Dalia et al.

, Egyptian Journal of Animal Health 4, 2 (2024), 259-267

Egyptian Journal of Animal Health

P-ISSN: 2735-4938 On Line-ISSN: 2735-4946
Journal homepage: https://ejah.journals.ekb.eg/

Impact of electolyzed water on B. cereus contaminating milk collection utensils

Dalia, Y. Youssef*; Asmaa Sh. Fayed*, Alaa Saad** Eman Nafei***
*
Reference Lab for Safety Analysis of Food of Animal Origin, Animal Health Re-
search Institute (AHRI), Agricultural Research Center (ARC), Giza, Egypt.
** Biotechnology Department, Animal Health Research Institute (AHRI), Agriculture
Research Center (ARC), Dokki, Giza, Egypt P.O. 12622, Giza.
*** Department of Food Hygiene and Control, Faculty of Veterinary Medicine, Benha
University, 13736 Mushtuhur, Toukh, Qaliobia, Egypt;

Received in 14/2/2024 ABSTRACT:

I
Received in revised from n the dairy industry, cleaning and disinfection of surfaces are important
7/3/2024
Accepted in 21/3/2024 issues, and the development of innovative strategies in this matter may
improve food safety. Biofilm formation on food-contact surfaces can
lead to recurrent contamination. To find an environmental friendly and ener-
Keywords: gy-efficient alternatives to acidic detergent for a milking system clean-in-
Bacillus cereus place (CIP) process, this study was planned to investigate the feasibility of
Biofilm applying Electolyed Water (EW) alone to clean and sanitize the soiled stain-
Disinfection less steel (304) pipes system as well as study the effect of EW on biofilm
Sanitizer formation process on stainless steel containers used in the dairy industry and
electrolyzed water (EW, to clarify the synergistic action between electrolyzed reduced water (ER)
EO, ER), and Acidic oxidized water (EO) to optimize the effect as alternative cleaners
and disinfectants to unsafe human health chemical cleans and disinfectants
on stainless steel plates (SSP) and examined the disinfection efficacy and
mechanism of electrolyzed water (EW) on Bacillus cereus biofilms. Acidic
(EO) with a pH ranging from 2 to 3 , alkaline electrolyzed water (ER) with a
pH ranging from 10 to 13 is regarded one of the most applicable in the anti-
microbial treatment of milk collecting containers and utensils. Both ER and
EO achieved a ˃5 log CFU/cm2 reduction of B. cereus to a non-detectable
level (< 1 log CFU/cm2) . The optimal effect was achieved by using ER as
cleaner followed by using EO as sanitizer (temperature 40 °C, contact time
10 min). and therefore, rendered EW as a promising cleaner and sanitizer to
be applied in the food industry. EW can be advantageous for environmental-
ly friendly, it considered also one of the promising novel antimicrobial
agents recently proposed as an alternative to conventional decontamination
methods such as heat and chemical sanitizers.

Corresponding author: Dalia, Y. Youssef , Reference Lab for Safety Analysis of Food of Animal Origin,
Animal Health Research Institute (AHRI), Agricultural Research Center (ARC), Giza, Egypt.
E. mail: Daliayousry1000 @gmail.com
DOI: 10.21608/ejah.2024.355623

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Dalia et al., Egyptian Journal of Animal Health 4, 2 (2024), 259-267

