College, Public
Health Laboratory/University of Basrah.
Preparation of sample
Before processing, swabs from each egg were taken from the shell using rinsed swab
method (10). Briefly, sterile cotton swab was soaked in 0.1% peptone, rubbed the surface of the
egg, kept in the nutrient broth medium, and incubated at 37°C for 24h. The swabs were then
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
Veterinary Medicine. University of Basrah. Iraq
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streak on each of the following media: Blood agar, mannitol salt agar, macConkey agar,
salmonella-shigella agar, eosin methylene blue agar, and tryptic soy agar to differentiate different
types of bacteria depend on its morphology and Gram's staining.
All culture media were prepared following the manufacturer’s instruction and sterilized
by autoclaving at 121°C for 20 min. The detection of organisms for genus and species were then
done based on biochemical characteristics using Gram-negative and Gram-positive cards by
VITEK® 2 system (bioMérieux)(Table1).
After processing of egg by rub the surface of the egg with lemon juice (a pH of around
2.2), swabs from each egg were taken from the shell using rinsed swab method (10).
Briefly, sterile cotton swab was soaked in 0.1% peptone, rubbed the surface of the egg,
kept in the nutrient broth medium, and incubated at 37°C for 24h. The swabs were then streak on
the on the selective media mentioned above to differentiate different types of bacteria based on
its morphology and Gram's staining. Detection genus and species of bacteria was then carried out
by the VITEK® 2 system (bioMérieux) based on biochemical characteristics using Gramnegative
and Gram-positive cards(Table 1).
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding o
Veterinary Medicine. University of Basrah. Iraq
Table 1: Biochemical tests on
Statistical analysis: PC LIS-compatible software
ournal of the 17th International
181
Gram-negative and Gram-positive cards
used to analyse the result of the current study
f Conference. College of
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RESULTS
This study revealed that the major contaminant of table eggs was with Gram-negative
bacteria and the minor contaminant was with Gram-positive bacteria. 6 of the bacteria present on
egg shells belong to Gram-negative and 2 belong to Gram-positive bacteria (Table 2). Gramnegative
bacteria present on egg shells were Cronobacter sakazakii, Klebsiella oxytoca,
Raoultella orinthinolytica, Enterobacter aerogenes, Moraxella group, and Serratia plymuthica.
the Gram-positive bacteria present on eggshells were Leuconostoc mesenteroides and Gemella
bergeri (Table 2). The present study also revealed that the major contaminant of table eggs was
with Raoultella ornithinolytica and the minor contaminant was with Gemella bergeri (Figure 1).
Table 2: Morphologic features of the bacterial present on the surface of egg.
Bacterial present on the shell Morphologic features
Cronobacter sakazakii group Gram-negative
rod glossy
yellow
Raoultella ornithinolytica Gram-negative
bacilli
smooth circular
Klebsiella oxytoca Gram-negative
rod, slightly mucoid
yellow to cream
Enterobacter aerogenes Gram-negative,
Straight rods, smooth
irregularly round to rough "cauliflower"
Moraxella group Gram-negative,
rods or cocci, smooth, convex, and circular
whitish
Serratia plymuthica Gram-negative,
rods, a smooth, round, convex
pale pink
Leuconostoc mesenteroides spss mesenteroides Gram-positive,
rods, spherical, oval, slimy, smooth, round
grayish in nature
Gemella bergeri Gram-positive
cocci
tiny convex
pink
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding o
Veterinary Medicine. University of Basrah. Iraq
Figure 1: Percentages of bacterial
Table 3: The bacterial present on
Note: (+) denotes for presence and (
Bacterial contamination of table eggs is a serious public health problem all over the world
due to increase the risks of food
is required to minimize the possibility egg contamination from
revealed that bacterial present on the surface of egg
0 5
10
15
20
25
30
35
40
Ovar all percentages (%)
Bacterial present on
Cronobacter sakazakii group
Raoultella ornithinolytica
Klebsiella oxytoca
Enterobacter aerogenes
Moraxella group
Serratia plymuthica
Leuconostoc mesenteroides spss mesenteroides
Gemella bergeri
ournal of the 17th International
183
detection on the surface of the
the surface of egg before and after processing
juice
(-) stands absence of bacteria
DISCUSSION
food-borne illness. Disinfection of table eggs by using natural biocide
shells.
are Cronobacter sakazakii
Identified bacteria
the shell Before
processing
+
+
+
+
+
+
+
+
f Conference. College of
table eggs
with lemon
The present study
sakazakii, Raoultella
After
processing
-
-
-
-
-
-
+
+
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orinthinolytica, Klebsiella oxytoca, Enterobacter aerogenes, Moraxella group, Serratia
plymuthica, Leuconostoc mesenteroides and Gemella bergeri. All these bacteria detected on shell
of table egg play an important role in spoilage of food product, leading to food poisoning in
consumers. This finding is in agreement with previous study, in which those bacteria identified
on the surface of table eggs are pathogenic and spoiled food product. It has been found that eggs
can be infected horizontally from cloaca to shell egg, which the excretion of faeces takes place or
vertically from the infected ovary or oviduct tissue to eggs prior- shell formation(4).
Contaminated with pathogenic bacteria might also be due to the environmental condition
combined with the poor hygiene (4). Cronobacter sakazakii (Enterobacter sakazakii) is a foodborne
pathogen causes a life-threatening to human, predominantly in neonates. In infant and
young children, Infection of C. sakazakii causes bacteremia , enterocolitis, meningitis,
septicemia, and urinary tract inflammation (11).Infections among
immunocompromised adults have also been found (12).