INTRODUCTION: of dairy products and equipment damage

Milk from the mammary glands of healthy (Gopal et al. 2015). The hydrophobic proper-
animals is initially sterile, but post-harvest han- ties of B. cereus endospores and their re-
dling like the milking personnel and milk han- sistance to heat, desiccation, and disinfectants
dling containers; remain to be the major allow strong biofilm formation on dairy uten-
sources of bacterial contamination of raw sils (Kumari and Sarkar 2014b). Biofilms
milk (Coorevits et al. 2008; Reta et al. formed from exopolysaccharides of mucous
2016). Therefore, milk should be produced substances produced by B. cereus protect bac-
in hygienic conditions so as to meet set terial cells from adverse environmental stress-
standards (Ahmad et al. 2015) which are es, such as chemical disinfectants, antimicrobi-
<106 colony forming units/ml in the case al agents, heat, and acid challenges, and act as
Kenya (KEBS 2010). a reservoir for recurrent contamination of dairy
products (Yuan et al. 2020). The most com-
Bacillus cereus is one of the most im- monly used disinfectants include sodium hypo-
portant endospores-forming spoilage microor- chlorite and quaternary ammonium com-
ganisms in the dairy environment, which is not pound (Peng et al.
only associated with foodborne outbreaks but 2002) ,glutaraldehyde (Simões et al.
also its growth may result in various dairy de- 2011), and peroxyacetic acid (Ryu and
fects (Guinebretiere et al. 2013). The milk se- Beuchat 2005), However, these disinfectants
creted fom a healthy animal's udder is sterile failed to eliminate B. cereus biofilms. The ina-
by nature, but it becomes contaminated by Ba- bility of these disinfectants to penetrate the
cillus spp. either through unhygienic milking biofilm’s matrix and their undesirable by-
practices, poor personal hygiene, and unsani- product residues in milk constitutes a consider-
tary utensils (Reta et al. 2016). B. cereus can able challenge to the dairy industry (Gil et al.
2009).
negatively affect product quality. It produces
various extracellular enzymes which can be
Electrolyzed water (EW) represents a green
responsible for undesirable effects in the or-
cleaning alternative, colorless, odorless, highly
ganoleptic quality of milk and dairy products
efficient, and inexpensive with a high broad
(Lücking et al. 2013). These enzymes are ther-
bactericidal action with less corrosion and no
mostable which resist the milk pasteurization
rinsing requirement for dairy utensils (Wang et
pocess, leading to degradation of milk compo-
al. 2019). In the USA, the (Environmental Pro-
nents, spoilage, and reduction of the shelf life
tection Agency (EPA) has approved the appli-
of milk and dairy products (Kumari & Sarkar
cation of EW as an effective food cleaning and
2014b). Spoilage is r elated to var ious fla-
sanitizing agent without leaving any traces of
vors, taste, smells, and textural defects such as
chlorine residues (Cl2) residues in the food
bitter, rancid, acidic, or sour taste, curdling or
manufacturing industry (Venturini 2013).
thickening, and ropy texture in milk and dairy
products (Lücking et al. 2013). Also, B. cereus
Electolyzed water contains non-dissociated
is involved in serious foodborne illnesses in-
cluding diarrheal and emetic syndromes, de- hypochlorous acid (HOCl) which considered
the main component obtained by the electroly-
pending on the ingested produced toxin. These
sis of water and sodium chloride at the anode
toxins are highly resistant to heat treatments,
side of the electolsis .It has been widely used
pH extremes, and proteolytic degradation in
to inactivate several foodborne pathogenic bac-
the digestive system (Jovanovic et al. 2021).
terial spores and biofilm formation in food
products, food processing surfaces, and non-
One of the biggest safety problems with
food-contact surfaces in a very short time
these organisms is their adherence to stainless
(Possas et al. 2021).
steel equipment surfaces in dairy plants and
EW was an effective disinfectant for the
form biofilms resulting in serious hygienic
elimination of biofilms of Staphylococcus au-
problems and economic losses due to spoilage
reus, Candida albicans and Streptococ-

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 Dalia et al., Egyptian Journal of Animal Health 4, 2 (2024), 259-267

cus mutans (Ozaki et al. 2012), and Listeria Heidelberg, Germany) agar plates from stocks
monocytogenes (Arevalos-Sánchez et al. solution stored in BHI broth containing 15%
2012). Most disinfectants may work better glycerol at – 80 o C and was incubated at 30 o C
against the initial attachment of microbes than for 24 h without shaking. Single colonies were
against developed biofilms so, eliminating the inoculated into tubes containing 10 mL of BHI
formed biofilms is an essential aspect in the broth and left to grow for 18 h at 30o C. This
dairy industry. The emergence of multidrug- overnight culture was contained an average of
resistant food poisoning microorganisms and 8.0 to 8.5 log10 CFU/ml. Seial dilution were
the demand for disinfection of heavily contam- made to obtain an initial concentration of 5.5 –
inated dairy equipment is expected to continue 6.0 log10 CFU/ml which used for expeimentall
growing in the future. There are few studies biofilm formation. Ethical approval was grant-
concerning the mechanism of EW disinfection ed by the Ethical Approval Committee of the
on B. cereus biofilms. Therefore, the objective Faculty of Veterinary Medicine, Benha Uni-
of this study was to investigate the inhibitory versity, Egypt (BUFVTM 01-01-24).
effect when spraying EW against B. cereus
biofilm and its effect on the expression of vi- 2.3. Preparation of Stainless-steel food con-
ulence genes (tasA and sipW) on dairy uten- tact surfaces according to the method
sils. recommended by Rosmaninho et al.
(2007):
2. MATERIAL and METHODS Stainless-steel plates (SSP) of 2 cm X 5 cm
2.1. Preparation of electrolyzed water Ac- dimensions (10 cm2) were cleaned by immers-
cording to Tolba et al. (2023) ing in neutral detergent (Hyclin-plus, Hycel,
Acidic electrolyzed water (AcEW) of pH 2 Ciudad de Mexico, Mexico) at 65o C for 5 min,
- 3 and Alkaline electrolyzed water (AlEW) of followed by rinsing with distilled water for 5
pH 10-13 were prepared using a current of 9- min. and then sterilized using dry heat at 180 o
10 VA passed through two separate electroly- C for 30 min.
sis cells containing potable water, sodium
chloride (NaCl) with two poles; anode (+) and 2.4. Design of The Experiment:
cathode (-). Upon the onset of the electrolysis 2.4.1. Preparation of milk contact sufaces:
process, at the anode side, water was oxidized
(EO) to give Oxygen gas (O 2), Chlorine gas Stainless steel plates (SSP) (304) materials
(Cl2), Hydrochloric acid (HCl), Hypochlorite used in milking systems were classified into 4
ions (OCl), and hypochlorous acid (HOCl) ac- groups; 1st group was control (SSP with inocu-
cording to the following formula: lum only without treatment), The 2nd (SSP
2 H2O (l) → 4 H +(ions) + O2 (gas) + 4 e─,2 with inoculum and sanitized with EO only),
NaCl → Cl2 (gas) + 2 Na+, (2 Cl- → Cl2 + 2e- while the 3rd one (SSP with inoculum and
), Cl2+ H2O (l) → HCl + HOC + Ocl cleaned with ER only.), The 4th one (SSP with
While at the cathode side, water was reduced inoculum and cleaned with ER followed by
(ER) to give Hydr ogen gas (H 2), chlorine ions sanitized with EO). To contaminate the Stain-
(Cl), and sodium hydroxide (NaOH) according less-steel plate sample, 0.1 mL of B. cereus
to the following formula: culture was evenly soiled on the whole surface
2 H2O (l) + 2 e─ →2 OH─ + H2 (gas) of each Stainless-steel plate with a sterile glass
2 NaCl + 2OH─ →2NaOH + Cl -coated rod. Then, the plates were dried using
laminar flow for 30 minutes to evaporate all
2.2. Preparation of B. cereus for experi- visible liquid. The initial concentrations of
mental inoculation bacteria on control stainless steel plates were
5.57 log10 CFU/cm2 and EW treatments were
The target bacterium for this study was B. applied for 10 min. (temp. of EW water was
cereus strain, which was obtained from Animal 40o C) based on the method of Yu Liu et al
Health Research Institute (AHRI) ,which has (2020).
the ability to biofilm formation. The strain was
streaked onto brain heart infusion (BHI; BD,