In adult, C. sakazakii infection causes appendicitis (appendix inflammation), biliary
sepsis, conjunctivitis (eye inflammation), pneumonia, and urosepsis (12). C. sakazakii can be
found in water, soil, and plant materials (12). A basic reason for hen's egg contamination in C.
sakazakii is the flies (environment vectors) (13). C. sakazakii has been isolated from a wide
range of food including egg, milk, meat, and seafood products (14).
Raoultella ornithinolytica is human pathogen causes a life-threatening, predominantly in
adult. It causes bacteraemia, pneumonia, Cholangitis (biliary tract infection). R. ornithinolytica
can be found in the soil, plants and water. It may also be found in the upper respiratory and
intestinal tracts of healthy chickens. A basic reason for hen's egg contamination in
R. ornithinolytica may be the fact that the egg excretes from the hen's chicken through the
cloaca, where faecal material is also excreted. The adherent faeces to the egg's shell contaminate
egg contents through shell penetration by microorganisms. In addition, the environmental factors
(temperature and humidity) play an important role in pathogenic penetration and increase the
frequency of contamination (15).
Klebsella oxytoca (Bacterium oxytocum) is a pathogenic bacteria now being isolated
more frequently. It causes neonatal infections of the bloodstream, urinary tract, central nervous
system, wound, lung, and soft. It is found in gut, mouth, nose of man and animals. It is also
Basrah Journal of Veterinary Research,Vol.19, No.3, 2020. Proceeding of the 17th International Conference. College of
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acquired from the environment (water, soil, and insect). This bacterium can live on the surface of
the housefly eggs (Musca domestica) and contaminate hen’s egg. In addition, adherent faeces to
the egg's shell contaminate egg table by this pathogen (16)
Enterobacter aerogenes (Klebsiella aerogenes) is a pathogenic bacterium for humans and
animal. It is widespread in nature, water, sewage, soil, and on plants. It is normal inhabitants in
gastrointestinal and mucosal surfaces of the animal. In animal, E. aerogenes causes mastitis
(inflammation of udder). In humans, E. aerogenes causes wound infections, septic shock,
bacteremia (the presence of bacteria in the blood), meningitis (inflammation of the membranes
brain), septicemia (blood poisoning), lower respiratory and urinary tract infections. E. aerogenes
infections are generally treated with antibiotics, but the effectiveness of treatments for infection
with E. aerogenes could compromise by development of bacterial resistance (17).
Moraxella group is Gram-negative bacteria in the Moraxellaceae family. They are
normal inhabitants the mucosal surfaces of human and other animals. The bacterium can be
transmitted by flies. In human, Moraxella causes tracheobronchitis (inflammation of the trachea
and bronch), pneumonia, otitis media (inflammation of middle ear), sinusitis (a sinus infection),
and blepharoconjunctivitis (inflammation of the eyelids and conjunctiva). In animal,
Moraxella causes infectious ovis keratoconjunctivitis (inflammation of the cornea and
conjunctiva)( 18).Serratia plymuthica is a significant pathogen in a case of chronic osteomyelitis
and in case of sepsis associated with infection of a central venous catheter (19). It has been found
in a variety of environments (soil, water, plants, animals and air).
Leuconostoc species are Gram-positive bacteria widely spread in water and soil and play
an important role in food fermentations and several industrial (20). Gemella bergeri is a normal
flora found in the oral cavity and digestive tract of human and other animals. It has been
recently implicated in endocarditis (an infection of the endocardium) in infancy with a bicuspid
aortic valve status post-intervention (21).
The current study revealed that Gram-positive bacteria detected on shell of table eggs
(Leuconostoc species and Gemella bergeri) were resistant to lemon juice. This finding is in
agreement with previous study, in which those Gram-positive bacteria are tolerant to acidity
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(22). The mechanisms used by those Gram-positive bacteria to survive in acidic environment are
F1F0-Type ATPase proton pumps inhibitor, macromolecular repair, cell membrane alteration,
alkali production, pathways induction by transcriptional regulators, metabolism change, and cell
density and signaling modification. However, Gram-negative bacteria detected on shell of table
eggs (Cronobacter sakazakii, Raoultella ornithinolytica, Klebsiella oxytoca, Enterobacter
aerogenes, Moraxella group, and Serratia plymuthica) were sensitive to the lemon juice. This
finding is in agreement with previous studies, in which those Gram-negative bacteria are
susceptible to organic acid (23, 24, 25, 26).
The most of food-borne pathogen is not grown in too acidic environment, making lemon
juice a natural biocide due to its rich acid content. The acid in lemon juice penetrates the cell
membrane of bacteria, prompting a release of protons, which causes cell death. The structure of
all macromolecules affects at high acidity. Lipid and large proteins of bacteria (enzymes) are
sensitive to the pH value in their environment. Usually, lipids are hydrolysed at high acidity and
an enzyme a shape alters brings about an alteration of the ionic charges on the molecule, lost the
catalytic properties of the enzymes and halts the metabolism. In addition, hydrogen ion bonds
holding together strands of DNA break up at high acidity. Furthermore, the production of ATP in
cellular respiration are collapsed and impaired at high acidity because the proton motive power
depends on the concentration of hydrogen ion across the plasma membrane. The ionization of
amino-acid functional groups and disrupt hydrogen bonding can also modify by moderate
alteration in hydrogen ion, promoting denaturation and destroying activity of bacteria’s cell ( 27)
Acknowledgements
The authors thank the Veterinary Medicine College, Basrah University for offering support to
achieve this research. The authors also thank all staff and technicians of the Department of
veterinary Public Health for their kindness and useful advices.
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