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2.4.2. Cleanliness Assessment and B. cereus (Applied Biosystem), were used to determine
Counting: the B. cereus biofilm genes (tasA and sipW)
Control plates of SSP were checked for expression (Table 1). 10 µL reaction vol-
their initial contamination levels, while the oth- umes with 0.5 µl of each primer and 1 µL
er treated plates were checked for effects of of RNA were used for the amplification pro-
EW of both types (EO & ER). The plates were cess. The subsequent thermal cy-
swabbed for microbiological analysis using cling parameters were used: 30 minutes
sterilized cotton swabs soaked with 0.1% pep- of reverse transcription at 55 °C, 5 minutes
tone water, The B. cereus counting process was of activation at 95 °C, 40 cycles of denatura-
calculated according to ISO/ 7932 (2004) tion at 95 °C for 10 s, 30 seconds of annealing
AMD 1 (2020). (60 °C for 16srRNA and tasA, and 54 °
C for sipW), and 30 seconds of extension at
60 °C. The synthesized oligonucleotide pri-
2.5. Nucleic Acid extraction: mers (OligoTM) were used in this study (Table
A).
Following the manufacturer's instructions,
mRNA was extracted using the FastPure®
DNA/RNA Mini kit. Using a HERA SYBR®
Green RT-qPCR Kit (Willowfort) and the Ap-
plied Real-Time PCR Detection System

Table A. Primer used for determine the B. cereus taget genes.

Gene Primer sequence (5’ to 3’) Reference

16S rRNA F- TCG AAA TTG AAA GGC GGC Priha et al. 2004

R- GGT GCC AGC TTA TTC AAC

tasA F- AGC AGC TTT AGT TGG TGG AG Caro-Astorga et al. 2015
R- GTA ACT TAT CGC CTT GGA ATTG

sipW F- AGA TAA TTA GCA ACG CGA TCTC

R- AGA AAT AGC GGA ATA ACC AAGC

2.6. Statistical Analysis Microsoft Excel was used to perform

The bacteria population was ex- the statistical and imaging analyses. Us-
pressed as log10 CFU/cm2. The mean ing the 2^(−ΔΔCt) technique, the rela-
values for B. cereus were calculated tive expression of target genes was ana-
from the independent triplicate trials. lyzed and compared with that of the dis-
Significant differences in mean values tilled water (d.d. H20) group. The aver-
of bacteria counts and reduction rates & age cycle threshold (Ct) of the target
percentages were analyzed using least genes was deducted from those of the
significant differences with analyses of endogenous control gene 16srRNA to
variance (ANOVAs) and a 95% confi- obtain the ΔCt value
dence interval in SPSS 21.0 (SPSS, Inc.,
Chicago, IL, USA).

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RESULTS:

Table 1. Mean B. cereus count (log10 CFU/m2), reduction rate, and % of control and treated plates

B. cereus Control ER EO ER & EO

Initial count 5.57A 1.3ab 1.1ab <1aB
Reduction rate (log10 CFU/m2) ---- 4.27 4.47 5.57

Reduction % 0.0 76.66 80.25 100

Significance difference between small and capital litters in the same raw
<1 log is represented by zero in estimating the significant difference

Fig 1. reduction percentage of different treatments as compared with the control plates

Fig (2): Relative tasA and sipW genes expression of B. cereus contaminated stainless steel surfaces after
cleaning by EW (ER, EO, and mix of ER&EO) compared to the control. Values are expressed as the
mean ±SD. Total RNA was extracted. Biofilm gene expression levels were measured by the 2−ΔΔCt
method with relative quantification by real-time quantitative reverse transcription polymerase chain
reaction (Real-time qRT-PCR).

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DISCUSSION: cfu/ml. at > 2 ppm (AC) and 30s exposure

Biofilm formation by Bacillus spp. on milk time. Bremer et al. (2002) and Parkar et al.
equipment is a most common phenomenon and (2004) also stated that the cleaning efficiency
it has caused contamination, substantial eco- of Clean-In-Place (CIP) systems significantly
nomic loss and safety hazards with about 60% depends on the exposure time, temperature,
of foodborne outbreaks. 78.5% of dairy prod- and cleaning agent concentration. Meanwhile,
ucts such as milk, ice cream and cheese con- stainless steel food contact materials treated
taminated with B. cereus strains from dairy with EW achieved a 5-log reduction in
utensils (Ibrahim et al. 2022). There is evi- B.cereus species, which corresponded with the
dence that electrolyzed water has been intro- definition of sanitization recommended by the
duced to food industries as a novel disinfecting Food and Drug Administration (2005).
agent can work better than water and chlorine Strong oxidizing effect of EO due to the pres-
solutions as a sanitizer of cutting boards and ence of HClO, ClO, and Cl2. HClO can kill
utensils.Table (1) and Fig (1) illustrate EW bacteria by destroying the membrane, leading
efficacy of both types (ER & EO) in eleminat- to leakage of the cytoplasmic content
ing B. cereus contaminating the milk contact Mokgatla et al. (2002), protein denaturation,
surfaces. The results cleared that the highest and stopping cellular metabolism Chen et al.
reduction rate and % of EW were observed (2001). Bactericidal Activities of EW was
when using ER followed by EW (5.57 greater than common sanitizers used in dairy
log10/100 %, followed by EO (4.47 log10 / industry (Jiménez-Pichardo et al. 2016).
80.25%) and finally, ER which recorded a re-
duction rate of 4.27 log10 (76.66%) as com- Fig. (2) Cleared the assay results of the
pared with the control plates . There were sig- relative expression of B. cereus tasA and sipW
nificant differences (P<0.05) between the con- genes contaminating stainless-steel surfaces
trol on one hand and all othe treatments on the after cleaning by using different types of elec-
other hand, such differences were also clear trolyzed water (EW). The expression of both
between both ER and EO separately as well as genes was significantly reduced (p < 0.05) as
between their combination together , while no for the relative expression of the tasA gene. It
significance difference was observed between was recorded as 0.75, 0.81, and 0.37 after us-
using ER and EO (P>0.05). The obtained re- ing ER, EO, and a mix of ER & EO respective-
sults in the current study were in line with Vo- ly, when matched with control one (1.1). In the
robjeva et al. (2004) who reported that five same way, sipW gene expression was recorded
minutes was sufficient to complete the decon- at 0.63, 0.87, and 0.35 by the same aforemen-
tamination of B. cereus from food contact sur- tioned water types, while the control one re-
face. Moreover, Kim et al. (2000) obtained the coded 1.02. The potential way in which EW
same log reduction of B. cereus within two affects gene expression is by suppressing enzy-
minutes by using EO water. In this respect, matic activity, causing the cell wall to become
AOAC (2000) recommended the use of EW less permeable, and allowing intracellular
containing 10 ppm available chlorine (AC) for components to escape (Rahman et al. 2016). In
30 s to reduce > 5 log cfu of aerobic plate this respect, Park et al. (2019) concluded that
count( APC). Similar results were recorded by there are multiple factors involved in biofilm
Al-Qadiri et al. (2019) who revealed that the formation by emetic toxin-producing B. cere-
exposure of food contact surfaces to the reac- us. Furthemore, Hussain & Oh (2017) stated
tive chlorine (60 mg/L) in acidic elyctrolyzed that B. cereus can form several types of bio-
water (AEW) for 5 min could reduce B. cere- films such as air-liquid, submerged, and float-
us by 2.11 log CFU/cm2 .Furthermore, the in- ing pellicles. Bacterial attachment to a surface
hibitory effect of EW against different micro- is influenced by several factors, including the
organisms was previously investigated by Are- physicochemical properties of the substratum
valos et al. (2012) against L. monocytogenes surface and the surface characteristics of the
and Rahman et al. (2016) against E. coli and L. strain used (Whitehead & Verran, 2009). In
monocytogenes (reduction rate of 6.9 log10 addition, bacterial motility is an important fac-

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tor that triggers biofilm formation or attach- mendárez BE. 2012. Effect of neutral elec-
ment of cells to a surface (Abee et al. 2011). trolyzed water and nisin on Listeria mono-
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CONCLUSION : Chinese with English Abstract)

I n conclusion, electrolyzed water is an ex-

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